Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the <Emphasis Type="Italic">Pax5</Emphasis> transgene

We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34⁺ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34⁺ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34⁺ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19⁺ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the κ/λ chain on Pax5-transduced CD19⁺ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors.     

Effects of L-leucine on the insulin production,oxidative metabolism and mitochondrial ultrastructure of isolated mouse pancreatic islets in tissue culture

Summary This study was performed to evaluate whether L-leucine is able to relieve the structural and functional alterations previously described in pancreatic islets exposed in vitro for a prolonged time to a subnormal glucose concentration (3.3 mM). It was found that both the impairment of secretion and the decreased rate of biosynthesis of insulin characteristic, of islets cultured for one week in 3.3 mM glucose were prevented by adding 15 mM L-leucine to the culture medium. Furthermore, the rates of tritiated water production and glucose or leucine oxidation were significantly enhanced after culture in the presence of L-leucine. The rate of DNA synthesis as estimated by the incorporation of tritiated thymidine was, however, unchanged by the presence of L-leucine in the culture medium. Leucine cultured islet cells displayed ultrastructural signs of high functional activity. A detailed morphometric examination revealed fewer but hypertrophic mitochondria. The present results suggest that L-leucine can replace glucose in several respects as a long-term stimulus of the pancreatic B-cells, possibly by acting as a metabolic substrate.Presented in part at the Congresses of the European Association for the Study of Diabetes, Munich, 1975 and Helsinki, 1976     

Haematogones in the peripheral blood of adults: a four-colour flow cytometry study of 102 patients

Haematogones have been extensively characterized in bone marrow, but not in the peripheral blood (PB). We studied 102 PB samples from adult patients with a sensitive flow cytometry method. Sixty-six of 102 samples (65%) contained detectable haematogones, ranging from 0.01% to 1.3% of white blood cells (median 0.06%, mean 0.13%). Of 66 cases with complete blood count data, 51 had absolute haematogone counts of 0.00037-0.105 x 10(9)/l (median 0.0054 x 10(9)/l, mean 0.012 x 10(9)/l). PB haematogones belonged exclusively to the most mature maturational stage. These findings have implications for PB analysis of minimal residual disease in acute lymphoblastic leukaemia and follicular lymphoma.     

Analysis of T-cell subsets in B-cell chronic lymphocytic leukemia: a correlation with the stage of disease

T-cell subsets were determined by the Leu monoclonal antibodies in the peripheral blood and/or bone marrow of 52 patients with B-cell chronic lymphocytic leukemia (B-CLL) not on therapy at the time of study. The diagnosis of B-CLL required that the leukemic cells expressed surface receptors for "la-like" antigen, Fc fragment of IgG, mouse red blood cells (MRBC), C3-coated red cells (EAC), and low density of monoclonal surface immunoglobulin. The Leu-3a+/2a+ ratio was applied to define the balance between the helper/suppressor subsets in the residual T-lymphocytes. Most patients showed a decrease in the Leu-3a+/2a+ ratios at all stages of disease. The decrease in ratio was mainly related to a decrease in the Leu-3a+ T-cell subset. The more advanced stages of B-CLL were associated with lower Leu-3a+/2a+ ratio, higher total white cell and percent lymphocyte counts. There was no correlation between the proportion of EAC or MRBC rosetting cells and stages of B-CLL. This analysis further suggests that B-CLL is an immunosuppressed state that becomes more pronounced in the advanced stages and is characterized by a progressive decrease in the Leu-3a+ (helper) T-cell subset.     

Treatment of systemic lupus erythematosus in two patients with extreme B-cell lymphopenia: importance of immunomonitoring and avoidance of B-cell targeted therapy

Introduction:?Because dysfunction of the B-cell compartment is thought to be important in the pathogenesis of systemic lupus erythematosus (SLE), there has been a recent focus on therapies that target humoral immunity via multiple mechanisms. The aim of this paper was to demonstrate the importance of immunomonitoring in two cases with class II lupus nephritis on steroids who presented with a flare-up of disease. After a thorough work-up for infectious triggers of disease activity, conversion to another histopathological class of lupus nephritis was suspected. Deterioration of the patients’ clinical condition made kidney biopsy impossible, and as B-cell targeted therapy was considered, we decided to perform an immunophenotypic analysis and to tailor therapy to the results of the lymphocyte profile. As we incidentally found extremely low B-cell counts, any B-cell–targeted therapy was prohibited, and cyclophosphamide (Cy) was considered a viable therapeutic option.

Methods:?We performed flow-cytometric lymphocyte (Ly) phenotyping (CD19, CD3, CD3CD4, CD3CD8, CD56/16) on two patients with class II lupus nephritis before and after two intravenous (i.v.) Cy pulse administrations. During all this time, patients were on steroids.

Results:?Both patients showed extreme B-cell lymphopenia, a marker of active SLE, which was not greatly impacted by the treatment over the follow-up period.

Conclusions:?As current therapies are aimed at targeting the B cell, an important component of adaptive immunity, caution must be exercised before their use. In addition, monitoring of Ly subsets is essential due to the occurrence of extreme B-cell lymphopenia.     

Atypical B Cell Receptor Signaling: Straddling Immune Diseases and Cancer

The B-cell receptor (BCR) signaling pathway plays an essential role in the survival, proliferation, differentiation and trafficking of lymphocytic. Recent findings associate aberrant BCR signaling with specific disease pathologies, including B-cell malignancies and autoimmune disorders. Inhibition of the BCR signaling pathway may therefore provide promising new strategies for the treatment of B-cell diseases. This special issue of International Reviews of Immunology focuses on atypical B-cell receptor signaling, its role in immune diseases and cancer, and its implications for potential therapeutic intervention.