Expression and function of CD40 on Hodgkin and Reed-Sternberg cells and the possible relevance for Hodgkin's disease

CD40 was originally described as a B-cell-restricted antigen and was subsequently found to be a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is also expressed on dendritic cells, thymic epithelium, monocytes, and some carcinoma cell lines, and plays a critical role in cell contact-dependent activation. Primary and cultured Hodgkin and Reed-Sternberg (H-RS) cells, the presumed malignant cells of Hodgkin's disease (HD); were found to express high levels of cell surface CD40. We found that recombinant CD40 ligand (CD40L) induced interleukin-8 (IL-8) secretion and enhanced IL-6, TNF, and lymphotoxin-alpha (LT-alpha/TNF-beta) release from cultured H-RS cells. These cytokines play a significant role in the clinical presentation and pathology of HD, a tumor of cytokine-producing cells. CD40L had no mitogenic activity for HD-derived cell lines. In contrast, CD40L enhanced expression of costimulatory molecules intracellular adhesion molecule-T and B7-1 on cultured H-RS cells, both of which are overexpressed on primary H-RS cells. In addition, CD40L induced a 40% to 60% reduction of the expression of the HD-associated CD30 antigen, another member of the TNF receptor superfamily. Primary and cultured H- RS cells express not only CD30, but also CD40. CD40L has pleiotropic biologic activities on H-RS cells, and the CD40-CD40L interaction might be a critical element in the deregulated cytokine network and cell contact-dependent activation cascade typical for HD.     

Differential CD26-mediated activation of the CD3 and CD2 pathways after CD6-depleted allogeneic bone marrow transplantation

Patients who have undergone allogeneic bone marrow transplantation (allo-BMT) are susceptible to a variety of opportunistic infectious complications in the months to years after engraftment. Impaired in vitro T-cell functions have been documented in these patients, and these T-cell dysfunctions contribute to the prolonged immune deficiency after allo-BMT. In the present study, we examined the expression of CD26 as well as the reconstitution of CD26-mediated T-cell costimulation via the CD3 and CD2 pathways at various times in patients aged greater than 18 years after CD6-positive, T-cell depleted allo- BMT. We found that the percentage of CD26- and CD3-positive cells, as well as the levels of expression of both antigens, was lower than in normal controls during the first 4 months after CD6-depleted allo-BMT. Subsequently, the amount of lymphocytes expressing CD3 and CD26 and the quantitative surface expression of CD3 and CD26 were not significantly different in patients and normal controls. Functional studies showed that CD26-mediated T-cell proliferation via the CD3 pathway was considerably improved and almost reached normal levels by 1 year, whereas recovery of CD26-mediated T-cell proliferation via the CD2 pathway was delayed for at least 2 years after CD6-depleted allo-BMT. As CD26 involvement in the regulation of human thymocyte activation is restricted preferentially to the CD3 pathway--unlike its involvement with both CD3 and CD2 pathways of peripheral T cells--our results suggest that the different effects of CD26-mediated costimulation via the CD3 and CD2 pathways after CD6-depleted allo-BMT may be a reflection of peripheral T-cell immaturity in those individuals, similar to that seen in mature medullary thymocytes or cord T lymphocytes.     

Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines

Group I Burkitt lymphoma (BL) cell lines, which retain the original biopsy phenotype, have been shown to enter apoptosis in response to a number of external stimuli including serum deprivation, thermal shock, addition of calcium ionophore, and ligation of surface immunoglobulin (Ig) by antibody. Transforming growth factor-beta 1 (TGF beta 1) is known to cause growth arrest in BL lines. Here we show that while it is by itself capable of promoting some degree of apoptosis in group IBL cells, TGF beta 1 cooperates with anti-immunoglobulin to this end. Trimeric soluble recombinant human CD40 ligand (sCD40L) was able to inhibit apoptosis induced by the combination of agonists to some degree, but such rescue proved to be short-lived. Both TGF beta 1 and anti-Ig individually caused BL cells to undergo growth arrest at the G1 phase of cell cycle before their entry into apoptosis: the consequence of sCD40L addition was to maintain the cells in cycle for longer. No induction of the apoptosis-protecting gene, bcl-2, occurred in the presence of sCD40L. These findings are discussed, particularly highlighting the relationship existing between survival and the cell cycle. The strong cooperative effects observed between anti-Ig and TGF beta 1 in promoting apoptosis and the inability of CD40 to signal for long-term rescue raise the potential for a novel therapeutic attack on B-cell lymphoma.     

Moderation of hemophilia A phenotype by the factor V R506Q mutation

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.     

Assessment of aldehyde dehydrogenase in viable cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.     

A Pantetheinase-Resistant Pantothenamide with Potent,On-Target,and Selective Antiplasmodial Activity

Pantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC₅₀], ∼20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of α-methyl-N-phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC₅₀, 52 ± 6 nM), target specificity, and low toxicity.