ALBERT

Description: CD157, a glycosylphosphatidylinositol (GPI)–anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca2+ concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]–ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)–differentiated (CD157+/CD38-) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and β2 integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.

Description: PNH is an acquired clonal disorder of the hematopoietic stem cell (HSC) characterized by intravascular hemolysis, venous thrombosis, and variable degrees of bone marrow failure. In PNH a somatic mutation of the X-linked PIG-A gene in HSC results in complete or partial deficiency of all proteins anchored by the glycosylphosphatidylinositol (GPI) on the membrane of the mutated HSC and in its mature progeny. The close association between PNH and Idiopathic Aplastic Anemia (IAA), and other lines of evidence support the hypothesis that auto-reactive T cells might be responsible for the expansion of hematopoietic PNH clone(s), which is required to cause clinical PNH. Stemming from our observation of a unique patient with PNH and with a large granular lymphocyte (LGL) leukemia with NKT phenotype (Karadimitris et al, Br J Haematol115:1010, 2001), we have measured systematically the percentage of NKT [CD3+ CD8+(bright) CD57+] cells in the peripheral blood of PNH patients. The proportion of NKT cells was quite variable and very similar in 18 patients (6.9±5.9; range: 0.8 – 22.3%) and in 18 healthy individuals (6.5±5.2; range: 0.9 – 21.2; P〉0.5). However, when we analyzed the size distribution of the complementarity-determining region 3 (CDR3) of the TCR-beta chain genes in sorted NKT cells, there was a sharp difference. In healthy individuals we observed a normally distributed ladder of bands of different sizes. By contrast, in 14 out of 15 PNH patients we found a markedly non-random (“oligoclonal”) pattern; and in each patient some clones were predominant. In 6 out of 6 patients followed-up longitudinally over 6–12 months the “oligoclonal” pattern was consistent and persistent. In each of 10 patients in whom we carried out systematic sequencing of the TCR-beta CDR3 of sorted NKT cells we have observed an average of 25 different TCR-beta CDR3 sequences (out of an average of 80 total sequences obtained): but only one or two sequences were predominant. Interestingly, an identical or quasi-identical (single amino acid difference) sequence was found in 4 patients; and in two of these the sequence belonged to one of the predominant clones. In addition, in 5 cases a sequence found in one patient was subsequently found also in another patient. These data are reminiscent of recent findings reported in patients with IAA (Risitano et al, Lancet364:355, 2004): in these patients, however, no identity of sequence was detected. We surmise that in both groups of patients specific T cells clones may be responsible for damage to normal HSCs. However, it is possible that in IAA a number of different antigens are recognized on HSCs in individual patients; whereas in PNH the range of potential target antigens is much more restricted, because they must be present on normal HSC but not on PNH HSCs, thus enabling them to survive the auto immune attack and to expand.

Description: The humanized anti-CD74 monoclonal antibody (mAb) hLL1 is under evaluation as a therapeutic agent. The effects of hLL1—at times in comparison with the CD20 mAb rituximab—were assessed on non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) cell lines and in tumor-bearing SCID mice. In vitro, hLL1 caused growth inhibition and induction of apoptosis in B-cell lines when cross-linked with an antihuman immunoglobulin G (IgG) second antibody. The sensitivity profile of the cell lines was different for hLL1 and rituximab, and antiproliferative activity was augmented when the 2 mAbs were combined. Unlike rituximab, hLL1 did not induce antibody-dependent cellular cytotoxicity or complement-mediated cytotoxicity. In xenograft models of NHL and MM, treatment with hLL1 yielded significant survival benefits without cross-linking agents. Efficacy was greater in the MM model, in which median survival time was increased more than 4.5-fold. Thus, hLL1 has therapeutic potential as a naked mAb for B-cell malignancies because of high antigen expression on malignant cells, specifically MM, with limited expression on normal tissue, and because of its antiproliferative activity. Further, hLL1 may be a therapeutic candidate for rituximab-resistant disease because the 2 antibodies apparently act through distinct mechanisms and exhibit different expression and sensitivity profiles, and activity can be augmented when the mAbs are combined.

Description: Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). Somatic mutations in thePIG-A gene result in the deficiency of several glycosylphosphatidylinositol-linked proteins from the surface of blood cells. This explains intravascular hemolysis but does not explain the mechanism of bone marrow failure that is almost invariably seen in PNH. In view of the close relationship between PNH and idiopathic aplastic anemia (IAA), it has been suggested that the 2 disorders might have a similar cellular pathogenesis, namely, that autoreactive T-cell clones are targeting HSCs. In this paper, we searched for abnormally expanded T-cell clones by size analysis of the complementarity-determining region 3 (CDR3) in the beta variable chain (BV) messenger RNA (mRNA) of the T-cell receptor (TCR) in 19 patients with PNH, in 7 multitransfused patients with hemoglobinopathy. and in 11 age-matched healthy individuals. We found a significantly higher degree of skewness in the TCR BV repertoire of patients with PNH, compared with controls (R2 values 0.82 vs 0.91,P 

Description: Methionine synthase (MS) is a cobalamin dependent enzyme that catalyses the remethylation of homocysteine to methionine. The methionine synthase reductase (MSR) maintains adequate levels of methylcob(III)alamin, the activated cofactor for MS. The aim of this study was to investigate the effect of MS A2756G and MSR A66G polymorphisms on total homocysteine (tHcy), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) levels in 390 pregnant women and their 292 newborns, from Sorocaba city, Brazil. Genotypes of two polymorphisms were determined by PCR-RFLP. Pregnant women with MS 2756AA genotype have higher tHcy and lower Cbl levels than those with 2756G alleles. The MMA values were increased in neonates with MS 2756AA genotypes (Table 1). There are no difference between the maternal values of Cbl, serum folate, tHcy, MMA and SAM according to MSR A66G genotypes.The values of SAM were lower in neonates with MSR 66G alleles than those with AA genotypes (Table 2). We conclude that MS 2756AA genotypes are associated with higher tHcy levels in pregnant women and higher MMA levels in neonates. The MSR 66GG genotypes is associated with lower SAM levels in neonates. Table 1- Distribution of geometric means and confidence intervals 95% (CI 95%) and numbers of subjects for maternal and neonatal values of cobalamin (Cbl), serum folate, total homocysteine (tHcy), methymalonic acid (MMA) and S- adenosylmethionine (SAM) according to genotypes for the polymorphism MS A2756G. Variables Genotypes for MS A2756G Student t Test AA AG + GG Pregnant Women Cbl (pmol/L) 139 (133 – 144) 235 156 (146 – 166) 129 P= 0.001 SF (nmol/L) 14.3 (13.6 – 15.0) 234 14.5 (13.6 – 15.5) 129 P= 0.667 tHcy( μmol/L) 6.8 (6.5 – 7.1) 235 6.2 (5.9 – 6.6) 128 P= 0.036 MMA(nmol/L) 234 (219 – 245) 194 241 (219 – 265) 106 P= 0.610 SAM(nmol/L) 81.8 (77.9 – 86,0) 229 83.1 (79.1 – 87.4) 124 P= 0.663 Neonates Cbl (pmol/L) 227 (212 – 244) 188 234 (213 – 257) 101 P= 0.646 SF (nmol/L) 32.0 (31.0 – 33.0) 186 32.0 (30.8 – 33.2) 99 P= 0.967 tHcy μmol/L) 5.8 (5.5 – 6.1) 185 5.5 (5.1 – 5.9) 100 P= 0.229 MMA(nmol/L) 383 (364 – 402) 183 342 (317 – 369) 100 P= 0.011 SAM(nmol/L) 188 (181 – 196) 178 182 (168 – 197) 98 P= 0.491 Table 2 - Distribution of geometric means and confidence intervals 95% (CI (%%) and number of subjects for neonatal values of cobalamin (Cbl), serum folate, total homocysteine (tHcy), methylmalonic acid (MMA) and S- adenosylmethionine (SAM) according to genotypes for the polymorphism MSR A66G. Variables Genotypes MSR A66G Student t Test AA AG GG Groups not sharing a common superscript letter are significantly different at P

Description: In the homocysteine metabolic pathway, several key enzymes, including methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), have been implicated in abnormal homocysteine accumulation in the presence of rare alleles. In previous study, we showed that lower maternal Cbl levels were associated with higher tHcy and lower S-adenosylmethionine/S-adenosylhomocysteine ratio in pregnant women and their neonates.The aim of this study is to investigate whether MTHFR and MTRR polymorphisms are involved in the risk for elevated total homocysteine (tHcy) and its interaction with low cobalamin (Cbl) or serum folate (SF) levels. Genotypes for polymorphisms MTHFR C677T and MTRR A66G were determined by PCR-FLRP. The serum levels of Cbl, SF and tHcy were determined in 377 pregnant women (37–42 weeks of gestational age), and cutoff values for Cbl and SF were considered the first quartile (low values). Four models of univariate logistic regression analyses were used (Table 1). Pregnant women with MTHFR 677T allele have high risk for elevated tHcy that is increased when 677T allele is associated with low Cbl. Increased risk for elevated tHcy is also met when MTRR 66G allele and low Cbl levels were associated. Women with low SF and common MTHFR and MTRR alleles have high risk for elevated tHcy, that is increased when in association with 677T allele or with 66G allele. Interaction between MTHFR C677T and MTRR A66G polymorphisms and vitamins levels in pregnant women Dependent variables Comparation levels (N) P value Odd Ratios 95% CI P for trend: (a) P115.8 pmol/L (145) 0.015 2.09 1.16 – 3.77 MTHFR 677CT and 677TT genotypes and≤Cbl 115.8 pmol/L (48) 0.001 4.63 2.22 – 9.65 tHcy〉8.3μmol/L MTHFR 677CC genotype and SF 〉 10.9 nmol/L (ref) (148) b 1.00 MTHFR 677CC genotype and≤SF 10.9 nmol/L (33) 0.008 3.20 1.35 – 7.59 MTHFR 677CT and 677TT genotypes and SF 〉 10.9 nmol/L (133) 0.035 1.95 1.05 – 3.61 MTHFR 677CT and 677TT genotypes and≤SF 10.9 nmol/L (59) 0.001 6.62 3.31 – 13.26 tHcy〉8.3μmol/L MTRR 66AA genotype and Cbl〉 115.8 pmol/L (ref) (96) c 1.00 MTRR 66AA genotype and ≤Cbl 115.8 pmol/L (23) 0.222 1.90 0.68 – 5.29 MTRR 66AG and 66GG genotypes and Cbl〉115.8 pmol/L (183) 0.418 1.29 0.70 – 2.39 MTRR 66AG and 66GG genotypes and ≤Cbl 115.8 pmol/L (69) 0.013 2.46 1.21 – 5.01 tHcy〉8.3μmol/L MTRR 66AA genotype and SF 〉 10.9 nmol/L (ref) (92) d 1.00 MTRR 66AA genotype and ≤SF 10.9 nmol/L (27) 0.006 3.83 1.47 – 9.96 MTRR 66AG and 66GG genotypes and SF 〉 10.9 nmol/L (186) 0.399 1.34 0.68 – 2.63 MTRR 66AG and 66GG genotypes and≤SF 10.9 nmol/L (65) 0.001 4.78 2.26 – 10.10 In conclusion, the interaction between MTHFR and MTRR polymorphisms and low folate and cobalamin serum levels may explain the increased risk for elevated tHcy found in pregnant women.

Description: Following the identification, in collaboration with the Spanish PETHEMA group, of distinct prognostic categories among APL patients receiving AIDA-like therapies (Sanz et al. Blood 2000) the Italian GIMEMA group designed a protocol for newly diagnosed APL (AIDA-2000) in which the intensity of post-remission treatment was adapted to the relapse risk. A total of 298 PML/RARa-positive patients with median age 40 yrs (range 1–60) were enrolled during the period January 2000 – February 2003 from 64 Italian institutions. After the standard AIDA-0493 induction (Mandelli et al, Blood 1997), patients with low- and intermediate-risk received 3 anthracycline-based consolidation courses with idarubicin, mitoxantrone, and idarubicin as in the PETHEMA LPA-96 (Sanz et al. Blood 1999), whereas patients with high-risk disease (WBC 〉 10 x 109/L) received the same 3 anthracycline courses with the addition of cytarabine, etoposide and cytarabine plus 6-thioguanine during the first, second and third course, respectively, as in the original AIDA. In addition, distinct from those in the AIDA-0493, all patients enrolled in the AIDA-2000 received concomitant ATRA 45 mg/m2 for 15 d during each consolidation course. The results of the AIDA-2000 series were compared to those obtained in 346 consecutive patients (median age 36 yrs, range 1–60) enrolled in the AIDA-0493 study during the period May 1997 – May 2000. All patients in either studies who tested PCR-negative post-consolidation received ATRA maintenance for a total of two years. After induction, 323/338 (96%) and 276/294 (94%) evaluable patients achieved CR in the AIDA-0493 and AIDA-2000, respectively (P=0.34). Molecular remission was obtained after consolidation in 291/296 (98%) and 235/238 (99%) patients (P=0.69). With a median follow-up of 4.5 and 2.0 yrs in the two studies, the DFS at 2.0 yrs for patients in the AIDA-0493 and AIDA-2000 was 84% and 90%, respectively (P=0.09), whereas the CIR rate at 2.0 yrs was 14% and 5%, respectively (P=0.04). Five and 9 patients died in CR in the two series and were equally distributed among risk groups. By comparing separately the distinct risk groups in the AIDA-0493 and AIDA-2000, there was no significant difference in the CIR rate for low- (3% vs. 2%) and intermediate-risk (11% vs. 9%) groups, while a significantly higher CIR was observed in the AIDA-0493 for high-risk (29% vs. 2%, P=0.0004). In line with recent PETHEMA results, our data confirm that anthracycline-based consolidation is equally effective as cytarabine-containing regimens for patients with low- and intermediate-risk and suggest that a risk-adapted strategy including ATRA for consolidation provides an outcome improvement in newly diagnosed APL. In addition, our results suggest a benefit in terms of relapse rate reduction using cytarabine coupled to anthracyclines and ATRA during consolidation in the high-risk group.