Occurrence of voriconazole-induced cutaneous squamous cell carcinoma in Japan: data mining from different national pharmacovigilance databases.

Long-term voriconazole use may increase the risk of cutaneous squamous cell carcinoma (cSCC), especially in immunocompromised patients. However, relatively little is known regarding voriconazole-induced cSCC in Japan. Thus, the purpose of this study was to evaluate the association between voriconazole use and cSCC in Japan using different national pharmacovigilance databases. First, using the Japanese Adverse Drug Event Report (JADER) database, we evaluated the association between voriconazole use and cSCC in Japan. Second, using the U. S. Food and Drug Administration Adverse Event Reporting System (FAERS) database, we examined regional differences in the occurrence of voriconazole-induced cSCC between Japan and other countries. We calculated reporting odds ratios (RORs) as disproportionality analysis to evaluate voriconazole-induced cSCC. In this study, cases in which one or more of "Bowen's disease", "Carcinoma in situ of skin", "Keratoacanthoma", "Squamous cell carcinoma in skin", or "Squamous cell carcinoma" were reported as adverse events were considered to be cSCC cases. The analysis based on the JADER database showed an association between voriconazole use and cSCC in Japan, with a ROR (95% confidence interval) of 35.37 (25.60-48.87). Further, the analysis based on the FAERS database revealed that signals were detected in Japan as well as in Western countries and Australia. This study is the first in which the association between voriconazole use and cSCC in Japan is assessed using national pharmacovigilance databases. Healthcare providers need to be fully aware of the potential for cSCC development owing to voriconazole use and in all countries, including Japan, ensure careful follow-up of patients' skin.

COPD as an independent risk factor for osteoporosis and fractures.

Fractures are common in individuals with COPD and occur at higher bone mass values than expected. COPD appears to be an important risk factor for bone fragility. INTRODUCTION: Patients with chronic obstructive pulmonary disease (COPD) have an increased risk of osteoporosis and fractures, but screening and prophylactic measures to prevent both disorders are often neglected in this population. This case-control study assessed the prevalence of osteopenia, osteoporosis, and fractures in patients with COPD, and identified potential risk factors for fractures in this population. METHODS: Overall, 91 patients with COPD (COPD group; COPDG) and 81 age- and sex-matched controls (control group; CG) were assessed with bone mineral density (BMD), thoracic/lumbar spine radiographs, and serum PTH and 25-hydroxyvitamin D (25[OH]D) levels. The occurrence of prior fractures was retrieved from clinical history. RESULTS: The prevalence of total fractures in the COPDG was 57.1% (odds of fracture 4.7 times greater compared with the CG), and the femoral neck T-score emerged as the best predictor of fractures. Compared with the CG, the COPDG had lower spine and femoral BMD (p ≤ 0.01) and 25(OH)D levels (p = 0.01) and 2.6 times greater odds of osteoporosis. Among men, vertebral fractures were more prevalent in the COPDG versus CG (25.9% vs. 6.5%, respectively, p = 0.01). The odds of fracture increased with femoral neck T-scores ≤ - 2.7 in the CG and ≤ - 0.6 in the COPDG. CONCLUSION: These results add robust evidence to an increased odds of osteoporosis and fractures in COPD. Fractures in the COPDG occurred at higher BMD values than expected, suggesting that COPD may be an independent marker of fracture risk, reinforcing a need for regular osteoporosis screening with BMD measurement and prophylaxis of fractures in patients with this disorder.

Pseudo-high affinity interleukin 2 (IL-2) receptor lacks the third component that is essential for functional IL-2 binding and signaling.

Functional studies of the interleukin 2 receptor (IL-2R) of two (ED515-D and Kit225) IL-2-dependent and three (ED515-I, 3T3-alpha beta 11, and Hut102) IL-2-independent cell lines were done. All of these cell lines appeared to express high as well as low affinity IL-2R. However, ED515-I and 3T3-alpha beta 11, which expressed the IL-2R beta chain, did not bind IL-2 at all when IL-2 binding to their IL-2R alpha chain was blocked with anti-Tac monoclonal antibody, whereas the intermediate affinity binding in ED515-D, Kit225, and Hut102 cells remained. We tentatively called the high affinity IL-2R of the former cells pseudo-high affinity IL-2R. The dissociation constant of pseudo-high affinity IL-2R was higher than that of ordinary high affinity IL-2R. Internalization of cell-bound 125I-IL-2 into ED515-I and 3T3-alpha beta 11 cells was less efficient than that into ED515-D cells. The addition of IL-2 neither promoted cell growth nor upregulated IL-2R alpha chain expression in ED515-I and 3T3-alpha beta 11 cells. Furthermore, tyrosine phosphorylation of the cellular proteins (p120, p98, p96, p54, and p38) was induced or enhanced in response to the addition of IL-2 in ED515-D and Kit225 cells, but not in the cell lines expressing pseudo-high affinity IL-2R. Finally, 125I-IL-2 crosslinking followed by SDS-PAGE analysis showed an 80-kD band corresponding to p65 + IL-2, in addition to bands corresponding to IL-2R alpha and beta chain + IL-2 in cells bearing ordinary high affinity IL-2R but not in cells with pseudo-high affinity IL-2R. Taken together, we consider that another protein whose molecular mass is approximately 65 kD is functionally important in IL-2 binding and subsequent signal transduction and may be the third component of IL-2R.

A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion.

Molecular level studies on platelets deficient in collagen-induced aggregation provide evidence for identifying possible platelet collagen receptors. We investigated platelets from a patient with mild bleeding time prolongation, but otherwise normal coagulation data. Her platelets lacked collagen-induced aggregation and adhesion, but retained normal aggregation and release by other agonists. Labeling her platelets with 125I or 3H and analysis by SDS-PAGE/autoradiography showed normal levels of glycoproteins Ia, Ib, IIa, IIb, IIIa, and IV. However, there were significantly decreased incorporations of both radioactivities into a 61-kD membrane glycoprotein (GP), which was identified as GPVI from its mobility on unreduced-reduced, two-dimensional SDS-PAGE. Sugiyama et al. (1987. Blood. 69: 1712) reported that the serum from an idiopathic thrombocytopenic purpura (ITP) patient contained an antibody against a 62-kD platelet protein. Our patient's platelets lacked the antigen for the ITP patient's antibody, demonstrating that the ITP serum contains a specific antibody against GPVI. The patient's parents' platelets contained approximately 50% the normal amount of GPVI, but still had normal collagen-induced aggregation and adhesion. The patient's platelets did not bind to types I and III collagen fibrils. Our results suggest that GPVI functions as a collagen receptor.

Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder.

Recent advances in molecular genetics have revealed the mechanisms underlying a variety of inherited human disorders. Among them, mutations in G protein-coupled receptors have clearly demonstrated two types of abnormalities, namely loss of function and constitutive activation of the receptors. Thromboxane A2 (TXA2) receptor is a member of the family of G protein-coupled receptors and performs an essential role in hemostasis by interacting with TXA2 to induce platelet aggregation. Here we identify a single amino acid substitution (Arg60-->Leu) in the first cytoplasmic loop of the TXA2 receptor in a dominantly inherited bleeding disorder characterized by defective platelet response to TXA2. This mutation was found exclusively in affected members of two unrelated families with the disorder. The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. These results suggest that the Arg60 to Leu mutation is responsible for the disorder. Moreover, dominant inheritance of the disorder suggests the possibility that the mutation produces a dominant negative TXA2 receptor.

Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation.

Thromboxane A2 (TXA2) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR alpha and TXR beta, respectively. These isoforms differ only in their carboxyl-terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR alpha activates adenylyl cyclase, while TXR beta inhibits it. The Arg60 to Leu mutant of TXR alpha, which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR beta retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA2-induced platelet responses such as shape change and phospholipase A2 activation which remain unaffected in the patients with this mutation.

Expression of alpha-(1,3)-fucosyltransferases which synthesize sialyl Le(x) and sialyl Le(a), the carbohydrate ligands for E- and P-selectins,in human malignant cell lines.

Transcripts of alpha-(1,3)-fucosyltransferases in human epithelial cancer and leukemia cell lines were analyzed by Northern blotting and reverse transcriptase mediated-polymerase chain reaction using specific probes and primers which can discriminate between the transcripts derived from the four alpha-(1,3)-fucosyltransferase genes Fuc-TIII, IV, V, and VI. Flow cytometric analysis of the sialyl Le(x) and sialyl Le(a) antigens was also performed on the same cell lines. The sialyl Le(x) antigen was expressed on 14 of 15 epithelial cancer cell lines, and the sialyl Le(a) antigen was detected on 8 of them. The message of Fuc-TIII was detected in most of the epithelial cancer cell lines (14 of 15), which correlated with the surface expression of these carbohydrate determinants. In addition, the messages of Fuc-TIV and Fuc-TVI were detected in most epithelial cancer cell lines, while the message of Fuc-TV was undetectable in most of them. On the other hand, all leukemia cell lines were positively stained for sialyl Le(x), but none of them was stained for sialyl Le(a) in flow cytometry. The messages of Fuc-TIV++ were detected in all leukemia cell lines tested. Small quantities of Fuc-TIII, V, and/or VI messages were also detected in some leukemia cell lines in reverse transcriptase mediated-polymerase chain reaction analysis. These studies indicate that alpha-(1,3)-fucosyltransferase activities in epithelial cancer and leukemia cell lines are mixtures of multiple molecular species of alpha-(1,3)-fucosyltransferases. It is natural that epithelial cancer cells contain a significant amount of Fuc-TIII mRNA and leukemia cell lines contain Fuc-TIV mRNA, since their normal counterparts, normal epithelial cells and leukocytes, respectively, are known to contain these fucosyltransferases. The unexpectedly frequent occurrence of Fuc-TIV mRNA in epithelial cancer cell lines may be related to their retro-differentiation associated with tumorigenesis. Another unexpected finding was a weak but significant expression of the alpha-(1,3)-fucosyltransferases Fuc-TIII, V, and/or VI in leukemia cell lines detected by reverse transcriptase mediated-polymerase chain reaction analysis. Since these enzymes are known to be capable of synthesizing the sialyl Le(x) determinant, this finding implies a possibility that some of them may be involved in the synthesis of sialyl Le(x) in leukemia cells.

Accelerated declining tendency of human T-cell leukemia virus type I carrier rates among younger blood donors in Kumamoto, Japan.

The annual age- and sex-specific human T-cell leukemia virus type I carrier rate of blood donors in Kumamoto, Kyushu, Japan from 1986 to 1990 revealed that the carrier rates of all the age groups below 50 years declined linearly in both sexes (P less than 0.005). Furthermore, the annual declining rates relative to the carrier rates of 16-19-year-old and 20-29-year-old males were higher than those of all of the older males (P less than 0.02), and all female age groups below 50 years had higher relative declining rates than 50-64-year-old females (P less than 0.05). Although several factors, such as a notification program at obstetric clinics, methodological and technical improvement of the assays, wider knowledge of human T-cell leukemia virus type I infection in the latter years, and immigration of individuals from a nonendemic area, might cause an absolute decline of the carrier rate of the blood donors, these factors could not explain the acceleration of the relative declining rate among younger donors. Therefore, this acceleration represents the tendency of the general population.

A recurring translocation, t(3;6)(q27;p21), in non-Hodgkin's lymphoma results in replacement of the 5' regulatory region of BCL6 with a novel H4 histone gene.

3q27 translocations affecting the BCL6 gene can involve not only immunoglobulin genes (IG) but also other as yet uncharacterized chromosomal loci as partners. Here, we describe cloning of the junctional area of a recurring translocation, t(3;6)(q27;p21), in non-Hodgkin's lymphoma of B-cell type and isolation of clones from 6p21; high resolution fluorescence in situ hybridization mapped the clones to sub-band 6p21.3. Nucleotide sequence analysis of a fragment from the junctional area of 6p21 revealed the presence of a novel H4 histone gene that was included in the histone gene cluster on this particular region, and the same fragment detected approximately 380-bp transcripts in hematological tumor cells. Breakpoints on 3q27 of two cases carrying t(3;6) were immediately 3' of the BCL6 exon 1, and the H4 histone gene was substituted for the 5' regulatory elements of BCL6. Because H4 gene expression is tightly coupled to DNA replication, this study suggested an immediate mechanism for deregulated expression of BCL6, leading to the development of non-Hodgkin's lymphoma.

Molecular features of a new human lymphoma cell line carrying both BCL2 and BCL6 gene rearrangements.

Chromosomal translocations and/or their molecular equivalents involving the BCL6 gene on 3q27 band have been suggested to be involved in the development of non-Hodgkin's lymphoma of B-cell type (B-NHL). The rearrangement of BCL6 sometimes coexists with other translocations specific to B-NHL. Here, we report a novel B-cell lymphoma cell line, YM, established from a patient with diffuse large cell lymphoma. The YM cells expressed B-cell-associated antigens in addition to mu delta/kappa monoclonal immunoglobulin. Southern blot analysis of DNA from YM cells demonstrated rearrangement of the BCL2 gene within the 5' flanking region (5'-BCL2). Polymerase chain reaction (PCR) using primer pairs for the BCL2 exons 1 and 2, and for the constant region of the immunoglobulin kappa light chain gene (IGkappa) revealed PCR products encompassing the 5'-BCL2/IGkappa fusion, indicating that the YM cells had a t(2;18)(p11;q21) translocation. The BCL6 gene was rearranged at a point within the first intron, and cloning of the rearranged BCL6 revealed unidentified sequences juxtaposed to the 5' side of the gene. The isolated clones were mapped to 16p11.2 by high resolution fluorescence in situ chromosomal hybridization. Thus, the YM cells carried a 3q27 translocation involving 16p11.2 as a partner. Chromosome painting of metaphase spreads confirmed that the YM cells had both t(2;18) and t(3;16). Northern blot analysis using a fragment immediately adjacent to the breakpoint on 16p11.2 revealed transcriptional activity within this locus. The YM cells expressed abundant transcripts with aberrant sizes from BCL2 and BCL6, indicating deregulated overexpression of the two genes resulting from the t(2;18) and t(3;16). The YM cell line will therefore be useful to study whether BCL2 and BCL6 genes collaborate in the pathogenesis of B-NHL.

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Platelet adhesion to collagen-coated wells: analysis of this complex process and a comparison with the adhesion to matrigel-coated wells.

The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.

Decreased messenger RNA of arachidonate 12-lipoxygenase in platelets of patients with myeloproliferative disorders.

On the basis of the previous work by Okuma and Uchino [Blood 54, 1258-1271, 1979], three patients with myeloproliferative disorders were investigated with a special reference to arachidonate 12-lipoxygenase in their platelets. The cytosol of the patients' platelets showed a markedly reduced activity of arachidonic acid oxygenation to 12-hydroperoxy acid. A peroxidase-linked immunoassay for the 12-lipoxygenase demonstrated only 7-12% of the normal level of the enzyme protein in the cytosol fraction of platelets. Furthermore, 12-lipoxygenase mRNA level was determined quantitatively by a reverse transcriptase-polymerase chain reaction with an internal standard cRNA which was synthesized by in vitro transcription of human platelet 12-lipoxygenase cDNA with a 105-bp deletion. The 12-lipoxygenase mRNA content was 4.7 +/- 2.0 (mean +/- S.D.) ng/10(11) platelets in 13 normal subjects. In contrast, the mRNA content was as low as 0.15, 0.11 and 0.10 ng/10(11) platelets in the three patients. Taken together, the 12-lipoxygenase deficiency in these patients was attributable to the decreased 12-lipoxygenase mRNA level and thus the impaired synthesis of the enzyme protein in their platelets.

Long distance polymerase chain reaction for detection of chromosome translocations in B-cell lymphoma/leukemia.

To establish a rapid and sensitive method to detect neoplastic cells carrying a specific chromosomal translocation in B-cell lymphoma/leukemia, we have developed a novel strategy based on long distance polymerase chain reaction (LD-PCR) amplification. Genomic DNA were extracted from tumor cells carrying a t(14;19)(q32;q13), a t(8;14)(q24;q32), a t(3;22)(q27;q11), a t(2;3)(p12;q27), and a t(3;14)(q27;q32). Oligonucleotide primer pairs were designed to be complementary to exons or flanking sequences of the BCL3, c-MYC and BCL6 oncogenes, and to constant region genes of the IG genes. LD-PCR with a newly available Taq polymerase for longer product synthesis successfully amplified fragments representing BCL3/C alpha junctional sequences for t(14;19); c-MYC/C mu, c-MYC/C gamma and c-MYC/C alpha for t(8;14); BCL6/C lambda for t(3;22); BCL6/C kappa for t(2;3); 5'-BCL6/C mu and 5'-BCL6/C gamma for t(3;14), respectively. The sizes of the amplified fragments were varied from 1.8 kb to 12 kb, which were specific to each material. Present study provides a useful tool for diagnosis and subsequent management of B-cell lymphoma/leukemia characterized with specific chromosomal translocation.

Marked apoptosis of human myelomonocytic leukemia cell line P39: significance of cellular differentiation.

Myelodysplastic syndrome (MDS)-derived leukemia cell line P39/Tsugane could be induced to apoptosis by a variety of agents including metabolic inhibitors, a calcium ionophore and differentiation-inducing agents. As evaluated by characteristic morphological changes and oligonucleosomal lengths DNA ladder, the levels of apoptosis in P39 cells induced by actinomycin D, or A23187, were far greater than in other myeloid lines examined in this study. When 22-oxa-1 alpha, 25(OH)2D3 (D3), dimethyl sulfoxide (DMSO) and all-trans retinoic acid (RA) were used as differentiation-inducers, varying degrees of apoptosis were seen. D3 induced monocytoid differentiation, but not apoptosis above the control level. On the other hand, RA induced profound apoptosis concomitant with the progressive expression of differentiation markers. Studies on morphology, functions and phenotypes of P39 cells exposed to differentiation inducers suggest that the incidence of apoptosis was not affected by the process of differentiation, but cells in the process of varying degrees of differentiation may die via apoptosis. Moreover, RA-treated P39 cells are unique in the simultaneous occurrence of profound apoptosis and differentiation. We propose that RA-treated P39 differentiation model is ideally suited for the study of MDS.

Rearrangement of the BCL6 gene in B-cell lymphoid neoplasms.

We report here a large series of B-cell neoplasms with regard to rearrangement of the BCL6 gene on chromosome band 3q27. Southern blot analysis using probes from the major translocation cluster (MTC) region of the BCL6 revealed rearrangement in 32 of a total of 222 patients with various subtypes of B-cell neoplasm. In non-Hodgkin's lymphoma (NHL), rearrangements of the BCL6 gene were not closely associated with a specific histopathologic subtype but distributed in subcategories in the Working Formulation. A comparative study between NHL associated either with BCL2 or BCL6 rearrangement showed that advanced disease and bone marrow involvement were more frequent in BCL2(+) NHL. In contrast, extranodal involvement was more frequently observed in the BCL6(+) NHL. The survival curve of BCL6(+) NHL was characterized by a rapid decline followed by a plateau. Of the total of 32 BCL6(+) patients, 6 carried both BCL2 and BCL6 rearrangements, and showed clinicopathological properties of follicular lymphoma. This study suggests that BCL6 rearrangement is primarily associated with large cell lymphoma, and that BCL2(-)BCL6(+) NHL could potentially be curable with modern combination chemotherapy.

Immunoglobulin heavy chain class switching, mu to gamma, in a human lymphoma cell line FL-318 carrying a t(14;18)(q32;q21) chromosomal translocation.

Previously, we reported the establishment of a human lymphoma cell line, FL-318, carrying a t(14;18)(q32;q21) chromosomal translocation. FL-318 cells had mu-heavy chain on the cell surface, while they expressed 'sterile' germ-line gamma transcripts, suggesting that the chromatin structure of the immunoglobulin heavy chain (IGH) locus was 'accessible' to class switch recombination. After several months of in vitro cell culture, we found a small population of FL-318 cells expressing the gamma-chain. Using a limiting dilution method, a mu-producing cell clone FL-318M, and gamma-producing FL-318G were isolated. Hybridization studies with various DNA probes from the IGH locus as well as the BCL2 gene demonstrated that the mu-constant gene was deleted on the functional IGH allele of FL-318G cells, and that the cells produced abundant productive gamma-chain messages. These studies indicated that FL-318 cells underwent spontaneous class switching during in vitro cell culture, unrelated to T cell interaction or antigenic stimulation.

High levels of Ca(2+)-independent endonuclease activity capable of producing nucleosomal-size DNA fragmentation in non-adherent marrow mononuclear cells from patients with myelodysplastic syndromes and acute myelogenous leukemia.

The endogenous endonucleases capable of producing nucleosomal-size DNA fragmentation are considered candidates of the key enzyme of apoptosis. We examined these activities in the nuclear fraction of non-adherent marrow mononuclear cells (NonAd-MNCs) from patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) using a nuclear autodigestion method. We detected Ca2+/Mg(2+)-dependent endonuclease activity in all samples examined. In contrast, Ca(2+)-independent activity with the ability to produce nucleosomal-size DNA fragmentation was found only in samples from a proportion of patients with MDS (12 of 26 consecutive cases) and all the patients with AML (n = 6), but not in the samples from control group patients (n = 10). This activity was correlated with the percentage of bone marrow (BM) blast cells to some extent. Although the levels of these endogenous endonuclease activities seem not to be correlated directly with the susceptibility of the cells to apoptosis, we postulate that the Ca(2+)-independent endonuclease activity may be associated with apoptosis and/or cell proliferation. Further follow-up study of these patients may be meaningful to clarify the prognostic significance of the Ca(2+)-independent endonuclease activity in patients with MDS.

Rearrangement of the 5' cluster region of the BCL2 gene in lymphoid neoplasm: a summary of nine cases.

Rearrangement of the BCL2 gene with the immunoglobulin (IG) genes is the most frequent genetic abnormality in B cell lymphoid neoplasms. In the majority of cases, breakages occur at two breakpoint cluster regions; major breakpoint cluster (MBR) and minor cluster region (mcr). In a minority of cases with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL), rearrangements involving the 5' flanking region of the BCL2 (5'-BCL2) have been reported. Here, we investigated 196 patients with NHL and 31 with CLL, with regard to rearrangement of the BCL2 gene. Hybridization analyses using probes representing the three cluster regions revealed that a total of 57 patients had a rearrangement of the BCL2; 42 (73.7%) were within the MBR, seven (12.2%) were within the mcr, and nine (15.8%) had a rearrangement at the 5'-BCL2. The nine patients with 5'BCL2 rearrangement included two with follicular lymphoma, four with diffuse large cell lymphoma and immunoblastic variant, two with leukemic phase of follicular lymphoma, and one with CLL. Comigration analysis with probes for the IG heavy chain gene (IGH), kappa-chain gene (IG kappa) and lambda-chain gene (IG lambda), demonstrated a 5'-BCL2/IGH junction at the JH region in four patients with NHL derived from follicular center B cell. Thus, the 5'flanking region is a third cluster for recombination between the BCL2 and IGH, which is closely associated with the development of follicular center cell lymphoma. Molecular cloning of a 5'-BCL2/IGH junction demonstrated recombination of the two affected genes in divergent orientation. A 5'-BCL2/IG kappa junction was observed in two patients with immunoblastic lymphoma, and one with CLL had a 5'-BCL2/IG lambda recombination. Two patients, including one with a BCL2-MBR/JH junction, lacked obvious recombination of the 5'-BCL2 with IG genes, suggesting the presence of a deletion at the 5'-BCL2. Our findings demonstrated heterogeneity not only in clinicopathological presentation of B cell disease with rearrangement of 5'-BCL2, but also in molecular lesions resulting from the rearrangement.

Leukotriene B4-activated human endothelial cells promote transendothelial neutrophil migration.

We explored the effect of leukotriene B4 (LTB4) on endothelial cells in LTB4-induced transendothelial migration (TEM) of neutrophils as an in vitro model of neutrophil extravasation. Chemotactic response of human neutrophils to LTB4 was significantly lower than that in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP), whereas the extent of TEM in response to LTB4 was significantly higher than that to fMLP. The study on random migration induced by LTB4 and fMLP also showed similar results, which indicated that LTB4 might affect the human umbilical cord vein endothelial cell (HUVEC) barrier. Neutrophil TEM was induced by pretreatment of HUVEC monolayer with LTB4 but not with fMLP. Treatment of endothelial cells by ONO-4057, a LTB4 receptor antagonist, abolished the effect of LTB4 almost completely whereas neutrophils treated with ONO-4057 could transmigrate through HUVEC treated with LTB4. These findings indicated that LTB4 could induce neutrophil TEM by acting on HUVEC.