Successful treatment of refractory anemia by high-dose methylprednisolone associated with an increment in CD68-positive cells in bone marrow.

Refractory anemia has a relatively low incidence of the subsequent development of acute leukemia and a relatively long survival among the myelodysplastic syndromes (MDS). We observed hematological improvement due to high-dose methylprednisolone in 9 of 18 patients with refractory anemia. The patients' age range was from 28 to 78 years old (mean age: 54), including 14 male and 4 females. A complete response was obtained in 5 patients, minimal response in 4 patients, and no response in 9 patients. Laboratory data of peripheral blood counts and differential counts of bone marrow aspirates were not different, except that fewer chromosomal abnormalities (P = 0.086) were observed in the responder group. Side effects were seen in two patients but were controllable. Overall survival was significantly longer in the responder group (Log-rank P = 0.040, Wilcoxon P = 0.045). The overall survival of responders did not reach the median and 85% of the patients were alive after 180 months, while the median overall survival of the nonresponders was 61.8 months. Disease progression was more frequently seen in the non-responder group (P = 0.045). Furthermore, we investigated retrospectively immunohistochemical bone marrow staining, and a significantly higher percentage of CD68-positive (22.6% +/- 7.1%) and CD45RA-positive cells was observed in the responder group compared to the non-responder group (6.5% +/- 1.3%). Our present results indicate that high-dose methylprednisolone is valuable as a primary treatment before other immunosuppressive treatments, because of its ease of use. High efficacy with high-dose methylprednisolone is expected, especially in patients in which increments in CD68-positive cells in bone marrow are observed.

Genomic structure and mutations in adipose-specific gene, adiponectin.

BACKGROUND: Adiponectin is a collagen-like plasma protein specifically synthesized in adipose tissue. Plasma adiponectin concentrations are decreased in obesity whereas it is adipose-specific. OBJECTIVE: To clarify the significance of the genetic variations in adiponectin gene on its plasma concentrations and obesity. SUBJECTS: Two hundred and nineteen unrelated adult Japanese subjects (123 men and 96 women, age: 20-83 y, BMI: 16-43 kg/m2) including 77 obese subjects (BMI>26.4 kg/m2). MEASUREMENT: Human adiponectin gene was isolated from PAC DNA pools. Mutations in the adiponectin gene were screened by direct sequencing or restriction-fragment polymorphism. The levels of plasma adiponectin were determined by the enzyme-linked immunosorbent assay (ELISA). RESULTS: Adiponectin gene spanned 17 kb on chromosome 3q27, consisting of three exons and two introns. Within 2.1 kb of the 5'-flanking region, there were two octamer elements present in the promoter of adipsin. Two nucleotide changes were identified. One was a polymorphism (G/T) occurring in exon 2, and the other was a missense mutation (R112C) in exon 3. The mean plasma adiponectin levels of the subjects carrying G allele were low (G/G: 4.5 microg/ml; G/T: 5.9 microg/ml; and T/T: 6.3 microg/ml), but were not statistically significant. The allelic frequency between the obese and the non-obese showed no significant difference. The subject carrying R112C mutation showed markedly low concentration of plasma adiponectin. CONCLUSION: Two nucleotide changes have been identified in the adiponectin gene. G/T polymorphism in exon 2 was associated with neither plasma adiponectin concentrations nor the presence of obesity. A subject carrying missense mutation (R112C) showed markedly low plasma adiponectin concentration.

Isolation and characterization of a new member of the human Ly6 gene family (LY6H).

The Ly6 family of genes encodes glycosylphosphatidylinositol-anchored cell surface glycoproteins expressed on various types of cells. Intriguing patterns of expression of Ly6 genes on specific subpopulations of lymphoid and myeloid cells suggest that Ly6 molecules may be involved in the development and homeostasis of hematopoietic cells. We have isolated a new member of the human Ly6 gene family, LY6H, from a human fetal brain cDNA library. Fluorescence in situ hybridization and radiation hybrid analyses assigned LY6H to chromosome 8, where other members of the Ly6 gene family are also located. Northern analysis revealed that LY6H is highly expressed in particular subdivisions of human brain and also in MOLT-3 and -4 acute lymphoblastic leukemia cells. These data suggest that LY6H may play a role(s) in both the central nervous system and the immune system.

Identification and characterization of a novel human phosphatidylinositol 4-kinase.

The extensive sequence homology that exists among the catalytic domains of phosphatidylinositol 3- and 4-kinases allowed us to clone a novel human gene encoding a putative phosphatidylinositol kinase, NPIK. Among other known phosphatidylinositol 3- and 4-kinases, NPIK was most closely related to yeast PIK1 phosphatidylinositol 4-kinase. Several forms of NPIK cDNAs were isolated, and expression of NPIK message was detected in a wide variety of tissues. Fluorescence in situ hybridization and radiation hybrid analyses assigned the NPIK gene to human chromosome 1. Recombinant NPIK protein catalyzed a conversion from phosphatidylinositol to phosphatidylinositol 4-phosphate. The catalytic activity of NPIK was augmented by Triton X-100, and was reduced in the presence of adenosine. Using green fluorescent protein system we determined that NPIK is localized in the cytoplasm. Taken together, the data suggest that NPIK may play a pivotal role in regulating the synthesis of phosphatidylinositol 4-phosphate at the site(s) accessible from cytoplasm.

A novel alcohol resistant metalloproteinase, vimelysin, from vibrio sp. T1800: purification and characterization.

We found a novel metalloproteinase, which has high activity at low temperatures and in the presence of organic solvents, in the culture supernatant of a marine bacterium, Vibrio sp. T1800. The metalloproteinase, named vimelysin, was purified from the culture supernatant by three column chromatographies. About 150 mg of purified vimelysin was obtained from 3.3 liters of the culture supernatant with a high yield of 57%. The purified vimelysin showed a single protein band on SDS-PAGE with molecular weight of 38,000. The isoelectric point of vimelysin was 4.3 by isoelectric focusing. The optimum pH of vimelysin was pH 8.0 or pH 6.5 using casein or furylacryloyl-glycyl-leucine amide (FAGLA) as substrates, respectively. The optimum temperature of vimelysin was 50 degrees C when casein was used as a substrate, but it was 15 degrees C when FAGLA was used as a substrate. Interestingly, vimelysin activity was completely retained after 48 h of incubation at 25 degrees C in the presence of 50% ethanol. Moreover, vimelysin showed 40% activity of the control even in the presence of 10% ethanol, while thermolysin showed only 5% activity under the same conditions.

Thrombopoietin induces megakaryocyte differentiation in hematopoietic progenitor FDC-P2 cells.

Thrombopoietin (Tpo) is a cytokine that specifically regulates megakaryocyte maturation and platelet production. Little is known about the molecular and cellular mechanism of the Tpo-induced megakaryocyte maturation process including polyploidization and platelet release. To study Tpo-induced megakaryocyte differentiation, a mouse cell line FD-TPO, which responds and grows with Tpo, was established from a interleukin-3-dependent hematopoietic progenitor cell line FDC-P2. The FD-TPO cells, expressing endogenous Tpo receptor, grew with Tpo in a dose-dependent manner. Further, Tpo stimulation dramatically induced expression of megakaryocyte/erythroid-specific transcription factors GATA-1 and NF-E2 in FD-TPO cells. Flow cytometry analysis demonstrated that expression of platelet-specific cell surface antigens including CD61 (GPIIIa) dramatically increased in Tpo-stimulated FD-TPO cells and that expression of myeloid-specific antigens, Gr-1 and Mac-1, decreased. Therefore, we concluded that the binding of Tpo to FD-TPO cells induces not only cell growth but also differentiation into mature megakaryocyte-like cells, and thus this cell line was found to be useful for the study of Tpo receptor-mediated growth and differentiation signals.

Characterization of the truncated thrombopoietin variants.

Thrombopoietin (Tpo) is a specific cytokine which regulates megakaryocyte differentiation and maturation. We isolated a truncated mouse Tpo cDNA, the product of which turned out to function neither as an active Tpo variant nor as an antagonist. To define the functional domains of the Tpo molecule further, various truncated and point-mutated Tpo molecules were prepared and their biological activity was assayed. It was found that deletion of the amino terminal side of a potential proteolytic cleavage site, Arg-Arg motif, caused complete loss of Tpo's activity, and that point-mutants lacking one of four conserved cysteine residues lost Tpo activity. We also noticed that Tpo activity was inhibited by the reducing agent. Thus, it was concluded that the amino terminal half of the Tpo is sufficient for Tpo activity, and that the cysteine residues, especially the last cysteine residue located two amino acids away from the Arg-Arg motif, are critical for this activity.

Biological nature of Cryptosporidium sp. isolated from a cat.

A small type of Cryptosporidium oocysts was isolated from a naturally infected cat and its biological nature was investigated. In cats experimentally inoculated with Cryptosporidium oocysts, long-lasting shedding of the oocysts occurred after a prepatent period of 8-10 days, and a number of peaks of oocyst count appeared at intervals of several days to a few weeks, earlier in the infection course. Cryptosporidium infection in cats is likely to pass from an acute to a chronic stage. During the chronic stage, prednisolone injection into the cats gave rise to a recurrence of proliferation of the parasite along with a marked increase in the number of oocysts shed. None of the infected cats showed clinical symptoms. Infection experiments using Cryptosporidium oocysts were unsuccessful in several species of animals such as mice, rats, guinea pigs, dogs, suckling mice and mice previously injected with prednisolone or hydrocortisone.