Successful therapeutic use of a single-dose of rituximab on relapse in adults with minimal change nephrotic syndrome.

Minimal change nephrotic syndrome (MCNS) usually is considered to have a good renal prognosis, but the frequency of relapses is a therapeutic challenge to physicians. The treatment of patients with multiple relapses remains a matter of controversy, because few controlled studies are available. We report the case of a 25-year-old man who experienced relapses of MCNS. Single-dose rituximab therapy (total dose 500 mg) was given during the fourth relapse. Complete remission occurred 10 days later, when no CD19/20-positive B cells were detected in the blood. This the first report of efficacy of single-dose rituximab therapy to treat multi-relapsing MCNS in an adult patient.

A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate.

A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.

Inducement of antibody that mimics insulin action on insulin receptor by insulin autoantibody directed at determinant at asparagine site on human insulin B chain.

We previously showed that purified human IgG1 insulin autoantibody (IAA) from the serum of male patient T.H. with insulin autoimmune syndrome is directed at a determinant at the asparagine site on the human insulin B chain. An anti-idiotypic antibody (anti-TH) that inhibited TH-IAA binding to human insulin was obtained by immunizing BALB/c mice with TH-IAA. Anti-TH bound to viable IM-9 cells and the purified insulin receptor from IM-9 cells. Anti-TH binding to IM-9 cells and the insulin receptor was inhibited by TH-IAA but not by human IgG. Moreover, incubation of HepG2 cells with anti-TH had an inhibitory effect on insulin binding to HepG2 cells. Anti-TH, like insulin, stimulated amino acid uptake in HepG2 cells. These findings indicate that the conformation of TH-IAA idiotope is a mirror image of the determinant on the insulin B chain, the binding site for TH-IAA on anti-TH is also related to the insulin binding site on the insulin receptor, and anti-TH mimics insulin action on the insulin receptor.

Effects of anti-insulin receptor autoantibodies on the metabolism of human adipocytes.

We studied the effect of sera from two patients who had an unusual form of diabetic syndrome with extreme insulin resistance on the metabolism of human adipocytes in vitro. The IgG fractions from sera A and B, which were obtained from two patients (1 and 2) with insulin-resistant diabetes, inhibited [125I] insulin binding to human adipocytes and, at the same time, stimulated glucose oxidation and inhibited the lipolysis induced by levarterenol in human adipocytes. On the other hand, the IgG fraction from the C serum, which was obtained from patient 2 after her diabetic syndrome had completely disappeared as a result of immunosuppressive therapy, did not inhibit [125I] insulin binding to human adipocytes, stimulate glucose oxidation, or inhibit lipolysis in human adipocytes. These facts suggest that these IgG fractions bind to or near the insulin receptor of human adipocytes, that they exhibit their insulin-like effect by binding to the insulin receptor in vitro, and, furthermore, that they are responsible for the extremely insulin-resistant diabetes. However, the apparent discrepancy between the effects of these IgG fractions on man in vitro and in vivo is puzzling and needs to be explained.

Reverse transcription-polymerase chain reaction for PML-RAR alpha fusion transcripts in acute promyelocytic leukemia and its application to minimal residual leukemia detection.

Chromosome translocation t(15;17) specifically found in acute promyelocytic leukemia (APL) results in cleavage in the introns of PML gene on chromosome 15 and in the intron of the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creation and expression of PML-RAR alpha and RAR alpha-PML fusion genes. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the PML-RAR alpha fusion transcripts rapidly in APL patients. The fusion transcripts could be detected in all of the 10 APL patients studied. Of the two breakpoints in the PML gene so far reported, seven APL patients had the fusion transcript compatible with the downstream (3') breakpoint, and the other three APL patients were considered to have the upstream (5') breakpoint. RT-PCR could detect the fusion transcripts from as little as 50 pg bone marrow RNA, and from as little as 0.5 pg bone marrow RNA with the nested PCR. This method was applied to detect minimal residual leukemia cells in an APL patient who had undergone allogeneic bone marrow transplantation, in whom the RT-PCR could not detect the PML-RAR alpha fusion transcripts at several post-transplant time points. This system could be useful to detect minimal residual leukemia cells and accordingly modify the treatment strategy, as well as to make a quick diagnosis with a small amount of clinical sample.

Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells.

To elucidate the role of phosphatidylinositol-3-kinase (Pl3K) in erythropoietin receptor (EPOR)-mediated signaling, we examined the effects of wortmannin (WT), a specific inhibitor of Pl3K on the proliferation of erythropoietin (EPO)-induced erythroid differentiation in K562 human erythroleukemia cells. Percentage of benzidine-positive cells synthesizing hemoglobin and level of glycophorin A expression in the cells were increased after EPO treatment. EPO-enhanced Pl3K activity was suppressed by WT treatment in a dose-dependent manner and constant inhibition of Pl3K by WT interfered with both hemoglobin synthesis and glycophorin A expression promoted by EPO. Wortmannin, however, did not inhibit hemin-induced erythroid differentiation. These findings in the present study suggest that Pl3K plays a crucial role in the transducing the erythroid differentiation signal through EPOR activated by EPO-binding ib K562 cells and that the signaling pathways involved in EPO-induced erythroid differentiation differ from those involved in hemin-induced differentiation.

Low-dose continuous subcutaneous infusion of granulocyte colony-stimulating factor for chemotherapy-induced neutropenia in acute myelogenous leukemia and its pharmacokinetics.

Granulocyte colony-stimulating factor (G-CSF) shortens the duration of chemotherapy-induced granulocytopenia in acute leukemia. G-CSF is administered by 30-min intravenous infusion at a dose of 200 micrograms/m2/day or 5 micrograms/kg/day in most studies. In this study, the efficacy of a reduced dose (33 micrograms/m2/day) of continuous subcutaneous infusion of G-CSF was compared with the effects achieved by 30-min intravenous infusion of the standard dose (200 micrograms/m2/day) in neutropenia after identical chemotherapy in seven patients with acute myelogenous leukemia who were in remission. The duration of granulocytopenia (< 0.5 x 10(9)/l), thrombocytopenia (< 50 x 10(9)/l); G-CSF administration and fever (> 38 degrees C) were 10.1 +/- 5.0 days, 16.5 +/- 9.3 days, 16.6 +/- 7.4 days and 3.1 +/- 5.4 days for 33 micrograms/m2/day continuous subcutaneous infusion, and 10.7 +/- 6.8 days, 16.7 +/- 9.9 days, 16.1 +/- 7.6 days and 2.0 +/- 2.5 days for 30-min intravenous infusion of the standard dose of G-CSF. In each parameter studied, there was no statistical difference between the two methods of G-CSF administration by paired t-test. The costs for G-CSF could be substantially reduced. In most patients, plasma G-CSF concentration rose to the highest level of 1.8-3.7 ng/ml 48-72 h after starting 33 micrograms/m2/day continuous subcutaneous infusion, and gradually decreased as the peripheral granulocyte count recovered, suggesting binding of G-CSF molecules to the specific receptors on the cells of granulocytic lineage.

DNA vaccine against hamster oral papillomavirus-associated oral cancer.

Previously we developed a carcinogenesis model involving the combination of 9,10-dimethyl-1,2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer.

Successful immunosuppressive therapy in insulin resistant diabetes caused by anti-insulin receptor autoantibodies.

A 45-year-old, non-obese female patient with no previous history of insulin administration was found to have extreme insulin resistance and abnormally high plasma immunoreactive insulin in the absence of anti-insulin antibodies in the serum. Clinically, there was no ketonuria. The patient also had evidence of Sjogren's syndrome with several immunologic features including hypergammaglobulinemia, positive antinuclear antibodies, accelerated erythrocyte sedimentation rate and leukopenia. Plasma pancreatic glucagon and C-peptide were elevated, but other endocrinologic abnormalties were not present. In this patient the insulin resistance appeared to be due to anti-insulin receptor antibodies which could be detected even in 1:500 dilution of serum. Immunosuppressive therapy with prednisolone and cyclophosphamide resulted in a decreased level of serum gamma globulin and a concomitant decrease of blood glucose level. After immunosuppressive therapy for eight months, the diabetic syndrome disappeared completely and anti-receptor antibodies in the serum were no longer detectable. Furthermore, insulin sensitivity returned to normal. However, the patient's glucose tolerance deteriorated after the temporary termination of cyclophosphamide treatment and the lowering of prednisolone dosage.

Aldose reductase inhibitors from the nature.

Aldose reductase (AR) is an NADPH dependent enzyme that catalyses the reduction of the aldehyde to the corresponding alcohols. Diabetic complications including neuropathy, nephropathy, cataracts and retinopathy are considerately caused by accumulation of sorbitol, which is produced from glucose by AR in polyol pathway. The aim of AR inhibitor therapy is to normalize the elevated flux of blood and sorbitol through the polyol pathway in the target tissue. A large number of inhibitors have been prepared synthetically, and some of them are used therapeutically. However, none of them is satisfactory. From the plants, many AR inhibitors have been found, which are discussed in this review. By the structure based functioning of AR and its inhibitors, some will be developed promising in the treatment of diabetic complications. The main structural features of the inhibitors will be a polar head group and a hydrophobic ring system. The plants that contain the AR inhibitors may prevent from diabetic complications.

Prokaryotic expression of an immediate-early gene of human herpesvirus 6 and analysis of its viral antigen expression in human cells.

Segments of an immediate-early (1E) protein (1E03; 958 amino acids (aa)), encoded by clone pSTY03, of human herpesvirus 6 (HHV-6) variant B strain HST were expressed as beta-galactosidase fusion proteins in Escherichia coli. Using Western blot analysis, and the serum of a patient having high titer anti-HHV-6 antibodies, an antigenic region of the IE03 protein was mapped between residues 340 and 505 (pUE03IE-M). The fusion protein expressed in E. coli harboring plasmid pUE03IE-M was purified after electrophoresis in SDS-PAGE, and then immunized in mice to obtain a monospecific antibody. Monospecific antibody raised against the fusion protein reacted with IE03 protein species with apparent molecular weights of 155 and 170 kDa, and was detected as granular fluorescence in nuclei of infected cells by an immunofluorescence antibody test. Furthermore, this antibody reacted only with HHV-6 variant B, but did not react with HHV-6 variant A. The IE03 protein was confirmed to be an IE protein, since the synthesis of this protein was observed in infected cells that were first treated with cycloheximide, which was then replaced with actinomycin D. Further, it was also detected as early as 4 h after infection.

Alkaline phosphatase activity in the human promyelocytic leukemia cell line, HL-60, induced by retinoic acid and recombinant human granulocyte colony-stimulating factor.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) not only enhanced the growth of HL-60 cells, but also significantly increased NBT-reducing ability and alkaline phosphatase (ALP) activity of the cells, which were enhanced by the treatment with retinoic acid (RA). Protein kinase C inhibitors (H-7 and staurosporine) significantly suppressed this induction of ALP. The pretreatment with RA followed by rhG-CSF treatment showed almost the same degree of ALP activity as that induced by the simultaneous treatment with RA and rhG-CSF. This study suggests that RA and rhG-CSF are the potent inducers of ALP activity of HL-60 cells and protein kinase C is supposed to have a role in this induction of ALP.

Synthesis and anti-influenza virus activity of novel pyrimidine derivatives.

Efficient synthetic routes of 2-amino-4-(omega-hydroxyalkylamino)pyrimidine derivatives were investigated in relation to the anti-influenza virus activity of these compounds. The derivatives in which cyclobutyl and cyclopentyl groups were introduced to the beta-position of the aminoalkyl group (especially the cyclobutyl group substituted by a phenylalkyl group at the 3'-position) resulted in improved antiviral potency: i.e. an average 50% effective concentration for inhibition of plaque formation (EC50, microM) of 0.1-0.01 microM for both types A and B influenza virus. The antiviral efficacies were in the order of amino group > hydroxyiminomethyl group > halogen substitution at the 5-position, and chlorine or methoxy group > hydrogen at the 6-position of the pyrimidine ring. The antiviral indices of these compounds were 2-6 with respect to the 50% inhibitory concentration for cell proliferation (IC50, microM) for growing cells, but > 500 to > 10(4) with respect to the IC50 for stationary cells, indicating that these compounds may be efficacious for the topical treatment of influenza virus infection.

Correlation between loss of E-cadherin expression and overexpression of autocrine motility factor receptor in association with progression of human gastric cancers.

Loss of intercellular adhesion and increased cell motility synergistically facilitate tumor cell invasion. We studied these factors in 90 patients with gastric cancers by using an immunohistochemical technique to detect strong or weak expression of E-cadherin (ECD) and autocrine motility factor receptor (AMFR). Normal gastric mucosa (control) reacted strongly for ECD and weakly for AMFR. In study cases, ECD was weak in 47 cases, and AMFR was strong in 39 cases. Weak ECD and strong AMFR expression were associated with tumor dedifferentiation. AMFR expression correlated positively with depth of invasion but not with lymph node metastasis. ECD expression correlated negatively with lymph node metastasis but not with depth of invasion. A strong inverse correlation was found between ECD and AMFR expression. Tumors with weak ECD and strong AMFR expression displayed a more aggressive phenotype than tumors with strong ECD and weak AMFR expression. The postoperative survival of patients with tumors with weak ECD and strong AMFR expression was significantly shorter than that of other groups. Since they are involved in the pathway to development of tumors with a more aggressive phenotype, ECD and AMFR should be examined to evaluate the biologic potential of gastric cancers.

Clinical application of malignancy potential grading as a prognostic factor of human esophageal cancers.

BACKGROUND: Various biologic markers have been reported to be prognostic factors in human esophageal cancers. In the current study, we established a new tumor-grading system representing the malignancy potential of cancer cells and compared it with the clinical-stage system. METHODS: Tumor samples from 77 patients with squamous cell carcinoma of the esophagus were immunohistochemically evaluated for the expression of 10 molecules: the cell cycle-related molecules of cyclin D1, Rb, p16INK4, p27KIP1, and PCNA; the cell-cell adhesion molecules of E-cadherin, alpha-catenin, and beta-catenin; and the heat shock proteins of HSP27 and HSP70. RESULTS: P27KIP1, beta-catenin, and HSP70 were selected for their high hazard ratio in multivariate analysis, and the number of their disordered molecules was used to define the malignancy grade (MG). Five-year survival rates were 83%, 54%, 17%, and 0% for MG1, MG2, MG3, and MG4. The gradation of survival curves was better for MGs than for clinical stages. MGs and clinical stages showed significant correlation; however, 55% of those in higher clinical stages (stage 3 or 4) had lower MG (MG1 or 2) and showed better prognosis than others in their group (stage 3 or 4 and MG3 or 4). The proportions of shorter survival span to cancer death patients (less than 1 year) were 0%, 33%, 75%, and 100% in MG1, 2, 3, and 4, but the clinical stage was not associated with the survival span. CONCLUSIONS: The grading of malignancy potential is clinically useful, especially for selecting patients who may show good prognosis in the advanced clinical stage and for predicting short survival span. These predictions are not possible with the clinical-stage system, which is based on the anatomic spread of cancer cells.

Both positive and negative portal venous and hepatic arterial glucose gradients stimulate hepatic glucose uptake after the same amount of glucose is infused into the splanchnic bed in conscious dogs.

We studied the effects of both positive and negative portal venous and hepatic arterial glucose gradients on hepatic glucose uptake after the same amount of glucose was administered into the portal vein and/or hepatic artery. Studies were performed on eight unrestrained conscious dogs with catheters in the portal vein, hepatic vein, gastroduodenal artery, superior mesenteric vein, and femoral artery and Doppler flow probes on the portal vein and hepatic artery. Glucose was infused as follows: protocol 1, 55.6 micromol/kg/min into the portal vein for the first 90 minutes; protocol 2, 27.8 micromol/kg/min into both the portal vein and hepatic artery for the next 90 minutes; and protocol 3, 55.6 micromol/kg/min into the hepatic artery for the last 90 minutes. The portal venous and hepatic arterial plasma glucose gradient was 2.1+/-0.3, -3.0+/-0.5, and -7.1+/-0.6 mmol/L, the rate of hepatic glucose uptake divided by the administered glucose load was 46%+/-11%, 42%+/-10%, and 57%+/-8%, net hepatic glucose uptake was 25.4+/-5.9, 23.5+/-5.6, and 31.6+/-4.6 micromol/kg/min; and the fractional hepatic extraction of glucose was 10.7%+/-2.2%, 11.6%+/-2.5%, and 15.0%+/-2.1%, respectively (mean+/-SEM of three points at 60, 75, and 90 minutes in each protocol). The rate of hepatic glucose uptake divided by the administered glucose load, net hepatic glucose uptake, and fractional hepatic extraction of glucose did not change significantly despite the various portal venous and hepatic arterial glucose gradients. We also studied the effect of the same amount of intraportal glucose infusion for 240 minutes on net hepatic glucose uptake. From 60 to 240 minutes, net hepatic glucose uptake did not change significantly. In conclusion, the liver took up a large amount of glucose administered into the portal vein and/or hepatic artery, regardless of positive or negative portal venous and hepatic arterial glucose gradients. Augmentation of hepatic glucose uptake is not dependent on the signal of the positive or negative portal venous and hepatic arterial glucose gradient.

Investigation of nosocomial infection caused by arbekacin-resistant, methicillin-resistant Staphylococcus aureus.

An outbreak of coagulase VII-producing, arbekacin (ABK)-resistant, methicillin-resistant Staphylococcus aureus (MRSA) occurred between September 1994 and December 1995, involving five different wards. Twenty-one patients developed skin, wound, drainage, or respiratory tract colonization with coagulase VII-producing, (ABK)-resistant MRSA. Phenotypic characteristics (production of enterotoxin and TSST-1, antimicrobial susceptibility) and molecular-typing procedure (plasmid DNA profile, pulsed-field gel electrophoresis [PFGE] and arbitrarily primed polymerase chain reaction [AP-PCR] of chromosomal DNA) in isolated strains were compared. Plasmid analysis identified four different profiles and 19 of 22 strains recovered had identical patterns. PFGE of chromosomal DNA identified three different subtypes and 18 (81.8%) isolates shared the same subtype. AP-PCR also demonstrated that most strains had the same phenotypic characteristics. Although traditional epidemiological methods; for example, coagulase typing, plays a central role in hospital infection control, combination of plasmid DNA profile, AP-PCR, and PFGE may prove to be a particularly informative means of tracking the nosocomial spread of MRSA.

Investigation of nosocomial respiratory infection due to Pseudomonas cepacia by arbitrarily primed polymerase chain reaction.

We used DNA fingerprinting by the arbitrarily primed polymerase chain reaction (AP-PCR) technique for an epidemiologic investigation of Pseudomonas cepacia nosocomial isolates obtained from patients attending our hospital. This approach was compared with conventional phenotypic typing and pulsed-field gel electrophoresis (PFGE). The patterns of gel electrophoresis of the products of AP-PCR differed significantly according to differences in the concentration of Mg2+ and in pH. AP-PCR and PFGE was identical in their resolving power, as the two methods generated four different profiles and identified the same group of strains. The AP-PCR method constitutes an easy alternative to the well-established PFGE method.

Immunohistochemical identification of corticotropin releasing factor-containing neurons projecting to the stalk-median eminence of the rat.

The site of origin of CRF-containing projections to the rat median eminence was studied with immunofluorescence for CRF in combination with the retrograde transport of True blue. After the injection of True blue into the median eminence, retrogradely-labeled CRF producing neurons were identified in the medial division of the paraventricular nucleus and the periventricular nucleus. CRF neurons in the preoptic region had no positive dye. The present findings demonstrate that CRF neurons in the paraventricular and periventricular nuclei project directly to the median eminence.