Establishment of a monoclonal antibody to plasma cells: a comparison with CD38 and PCA-1

A new monoclonal antibody which recognizes plasma cells was developed by utilizing two myeloma cell lines, KMS12PE (12PE) and KMS12BM (12BM). established from the pleural of fusion and bone marrow, respectively, of the same patient. Since I2BM expresses CD20, CD38, and PCA-I antigens, while 12PE has lost CD20. 12PE is considered to be phenotypically more mature than 12BM. The 12PE cells were used to immunize a BALB/c mouse and a MoAb was produced which was more reactive to 12PE than to 12BM. Thus, a clone, D2, was obtained. On Western blotting, D2 detected a single band of 54 kD under both reduced and non-reduced conditions. This antigen was not detected by Western blotting in peripheral blood lymphocytes that had been stimulated with pokeweed mitogen (PWM) for 7 days or in those not so stimulated. On flow cytometry, D2 detected a myeloma cell line, RPMI 8226. Another myeloma cell line, U266, was negative for D2 antigen. Staining various cell lines by D2 and other antiplasma cell antibodies, PCA-1 and CD38, showed that D2 is distinct from PCA-1 and CD38. The fresh myeloma cells of 14 myeloma patients were stained by D2 and for other plasma cell antigens. D2 strongly stained three samples obtained from patients with clinically aggressive myeloma, while CD38 stained all cases except one. PCA-1 was positive in nine samples and negative in five. PCA-1 expression was observed in plasma cells obtained from pleural effusion and peripheral blood, while PCA-1-negative cases were not found in such samples, suggesting that PCA-I expression was related to extramedullary invasion. The morphology of the myeloma cells, classified according to Greipp's criteria, showed that there was no correlation between plasma cell antigen expression and plasma cell morphology. Analysis of D2 antigen expression should provide more information about the heterogeneity of myeloma cells.     

α2-Antiplasmin is involved in the production of transforming growth factor β1 and fibrosis

BACKGROUND: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)-beta is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. METHODS AND RESULTS: We examined experimental fibrosis in mice with a deficiency of alpha(2)-antiplasmin (alpha2AP), which is a potent and specific plasmin inhibitor. We found that the lack of alpha2AP attenuated bleomycin-induced TGF-beta(1) synthesis and fibrosis. In addition, the production of TGF-beta(1) from the explanted fibroblasts of alpha2AP(-/-) mice decreased dramatically as compared to that in wild-type mice. Moreover, we found that alpha2AP specifically induces the production of TGF-beta(1) in fibroblasts. CONCLUSION: The lack of alpha2AP attenuated TGF-beta(1) synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that alpha2AP plays in TGF-beta(1) synthesis during the process of fibrosis. Our results provide new insights into the role of alpha2AP in fibrosis.     

Effect of Arterial Infusion of Bleomycin on Esophageal Carcinoma -- An Evaluation by Nuclear Cytophotometry --

In order to determine the histological changes after a singleinfusion of bleomycin (BLM) into the aortic esophageal artery(BLM A.I.), resected specimens from 58 patients with esophagealcarcinoma (27 cases with BLM A.I., 14 untreated cases, 13 caseswith radiation therapy and four cases with combined therapyof radiation and BLM) were examined and evaluated by Feulgenmicrospectrometry for nuclear deoxyribonucleic acid (DNA) content.The histological changes following the BLM A.I. were characterizedby degeneration and necrosis at the front of the invading carcinomatissue accompanied by inflammatory cell infiltration with foreignbody giant cells and fibrosis. In comparison with untreatedcases, BLM-treated cases showed an increase in nuclear DNA contentand a wide dispersion of the DNA values especially at the perinecroticarea and at the front of advancing carcinoma nests. Therefore,a histological effect of the BLM A.I. was shown by the measurementof the nuclear DNA content of carcinoma cells.     

A synthetic peptide derived from staphylokinase enhances plasminogen activation by tissue‐type plasminogen activator

Summary. Background: A synthetic nonadecapeptide (SP; GPYLMVNVTGVDGKGNELL) previously enhanced the activation of plasminogen by the SAK/plasmin complex. Objectives: To identify the binding site for SP on plasminogen and elucidate the effects of SP on plasminogen activation by the tissue‐type plasminogen activator (t‐PA). Methods: The effects of SP on plasminogen activation were estimated using a chromogenic substrate and from the cleavage of plasmin on SDS‐PAGE under reduced conditions. The binding to SP of various peptides derived from the amino acid sequence of plasminogen was analyzed with an IAsys biosensor. The SP‐mediated structural change to plasminogen was analyzed by circular dichroism (CD) spectroscopy. The thrombolytic effects of SP were examined using a mouse model of thrombosis. Results: SP enhanced the activation of plasminogen by t‐PA. The catalytic efficiency (k_cat/K_m) of Glu‐plasminogen activation by t‐PA was 11.4‐fold higher in the presence than absence of SP. The binding of SP to plasminogen was greatly inhibited by a synthetic peptide, FEKDKYILQGVTSWGLG, located close to the C‐terminal of the plasminogen B region. Near‐ultraviolet CD spectra of the complex between SP and Glu‐plasminogen significantly differed from those of Glu‐plasminogen. When SP was administered in a mouse model of thrombosis, early recanalization was observed in a dose‐dependent manner. However, SP did not cause recanalization in t‐PA gene‐deficient mice. Conclusions: SP bound to the B region and promoted the activation of plasminogen by t‐PA, and then induced effective thrombolysis.     

Antiviral effect of lymphoblastoid interferon-alpha on hepatitis C virus in patients with chronic hepatitis type C

Abstract To study the antiviral effect of lymphoblastoid alpha interferon (IFN) on hepatitis C virus (HCV) we conducted a randomized, controlled trial on 80 patients with chronic hepatitis C using three different doses. Patients were randomly assigned to treatment with 1, 3 or 6 million units of lymphoblastoid IFN-alpha daily for 2 weeks. To assess the antiviral effect of IFN, the amount of HCV present in the serum was estimated by competitive nested polymerase chain reaction (PCR) before and after 2 weeks of treatment. The multiple logistic analysis was used to evaluate factors associated with virus clearance, adjusting the imbalance in predictive factors among patients. Hepatitis C virus became negative as assessed by nested PCR after therapy in 26, 50 and 63% of patients receiving 1, 3 and 6 mega units, respectively. Hepatitis C virus was cleared more often in patients having initially low (< 10⁵/mL) amounts of virus. No significant decrease in the amount of virus was observed in the untreated, control group. Patients without bridging fibrosis in liver histology and with HCV genotypes other than K1 (type II) tended to respond well. These results indicate that lymphoblastoid IFN-alpha suppresses HCV in a dose dependent manner. Higher initial virus amounts, bridging fibrosis and genotype K1 were factors associated with poor response.     

Urinary podocytes in childhood IgA nephropathy

SUMMARY: We have previously demonstrated the presence of detached podocytes in the urine in various glomerular diseases. In this study, we investigated the pathological significance of detachment of urinary podocytes (u-podocytes) in childhood IgA nephropathy. We investigated 28 children with IgA nephropathy. U-podocytes were detected in the urinary sediment by immunofluorescence. Renal pathological changes and urinalysis, including the u-podocyte test, were analysed. The expression of glomerular basement membrane (GBM) components, integrins and cytoskeleton components on urinary podocytes was investigated using dual immunofluorescence in 12 patients. The u-podocyte score paralleled the acute glomerular changes in renal biopsy samples. The infiltration of glomerular macrophages and polymorphonuclear leucocytes, and the number of urinary leucocytes, correlated highly with the u-podocyte score. Some of the u-podocytes expressed integrins and also components of the GBM. Detachment of podocytes reflects the presence of acute glomerular lesions. Infiltration of glomerular leucocytes is considered to be the cause of the detachment, partly because of the destruction of the GBM.     

The effects of transjugular intrahepatic portosystemic shunt on portal hypertensive gastropathy

In addition to variceal bleeding, haematemesis may occur due to haemorrhagic gastritis in patients with portal hypertension. This has been known as portal hypertensive gastropathy (PHG). We have evaluated the effects of the transjugular intrahepatic portosystemic shunt (TIPS) on portal venous pressure (PVP) and endoscopic gastric mucosal changes observed in patients with portal hypertension. We performed TIPS in 12 patients with complications due to portal hypertension as follows: variceal bleeding in nine patients (bleeding from oesophageal varices in seven and gastric varices in two), refractory ascites in three and haemorrhage from severe PHG in one. Endoscopic examinations were performed before and after TIPS for all patients. Changes of PVP and gastric mucosal findings on endoscopy were analysed. Before TIPS, PHG was seen in 10 patients. Portal venous pressure decreased from an average of 25.1 ± 8.8 to 17.1 ± 6.2 mmHg after TIPS ( P < 0.005). On endoscopy, PHG improved in nine of 10 patients. Oesophagogastric varices improved in eight of 11 patients. In one patient with massive haematemesis, haemorrhage from severe PHG completely stopped after TIPS. Because TIPS effectively reduced PVP, this procedure appeared to be effective for the treatment of uncontrollable PHG.     

Biochemical and histological features of hepatitis G virus infection

To assess the biochemical and histological characteristics of hepatitis G virus (HGV) infection, we examined four patients who were infected with HGV only (HGV group), and compared them with 16 patients infected with both HGV and hepatitis C virus (HCV; HGV + HCV group) and 18 patients infected with HCV only (HCV group). Biochemical examination showed a significantly low level of serum alanine aminotransferase (ALT) in the HGV group, and that the gamma-glutamyl transpeptidase (γ-GTP)/ALT ratio in the same group was significantly higher than in the other two groups. Although all three patient groups had a similar degree of liver fibrosis, both the degree of periportal inflammation and total histological activity index were significantly lower in the HGV group than in the other two groups. Fibrous enlargement of the portal tract without lymphoid infiltration and thin fibrous septa was characteristically observed in the HGV group. No significant difference was found between the HGV + HCV group and HCV group. Our results suggest that biochemical and histological changes in HGV infection are very mild and quite different from those of HCV infection.     

Lack of α2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions

BACKGROUND: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. METHODS AND RESULTS: We performed experimental cutaneous wounding in mice with a deficiency of alpha(2)-antiplasmin (alpha(2)AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in alpha(2)AP-deficient (alpha(2)AP-/-) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in alpha(2)AP-/- mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in alpha(2)AP-/- mice than in WT mice. In addition, the amount of released-VEGF from the explanted fibroblasts of alpha(2)AP-/- mice increased dramatically more than in the WT mice. Finally, the intra-jugular administration of anti-VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of alpha(2)AP-/- mice. CONCLUSION: The lack of alpha(2)AP markedly causes an over-release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated-wound closure. CONCLUSIONS: This is the first report to describe the crucial role that alpha(2)AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of alpha(2)AP on cutaneous wound healing.