Intra-articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII-deficient mice.

BACKGROUND: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. METHODS AND RESULTS: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor-1 (PAI-1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI-1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI-1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII-deficient mice. Interestingly, intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII-deficient mice. The therapeutic effects of a single intra-articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. CONCLUSIONS: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra-articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.

Immune response against serial infusion of factor VIII antigen through an implantable venous-access device system in haemophilia A mice.

Haemophilia A is a life long bleeding disorder caused by an inherited deficiency of factor VIII (FVIII). About 30% of haemophilia A patients develop neutralizing antibodies as a consequence of treatment with FVIII concentrates. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII. We evaluated the immune responses to serial intravenous administration of FVIII in preimmunized haemophilia A mice. We introduced an implantable venous-access device (iVAD) system into haemophilia A mice to facilitate sequential infusion of FVIII. After preimmunization with FVIII, the haemophilia A mice were subjected to serial intravenous administration of FVIII through the iVAD system. In all mice with serial infusion of FVIII, high titers of anti-FVIII inhibitory antibodies developed at 10 exposure days (EDs). However, the anti-FVIII IgG titers were decreased after 150 EDs of sequential low-dose infusion of FVIII [0.05 U g(-1) body weight (BW) five times per week]. Proliferative response to ex vivo FVIII stimulation was significantly suppressed in splenic CD4(+) T cells from mice with serial low-dose FVIII infusion compared with those from mice with high-dose FVIII infusion (0.5 U g(-1) BW five times per week) or preimmunized mice. Moreover, splenic CD4(+) T cells from mice with serial low-dose infusion of FVIII failed to produce interleukin-2 and interferon-γ. These data suggest that serial infusion of FVIII could induce T-cell anergy in haemophilia A mice with inhibitor antibodies.

Tolerance to solid organ transplants through transfer of MHC class II genes.

Donor/recipient MHC class II matching permits survival of experimental allografts without permanent immunosuppression, but is not clinically applicable due to the extensive polymorphism of this locus. As an alternative, we have tested a gene therapy approach in a preclinical animal model to determine whether expression of allogeneic class II transgenes (Tg's) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tg's, in combination with a short treatment of cyclosporine A, prolonged graft survival with DR and promoted tolerance with DQ. Class II Tg expression in the lymphoid lineage and the graft itself were sequentially implicated in this tolerance induction. These results demonstrate the potential of MHC class II gene transfer to permit tolerance to solid organ allografts.

Assessment of transduction rates of porcine bone marrow.

Although drug resistance is commonly used as an indicator of gene transfer in various cellular contexts, the assessment of drug resistance is often imprecise and over-estimated. To measure accurately transduction efficiencies of the retroviral-mediated transfer of genes encoding the neomycine phosphotransferase (Neo(r)) and porcine major histocompatibility (MHC) class II in pig bone marrow cells (BMC), the fraction of targeted progenitors was evaluated by both colony-forming unit granulocytes/macrophages assays (G418r CFU-GM) and by PCR analysis of the transgenes (Tg). Transduced and untransduced BMC were selected at different concentrations of G418 and revealed high individual variability of drug sensitivity. Comparison of the results obtained by estimating the CFU frequency and the PCR assays on drug-resistant colonies demonstrated a marked overestimation of BM transduction rates when determined by G418 resistance alone, because only approximately one-third of individual colonies were positive for both the Neo(r) and the class II Tg. Because this discrepancy is likely to affect the overall assessment of transduction rates using drug resistance markers, our data attest for the need of a combination of molecular assays to determine transduction efficiencies accurately.

Relationship between chimerism and tolerance in a kidney transplantation model.

The persistence of donor leukocytes in recipients of organ allografts has been associated with long-term graft acceptance. However, it remains unclear whether this peripheral donor cell microchimerism plays an active role in graft acceptance or is simply a consequence of the maintenance of sufficient immunosuppression to avoid rejection. A model of kidney transplantation between swine leukocyte Ag (SLA)-matched miniature swine, in which tolerance can be established with or without immunosuppressive treatment, has been used to study the correlation between donor leukocyte chimerism and kidney graft acceptance. SLA-identical kidney transplants were performed from animals positive for an allelic pig leukocyte Ag to animals negative for this marker. SLA-identical kidney transplant recipients given a 12-day course of cyclosporine (CyA) (n = 3) became tolerant, showing stable serum creatinine levels (1-2 mg/dl) after cessation of CyA treatment. Donor cell chimerism (0.2-0.7%) was present by FACS in all three animals with peak levels detected at 3 wk. Two control animals receiving SLA-identical kidney grafts without CyA also showed stable serum creatinine levels and became tolerant. However, in neither of these animals could donor leukocytes be detected in the peripheral blood beyond 1 wk following transplantation. In one additional control animal, ureteral obstruction occurred at day 10, and was associated with additional peripheral chimerism, presumably related to inflammation rather than to immune status. These results indicate that the persistence of donor cell chimerism is not a requirement for the maintenance of tolerance to organ allografts in this model.

Importance of positive peritoneal lavage cytology findings in the stage grouping of gastric cancer.

Although peritoneal lavage cytology is widely performed during surgery for gastric cancer and the results have been reported to be one of the accurate prognostic factors, the cancer stage is determined independent of the results of lavage cytology according to the First English Edition of Japanese Classification of Gastric Carcinoma. In this study we demonstrated the validity of lavage cytology for accurately staging gastric cancer. Between 1988 and 1996, peritoneal lavage cytology was performed in 347 patients with resectable gastric cancer. Among them, cytology was positive in 29 cases (8.4%). The survival rate of the cytology-positive patients in each stage was worse than that of all patients in the same stage. The prognosis of patients with positive cytology findings and serosa-exposed gastric cancer was significantly worse than that of negative cytology findings and serosa-exposed gastric cancer, and similar to that of negative cytology findings and serosa-infiltrating gastric cancer. Our data indicated that positive cytology findings thus indicated a poor prognosis, and the prognostic difference between positive and negative cytology findings was approximately a one-stage difference in the Japanese stage grouping. Based on our findings, the results of peritoneal lavage cytology should thus be included in the factors for staging gastric cancer.

Impact of preoperative serum carcinoembryonic antigen, CA 19-9 and alpha fetoprotein levels in gastric cancer patients.

The relationship between preoperative serum carcinoembryonic antigen (CEA), CA 19-9 and alpha-fetoprotein (AFP) levels and their clinicopathological features were evaluated in gastric cancer patients. The positive rates of CEA, CA 19-9 and AFP were 24.8, 27.6 and 12.7%, respectively. Gastric cancer with deeper tumor invasion was significantly more common among patients positive for these tumor markers. Patients with positive CEA or CA 19-9 values had a significantly high risk of lymph node metastases (p = 0.045 and p = 0.002, respectively). Synchronous liver metastases was more commonly found in patients with a positive CA 19-9 value. A significant difference (p < 0.001) in survival rate was found between patients with positive CA 19-9 values and those with negative values. CA 19-9 is useful for the prognosis of gastric cancer patients, whereas CEA, although unsuitable for prognosis, contributes to the prediction of cancer invasion.

Evaluation of a new staging system by the Japanese Research Society for Gastric Cancer.

The feasibility of the new classification of stage grouping by the Japanese Research Society for Gastric Cancer was evaluated. During the 22-year period between January 1975 and December 1996, a total of 1294 patients with primary gastric cancer underwent laparotomy at the Department of Surgery, Chiba University; 1222 had their lesions removed during the gastrectomy and 72 remained nonresected. Cases of direct operative death totaled 17 (1.3%). Five hundred patients (38.6%) died of a relapse of the original cancer and 42 (3.2%) died of other diseases within the followup period. Six patients (0.5%) were lost during the followup. The 5-year cumulative patient survival rates of the seven stages of the new stage grouping were distinctly proportional, and the differences were also statistically significant except between stages IIIb and IVa. The two major revised points in the new stage grouping, new classification of the depth of cancer invasion, and new stage grouping by a mosaic combination of the degree of invasion and lymph node metastasis were thus found to be reasonable based on the actuarial 5-year survival rates of the subgroups in the same stage. The present study also showed that the classification of stage IV still requires further discussion.

Enhanced gene expression of transforming growth factor-alpha and c-met in rat urinary bladder cancer.

To investigate the roles of growth factors in bladder cancer, changes in the expression of messenger RNAs (mRNAs) for several growth factors and their receptors were examined during rat bladder carcinogenesis induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Northern blot analysis showed that the contents of mRNAs for transforming growth factor-alpha (TGF-alpha) and c-met/hepatocyte growth factor (HGF) receptor increased with BBN treatment. Epidermal growth factor (EGF) receptor mRNA was hardly affected by the treatment; while mRNA for fibroblast growth factor (FGF) receptor 1 and transforming growth factor-beta (TGF-beta) type II receptor decreased with BBN treatment. A rat bladder tumor cell line, NBT-II, expressed both TGF-alpha and c-met mRNAs, and HGF showed apparent scattering and growth-stimulating effects on the cells. These results indicate the possibility that TGF-alpha produced by a bladder cancer, in addition to urinary EGF, plays a role in the development of bladder cancer, and that enhanced cell motility due to activation of the c-met/HGF receptor participates in the invasion and metastasis of the cancer cells.

[Giant intrascrotal lipoma: a case report].

A case of giant intrascrotal lipoma is presented. The patient was a 72-year-old man with the chief complaint of painless swelling in the scrotum which had been noticed about 10 years previously. In the right scrotum, an elastic soft mass with negative transillumination was palpated. Ultrasonography and X-ray film demonstrated that it was solid mass. Under the diagnosis of intrascrotal tumor, an operation was performed. As the testis, epididymis, and spermatic cord was intact, the tumor was removed. The pathological diagnosis of the tumor was a well-capsulated benign lipoma. Including the present case, 29 Japanese cases of intrascrotal lipoma are reviewed.

[Renin expression in rat testis is controlled by gonadotropin].

The effect of human chorionic gonadotropin (HCG) on renin concentration and renin mRNA was studied using the testis and kidney of rats. Forty-five rats were divided into three groups. The first group (25 rats) was used for short time course study with single injection of 150 IU HCG and the second group (10 rats) for long term injection of 150 IU HCG daily for 3 weeks. In both groups, serum and testicular sample were taken after 24 hours both nephrectomy in order to exclude the influence of renal renin and angiotensin. The third group (10 rats) was used to find the effect of HCG on renal renin. Plasma and testicular renin concentration were measured by radioimmunoassay of angiotensin I. The specificity of renin concentration was determined using a specific renin antibody. Renin mRNA of the kidney and testis were measured by a sensitive RNAase protection assay method. Testicular renin mRNA increased and reached the maximum level at 8 hrs after 150 IU HCG single injection. And also testicular renin concentration significantly increased and it showed the maximum levels at day 3. However long term HCG administration in rats showed higher in levels in testicular renin concentration, however renin mRNA level was not increased. Non-nephrectomized rats showed low levels of plasma renin concentration and no changes in mRNA after HCG administration. The results suggest that renin angiotensin results suggest that renin angiotensin system independently exists in the testis and testicular renin mRNA is increased by HCG administration.

[Analysis of antigen non-specific suppressor T cells factor (TsF) which suppresses the generation of cytotoxic T lymphocytes].

Anti-mouse melanoma suppressor T cells clone (F12) secreted a functional factor (TsF) which suppresses the generation of cytotoxic T lymphocytes (CTL). Ts clone F12 secreted TsF into culture supernatant by stimulating for more than 12 hours with anti-CD3 monoclonal Ab. In syngeneic CTL induction system, TsF suppresses the generation of both anti-melanoma and anti-lymphoma CTL. Moreover, TsF suppresses allogeneic CTL not only from C57BL/6 mice but also from C3H mice. Analysis of the biochemical and physiochemical character of TsF shows that TsF is stable for variance of pH and freeze and thawing, but unstable for heat in region of 100 degrees C. and in acetonitorile. It is also suggested that the molecular weight of TsF is in the vicinity of 12,000 daltons on the analysis in high performance liquid chromatography. F12 clone secretes other suppressive cytokine, TGF-beta and INF-gamma. However, TsF seems different from these suppressive cytokine, because anti-TGF-beta serum which blocks the suppressive function of TGF-beta 1 for CTL generation does not block TsF function. Moreover, recombinant INF-gamma does not suppress the generation of CTL.

Autoreactive T-cell clones which suppress cytotoxic T cell responses.

Autoreactive T-cell clones (Thy 1+, CD4+, CD3+) which suppress generation of cytotoxic T lymphocytes (CTL) were established in long-term in vitro culture by stimulation with GM3-liposomes or soluble melanoma (B16) antigen composed of GM3. The T-cell receptors (TCR) of two representative clones analyzed used the same TCR alpha- and V13+ beta-chains. The clones produce only interferon gamma(IFN-gamma) but not interleukins (IL)2 and 4, despite their CD4+ phenotype, suggesting that they are not a typical TH1 or TH2 type. The clones are effectively stimulated by IFN-gamma treated (I-Ab/GM3+) B16 melanoma or I-Ab-transfected GM3+ L cells, but not by GM3-/I-Ab mutant melanoma, EL 4, or I-Ad/k-transfected L cells. This strongly suggested the involvement of GM3/class II in T-cell recognition. Antigen specificity was required for stimulation of the clones. However, once stimulated, they suppressed CTL generation in an antigen non-specific fashion. As class II+ B16 melanoma cells effectively function as antigen-presenting cells to stimulate the autoreactive suppressor T cell (Ts) clones of this type, this negative circuit between class II+ tumor cells and IFN-gamma-producing Ts would be a possible mechanism whereby tumor cells could escape the immune system.

Effects of combination treatment with FK506 and cyclosporine on survival time and vascular changes in renal-allograft-recipient dogs.

In our previous experiments studying the effects of FK506 on renal allografting in the dog, we encountered two major problems. One problem was anorexia and the other problem was vascular changes mainly in the recipient heart. Anorexia was generally dose dependent, but the vascular changes were seen to be more prominent at lower doses rather than at higher immunosuppressive doses. The present study was undertaken to study these two problems. A nonanorexic, vascular change-related, nonimmunosuppressive dose of FK506 was combined with a low dose of cyclosporine or prednisolone in beagle dogs after renal allografting. Treatment with either FK506 alone at a dose of 0.32 mg/kg or cyclosporine alone at 2.5 mg/kg was not effective in prolonging renal recipient survival. The recipient dogs died of rejection, and a variety of vascular changes were observed in the hearts of both groups. Combined treatment with FK506 and cyclosporine at these same doses resulted in statistically significant prolongation of the survival time of the renal recipient (P less than 0.01), and histologic studies showed that the frequency and severity of the vascular changes were suppressed in the recipient receiving the combined treatment. The combination of FK506 and prednisolone at 0.5 mg/kg was not effective in prolonging survival. Furthermore, the extent of vascular changes was similar to those found in recipients receiving FK506 alone. The data suggest that combined treatment with low doses of both FK506 and cyclosporine acted synergistically in prolonging canine renal allografts and that the vascular changes frequently seen at low doses of FK506 were reduced by additional immunosuppression with a low dose of cyclosporine.

[Clinical feature of male non-gonorrhoea urethritis and minocycline treatment of Chlamydia or Ureaplasma-infected urethritis].

Thirty-nine male patients with urethritis were studied for gonorrhoea or non-gonorrhoea infections. Only 2 patients were infected with N. gonorrhoeae, the other 37 patients were non-gonorrhoea urethritis (NGU). In 9 of these patients, C. trachomatis was identified and in 6 patients, U. urealyticum was isolated. No chlamydial urethritis was combined with ureaplasma. There was no clinical difference between chlamydia and ureaplasma infection, such as serous urethral discharge or mild pyuria. Minocycline was given orally at the dose of 200 mg daily for 7 to 42 days to these patients. Seven of the 9 patients (78%) with C. trachomatis and 7 of the 6 patients (67%) with U. urealyticum infection showed improvement of subjective and objective symptoms after minocycline. In no case, was an adverse reaction noted. Minocycline was effective in the treatment of both C. trachomatis and U. urealyticum urethral infection.