Clinicopathological significance of laminin-5γ2 chain expression in superficial esophageal cancer.

The glycoprotein laminin 5γ2 chain (LN-5γ2) has recently become a focus of increased interest and investigation as a marker of invasion in gastrointestinal malignancies. We investigated the significance of LN-5γ2 expression as a prognostic factor in superficial esophageal cancer. The study population consisted of 87 patients who had undergone a transthoracic esophagectomy and three-field lymphadenectomy for the treatment of superficial esophageal cancer at Tokai University Hospital. Formalin-fixed, paraffin-embedded sections of the resected specimens were examined using immunohistochemical staining and hematoxylin and eosin staining to assess the correlations between the LN-5γ2 expression pattern and the clinicopathological factors (age, sex, T-factor, N-factor, ly-factor, v-factor, degree of differentiation, infiltrative growth pattern, tumor node metastasis classification of malignant tumors [TNM] stage, etc.) and the patient outcome. The expression pattern of LN-5γ2 was classified into an extracellular type (E type), characterized by the staining of extracellular matrix such as the basement membrane and the stroma (31 cases, 35.6%), and a cytoplasmic type (C type), characterized by the staining of the cytoplasm in the cancer cells (56 cases, 64.6%). The expression pattern was not correlated with any of the clinicopathological factors that were assessed. However, univariate analyses of the survival analysis data showed that the N-factor (P = 0.011), TNM stage (P = 0.011), and LN-5γ2 C type (P = 0.017) were prognostic factors. A multivariate analysis revealed that the N-factor (P = 0.049) and LN-5γ2 C type (P = 0.048) were prognostic factors. In the survival analysis, a univariate analysis of the 75 T1b cases also showed that the N-factor (P = 0.048), TNM stage (P = 0.048), and LN-5γ2 C type (P = 0.029) were prognostic factors, while a multivariate analysis showed that the LN-5γ2 C type (P = 0.035) was a prognostic factor. The C type expression of LN-5γ2, i.e. confined to the cytoplasm, was correlated with an unfavorable outcome among the patients with superficial esophageal cancer in the present series. Observation of the LN-5γ2 expression pattern may be useful for the diagnosis of highly malignant tumors.

Accumulation of p53 in esophageal squamous cell carcinoma.

p53 is one of the most important tumor suppressor genes. Mutation of the p53 gene can be detected immunohistochemically as over-expression of its protein in the nucleus. The p53 gene product is known to regulate cell growth and proliferation. Genetic alterations related to the carcinogenesis or progression of esophageal cancer are poorly understood. We examined accumulation of p53 protein in esophageal squamous cell carcinomas including early-stage cancers, and its clinicopathological significance. p53 immunoreactivity in the cancer tissues was found in 61 (79.2%) of 77 esophageal squamous cell carcinomas. Over-expression of p53 protein (diffusely and focally positive staining) was seen in 70.1% (54/77). p53 over-expression was detected not only in the cases of in situ or intramucosal carcinomas, but also in invasive carcinomas. No significant correlations were found between p53 over-expression and clinicopathological features such as depth of tumor invasion, lymph node metastasis or venous/lymphatic invasion. These results suggested that p53 mutations may be closely associated with the early-stage of pre-invasive esophageal carcinoma, and p53 gene mutations may play an important role in the carcinogenesis of human esophageal cancer.

Pancreatic acinar cell carcinoma with endocrine differentiation: immunohistochemical and ultrastructural analyses.

The majority of pancreatic malignant tumors are adenocarcinomas of the ductal type (ductal cell carcinomas) and combined tumors consisting of different tumor components are very rare. We present here a rare case of acinar cell carcinoma with apparent foci of endocrine differentiation. A 46-year-old man underwent pylorus-preserving pancreatoduodenectomy under the diagnosis of pancreatic tumor. The pancreatic tumor was mainly composed of typical acinar cell carcinoma, but some tumor cells were positive for both acinar and endocrine cell markers such as pancreatic amylase, trypsin, lipase and chromogranin A. At the electron-microscopic level, the tumor cells were seen to have numerous electron-dense neuroendocrine, as well as a few zymogen-like, granules. The tumor part positive for both acinar and endocrine cell markers originated from a subclone (dis-differentiated tumor cells) of the typical acinar cell carcinoma tissue of the pancreas.

MUC1 and MUC2 expression in human gallbladder carcinoma: a clinicopathological study and relationship with prognosis.

Mucins are a group of high molecular weight glycoproteins consisting of a mucin core protein and O-linked carbohydrates. To date, nine apomucins (MUC1-8, and MUC5B) have been identified. Recent studies have demonstrated that MUC1 is expressed in tumors of various human organs, and may function as an anti-adhesion molecule that inhibits cell-to-cell adhesion, inducing tumor metastasis. MUC2 is a major secreted mucin of colon and is known to be expressed in cells showing intestinal metaplasia in the stomach and other organs. MUC2 expression in the mucosal epithelia is an apparently abnormal phenomenon related to the neoplastic process. In this study, we examined MUC1 and MUC2 expression in human gallbladder adenocarcinoma and its clinicopathological significance and relationship with the prognosis of the patients. MUC1 immunoreactivity was detected not only in the cancer cells but also in the cancer stroma. Cytoplasmic MUC1 expression was significantly relation to lymphatic invasion and lymph node metastasis (p < 0.001), and was associated with a poor outcome. In contrast, MUC2 was rarely expressed in gallbladder carcinomas, and its immunoreactivity was detected only in the cancer goblet cells. Overexpression of MUC2 was not significantly related to lymphatic invasion or lymph node metastasis, or prognosis of patients. These observations suggested that MUC1 expression plays a more important role than MUC2 expression in cancer cell growth and metastasis of human gallbladder adenocarcinomas.

Rapid ex vivo expansion of human umbilical cord hematopoietic progenitors using a novel culture system.

Cell numbers limit the widespread clinical use of cord blood (CB) for gene therapy and marrow replacement in adults; a simple and effective method for ex vivo expansion of CB primitive progenitor cells (PPC) is required. Recently, the combination of thrombopoietin (TPO) and Flk-2/Flt-3 ligand (FL-2) was reported to support slow proliferation of CB-PPC in stroma-free liquid culture. We established a novel culture system in which the murine stromal cell line HESS-5 dramatically supports the rapid expansion of cryopreserved CB-PPC in synergy with TPO/FL-2. Furthermore, while HESS-5 cells directly adhered to human progenitors during culture, the cultured human cells could easily be harvested without contamination by HESS-5 cells. Within 7 days of culture, a 100-fold increase in CD34bright/CD38dim cells was obtained in serum-containing culture. When HESS-5 cells were physically separated from human progenitor cells in the presence of TPO/FL-2, synergy was blocked, suggesting that HESS-5 cells support proliferation of PPC by direct cell-to-cell interaction. The hematopoietic-supportive effects of this xenogeneic coculture system were then assessed in a very short-term (5 days) serum-free culture. Expansion was further enhanced by addition of stem cell factor (SCF) or interleukin-3 (IL-3). As a result, a 50- to 100-fold increase in CD34bright/CD38dim cells was noted. Colony-forming units in culture (CFU-C) and mixed colonies (CFU-GEMM) were enhanced by 10- to 30-fold and 10- to 20-fold, respectively. Moreover, generation of long-term-culture-initiating cells (LTC-IC) from CD34bright/CD38dim cells was amplified by 25-fold. The severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. These results indicate that this xenogeneic coculture system, in combination with human cytokines, can rapidly generate PPC from cryopreserved CB.

An in vivo model of human skin acute graft-versus-host disease: transplantation of cultured human epidermal cells and dermal fibroblasts with human lymphocytes into SCID mice.

The ability of mixed epidermal cell-lymphocyte reactions to detect allogeneic reactivities in an in vivo model was investigated by developing an in vivo model of acute graft-versus-host disease (GVHD), using SCID mice with a C.B-17 background in which human skin structures were generated by transplantation of cultured human epidermal cells (HEC) with dermal fibroblasts (HDFC). Suspensions containing cultured HEC and HDFC from a single donor were mixed with autologous peripheral blood mononuclear cells (PBMNC) or with PBMNC from unrelated individuals, and were injected into the flanks of C.B-17-SCID mice. Ten and 21 days after injection, subcutaneous nodules generated in the mice were examined histologically and immunohistochemically. Cystic structures developing after injection of HEC and HDFC without human PBMNC showed normal epidermislike tissue. Human skin generated in SCID mice injected with HEC and HDFC with auto-PBMNC showed no graft-versus-host reaction (GVHR) histologically, whereas those mice injected with PBMNC from siblings that shared an HLA haplotype showed mild GVHR. Human skin in SCID mice injected with HEC and HDFC with histoincompatible unrelated PBMNC showed moderate to severe GVHR. The severity of GVHR paralleled the dose of unrelated PBMNC, and GVHR was prevented by peroral treatment with cyclosporine A. Immunohistochemically, inflammatory cells infiltrating human cutaneous tissue formed in the SCID mice were stained by an anti-human CD45RO antibody that reacts with human T cells but not with murine lymphocytes, and most T cells were stained by an anti-human CD8 antibody recognizing HLA class I antigens. These findings are similar to those in clinical skin graft-versus host disease (GVHD) observed in patients undergoing allogeneic bone marrow transplantation. This experimental system should be useful as an in vivo model of human skin GVHD.

A simple and efficient purification of transduced cells by using green fluorescent protein gene as a selection marker.

BACKGROUND: Simple and efficient method for the selection of transduced cells would greatly facilitate the clinical utilization of retrovirus vectors. We developed a therapeutic bicistronic retrovirus vector for Gaucher disease, MFG-GC-GFP, which contains the human glucocerebrosidase (GC) gene and the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria as a vital selection marker, and investigated its applicability as gene therapy for Gaucher disease. METHODS AND RESULTS: A packaging cell line, GP + envAM12, was transfected with MFG-GC-GFP and, thus, produced a high titer recombinant virus (1.0 x 10(6) c.f.u./mL) in the culture supernatant. The expression level of GFP was correlated with the virus production in cells. The recombinant virus infected skin fibroblasts from a Gaucher patient and a sorted fraction of the cells expressing GFP by flow cytometry exhibited almost a six-fold higher activity of GC than normal fibroblasts. CONCLUSIONS: These data indicate that MFG-GC-GFP enables the one-step purification of a transduced fraction of target cells and is, therefore, considered to be a useful therapeutic vector for the experimental gene therapy of Gaucher disease.

[Umbilical cord blood transplantation for a patient with Diamond-Blackfan syndrome].

A five-year-old girl with Diamond-Blackfan syndrome received cord blood transplantation from an HLA-identical sibling. The patient showed pale face at birth, and was diagnosed to have Diamond-Blackfan syndrome. She had been treated with prednisolone (PSL), high dose of methylprednisolone, erythropoietin, and anti-lymphocyte globulin. Despite of these intensive therapies, erythropoiesis did not entirely improve, and transfusion of red blood cells had been required every third or fourth week until cord blood transplantation. Conditioning regimen consisted of thoraco-abdominal irradiation (TAI; 8 Gy), cyclophosphamide (CY; 50 mg/kg x 4), and anti-thymocyte globulin (ATG; 2.5 mg/kg x 4), Cyclosporin (CyA 3 mg/kg) was administered for the prophylaxis of graft-versus-host disease (GVHD). 4.14 x 10 (7)/kg of cord blood mononuclear cells were infused to the patient. White blood cell (WBC) and reticulocyte counts increased promptly, but recovery of platelet count was delayed. Skin GVHD (grade I) appeared on day +9, which responded to the administration of PSL (2 mg/kg). Chromosomal analyses of bone marrow cells for sex mismatch revealed complete chimerism on day +14, on day +28 and thereafter. Umbilical cord blood cells can be an alternative source of hematopoietic stem cells for allogeneic transplantation.

Construction and characterization of a recombinant adenovirus vector carrying the human preproinsulin gene under the control of the metallothionein gene promoter.

A new adenovirus vector carrying human-preproinsulin (h-PPI) genomic DNA, which was placed under the control of the mouse metallothionein gene promoter, was constructed. In the recombinant virus-infected cells, h-PPI gene expression increased as a function of ZnSO4 concentration. Reversed-phase high-performance liquid chromatography analysis revealed that the recombinant adenovirus-infected cells secreted immature insulin containing proinsulin and incorrectly processed insulin. Tyrosyl phosphorylation of human insulin receptor substrate 1 occurred when HepG2 cells were treated with the cultured medium, indicating that the h-PPI gene product was functionally active in vitro. We also examined the biological activity of the product using diabetic severe combined immunodeficient mice and confirmed that the h-PPI gene product reduced the blood glucose concentration in vivo. This study suggests that the adenovirus vector can be used to express a foreign gene under the control of an external promoter in various human cells.

Establishment and characterization of a villous lymphoma cell line from splenic B-cell lymphoma.

A new B-cell line (VL51) with cytoplasmic villi was established from a female patient with splenic lymphoma with circulating villous lymphocytes (SLVL). The patient exhibited a clinical picture characteristic of SLVL, including massive enlargement of the spleen. Tartrate-resistant acid phosphatase (TRAP)-negative villous lymphocytes were seen in the peripheral blood, bone marrow (BM) and both red and white pulps of the spleen. Monoclonality of the VL51 cell line was confirmed by clonal genotype abnormalities in the immunoglobulin heavy chain (IgH) gene and the T-cell receptor beta (TCR beta) gene. Evidence for commitment of phenotype of the VL51 cell line to the B lineage was also shown by the immunophenotype, including expression of CD10, CD19, CD20 and surface immunoglobins. The VL51 cells were positive for Epstein-Barr virus nuclear antigen (EBNA). The VL51 cell line is the first SLVL cell line to be established, and it is expected to be useful in clarifying the leukemogenesis of SLVL.

Effects of fibroblasts of different origin on long term maintenance of xenotransplanted human epidermal keratinocytes in immunodeficient mice.

We examined effects of fibroblasts of different origin on long-term maintenance of xenotransplanted human epidermal keratinocytes. A suspension of cultured epidermal cells, originating from adult human trunk skin, was injected into double mutant immunodeficient (BALB/c nu/scid) mice subcutaneously, with or without cultured fibroblastic cells of different origin. At one week after transplantation, the epidermal cells generated epidermoid cysts consisting of human epidermis-like tissue. When the epidermal cells were injected alone or together with fibroblastic cells derived from human bone marrow, muscle fascia, or murine dermis, organized epidermoid cysts regressed within 6 weeks. In contrast, when the epidermal cells were injected together with human dermal fibroblasts, generated epidermoid cysts were maintained in vivo for more than 24 weeks. Histological examination showed that the reorganized epidermis, after injection of both epidermal keratinocytes and dermal fibroblasts, retained normal structures of the original epidermis during 6 to 24 weeks after transplantation. The results indicate that human dermal fibroblasts facilitate the long-term maintenance of the reorganized epidermis after xenotransplantation of cultured human epidermal keratinocytes by supporting self renewal of the human epidermal tissue in vivo.

Immortalisation of human oesophageal epithelial cells by a recombinant SV40 adenovirus vector.

We introduced the origin-defective SV40 early gene into cultured human oesophageal epithelial cells by infection of a recombinant SV40 adenovirus vector. The virus-infected cells formed colonies 3-4 weeks after infection in medium containing fetal calf serum. When the cells derived from 'serum-resistant' colonies were then maintained in the serum-free medium with a low calcium ion concentration, some of them passed the cell crisis and kept growing for over 12 months. These cells, regarded as immortalised cells, resembled the primarily cultured oesophageal epithelial cells in morphology and had some of their original characteristics. Treatment of the cells with a high calcium concentration induced phenotypic changes. These cells still responded to transforming growth factor beta. When the immortalised cells were injected into severe combined immunodeficient mice, they transiently formed epithelial cysts, although the typical differentiation pattern of the oesophageal epithelium was not observed. These cysts regressed within 2 months without development into tumours. The results indicated that human oesophageal epithelial cells were reproducibly immortalised by infection with a recombinant SV40 adenovirus vector at relatively high efficiency. The immortalised cells should be useful in studies on oesophageal carcinogenesis and in assessing the cooperative effects with other oncogene products or carcinogens.

[A case report of left limb compartment syndrome associated with laparoscopic surgery].

A 63-year-old male patient with early gastric cancer was attempted for laparoscopic wedge resection of the stomach. After the introduction of anesthesia, bandage of bilateral leg was carried out to prevent deep venous thrombosis during laparoscopic surgery. Although the procedure was converted to open surgery, the bandage has been continued throughout the surgery for 6 hours. After the operation, the swelling and severe tenderness at his left leg was observed. MRI revealed remarkable edema in left deep posterior compartment. Under diagnosis of left limb compartment syndrome, fasciotomy was carried out. Postoperatively the patient did well without any functional disturbance.

Hematopoietic supportive function of human bone marrow stromal cell lines established by a recombinant SV40-adenovirus vector.

We have previously reported the establishment of a variety of human bone marrow stromal cell lines using a recombinant SV40-adenovirus vector. Using this vector, we obtained more clonal stromal cells. Here, we have characterized these cells and analyzed their capacity to support the proliferation and differentiation of hematopoietic cells. The stromal cells were cocultured with nonadherent human bone marrow cells used as hematopoietic cells. The total numbers of hematopoietic cells and CFU-GM in culture were counted every week. Two of the six stromal cell lines, AA101 and HAS303, supported the proliferation and differentiation of hematopoietic cells and CFU-GM for more than 9 weeks. Further, granulocytes, macrophages, and megakaryocytes were detected when cocultured with these cells. When hematopoietic cells were cocultured but separated from the two stromal cell lines by a 0.45-microns millipore membrane to prevent their attachment, almost all CFU-GM disappeared within 7 weeks. The supportive stromal cells produced GM-CSF and IL-6. However, other cell lines producing these humoral factors did not support hematopoietic cell proliferation for such a long time. These findings suggest that these established human bone marrow stromal cell lines will be useful, in that analysis of their supportive function in hematopoietic cell proliferation and differentiation through cell-to-cell interaction will shed some light on this area.

A case of nephrotic syndrome associated with hepatic glomerulosclerosis and diabetic nephropathy.

We report here an adult case of nephrotic syndrome associated with hepatic glomerulosclerosis with the hepatitis C virus (HCV) antigen and diabetic nephropathy. To clarify the etiology of the nephrotic syndrome, we performed a renal biopsy and obtained the histological findings of hepatic glomerulosclerosis, glomerular mesangial cell proliferation and mesangial expansion. Electron dense deposits and deposits of IgA and C3 were also noted in the glomerular mesangial areas. Histological findings of diabetic nephropathy included thickening of the glomerular basement membrane and aneurysmal change of glomerular capillary walls in light microscopy. In immunofluorescence, linear staining of IgG was observed in the glomerular capillary walls, Bowman's capsules and tubular basement membranes. HCV was also detected focally in the glomerular capillary walls by immunofluorescence. Association of these two diseases has not been reported in any of the previous manuscripts that we reviewed. Thus, this patient seems to be a case of very rare association of the two diseases.

Use of cultured human epidermal allografts for the treatment of extensive partial thickness scald burn in children.

In spite of recent progress in burn treatment, the early surgical therapy of partial thickness scald burns in children is still controversial. Early tangential excisions is not easily applicable for these patients because of difficulties in determination of the burn depth and probable physiological derangement after surgery. Hypertrophic scar formation and wound contraction after meshed autografts are other limitations. For these reasons, conservative treatment, not early excision therapy, has been chosen initially for these injuries. We used cultured epidermal allografts for extensive, partial thickness scald burns, during the early post-burn period without escharectomy. Fifty to 100% of the engrafted superficial dermal burns were epithelialized within 7 days. In contrast, untreated identical wounds remained open. Repeated grafting of cultured allografts on unexcised wound granulations of dermal burns also enhanced epithelialization. Long term results showed that hypertrophic scar formation in the mixed superficial and deep dermal burns was reduced when cultured allografts were used. Allografting of the cultured epidermis without surgical excision apparently promoted the rapid regeneration of the partial thickness burns. Procedural complications did not occur. Cultured allografts should be used as an effective and safe biological dressing for partial thickness scald burns in children.

Calcium induced differentiation of SV40 immortalized human epidermal keratinocytes cultured in a defined medium.

Human epidermal keratinocytes, immortalized by the introduction of SV40-adenovirus recombinants (9), were maintained in a low calcium (0.15 mM) defined medium. When differentiation was induced by a higher extracellular calcium concentration (1.5 mM), these cells altered in shape and developed stratification with formation of desmosomes, while they demonstrated limited terminal keratinization. The expression of involucrin, one of the precursor proteins of the cornified envelope, became elevated as the cell density increased, but was not affected by calcium. These results indicate that the SV40 immortalized human keratinocytes, maintained in a low calcium defined medium, partially respond to changes in extracellular calcium concentration.

Establishment of a variety of human bone marrow stromal cell lines by the recombinant SV40-adenovirus vector.

Various human bone marrow stromal cell lines were established from the adherent cell populations by introduction of the recombinant SV40-adenovirus vector with an infection or electric poration procedure. As compared with DNA transfection, the vector introduction was able to immortalize the cells with more than 100 times higher efficiency. Morphological and cytochemical analyses indicated that various cloned cell lines with different properties were isolated by the vector introduction. All the established cell lines expressed SV40 large T antigen. These results provided the evidence indicating that the recombinant SV40-adenovirus vector was a useful tool to establish a variety of cell lines with different biological activities from human bone marrow stroma.

Antigen receptor-mediated regulation of sustained polyphosphoinositide turnover in a human T cell line. Evidence for a receptor-regulated pathway for production of phosphatidylinositol 4,5-bisphosphate.

Stimulation of the human T cell line, Jurkat, by the addition of monoclonal antibodies reactive with the T cell antigen receptor complex (CD3/Ti) leads to sustained increases in levels of inositol 1,4,5-trisphosphate. To investigate the possibility that the production of polyphosphoinositides is regulated during CD3/Ti stimulation, we studied Jurkat cells whose inositol phospholipids had been labeled to steady state with [3H]inositol, as well as Jurkat cells during nonequilibrium labeling with [32P]orthophosphate. The addition of CD3 monoclonal antibodies led to a 4-5-fold increase in [3H]inositol trisphosphate that was sustained for greater than 20 min. Within 60 s of CD3/Ti stimulation, [3H] phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) decreased by 65 and 35%, respectively. This change in [3H]PtdIns(4,5)P2 persisted for greater than 20 min. The decrease in [3H]PtdIns4P, however, was transient, and, after 5 min, the levels of [3H]PtdIns4P were comparable in stimulated and unstimulated cells. To examine the rate of flux through inositol phospholipids, we measured the CD3/Ti-stimulated changes in the ratio, 32P cpm/3H cpm, in each inositol phospholipid. CD3/Ti stimulation led to accelerated fluxes through PtdIns(4,5)P2 and phosphatidylinositol (PtdIns) that were maintained for greater than 20 min. After the initial 30 s, however, there was no detectable effect of anti-CD3 on flux through Ptsins4p. This observation suggested that, during CD3/Ti stimulation, production of PtdIns(4,5)P2 from PtdIns might occur via a small pool of PtdIns4P with a very high turnover. The existence of such a pool was established by determining that, in stimulated cells, the 32P-specific activity of the 1-position phosphate of PtdIns(4,5)P2 was 8-10-fold that of PtdIns4P. We conclude that, during the initial 60 s of CD3/Ti stimulation, there is a substantial depletion of cellular PtdIns(4,5)P2 and PtdIns4P. Thereafter, a CD3/Ti-regulated pathway generates PtdIns(4,5)P2 from PtdIns through a small, but highly labile, pool of PtdIns4P.