Antiangiogenic activity of BAI1 in vivo: implications for gene therapy of human glioblastomas.

Glioblastomas are the most common primary brain tumors in adults. These tumors exhibit a high degree of vascularization, and malignant progression from astrocytoma to glioblastoma is often accompanied by increased angiogenesis and the upregulation of vascular endothelial growth factor and its receptors. In this study, we investigated the in vivo antiangiogenic and antitumor effects of brain-specific angiogenesis inhibitor 1 (BAI1) using human glioblastoma cell lines. Glioblastoma cells were transduced with an adenoviral vector encoding BAI1 (AdBAI1), and Northern and Western blot analyses, respectively, demonstrated BAI1 mRNA and protein expression in the transduced tumor cells. Using an in vivo neovascularization assay, we found that angiogenesis surrounding AdBAI1-transduced glioblastoma cells transplanted into transparent skinfold chambers of SCID mice was significantly impaired compared to control treated cells. Additionally, in vivo inoculation with AdBAI1 of established subcutaneous or intracerebral transplanted tumors significantly impaired tumor growth and promoted increased mouse survival. Morphologically, the tumors exhibited signs of impaired angiogenesis, such as extensive necrosis and reduced intratumoral vascular density. Taken together, these data strongly indicate that BAI1 may be an excellent gene therapy candidate for the treatment of brain tumors, especially human glioblastomas.

Significant expression of G-CSF-induced gene-1 (GIG-1) protein in myeloid cells and NK cells.

The granulocyte colony-stimulating factor (G-CSF)-induced gene, GIG-1, was originally cloned from G-CSF-stimulated bone marrow mononuclear cells obtained from a patient with chronic myelogenous leukemia (CML). We have characterized the GIG-1 gene and its protein product. Expression of GIG-1 mRNA was elevated by treatment with G-CSF in normal bone marrow mononuclear cells, as well as in some cases of blast cells obtained from patients with acute myelogenous leukemia (AML) and CML. Western blot analysis with anti-GIG-1 peptide antiserum showed the molecular mass of GIG-1 product was about 17 kDa. Immunostaining of the hematopoietic cells demonstrated that GIG-1 product was mainly localized to the cytoplasm of both myeloid and natural killer (NK) cells. These results suggested that GIG-1 protein is an integral component that is accumulated during the differentiation of myeloid cells toward the stage of mature neutrophils. Expression of GIG-1 gene in mature neutrophils was tightly regulated and reactivation of GIG-1 gene by G-CSF in mature neutrophils may represent a compensation process for the protein lost through the activation of these cells, thus implying an important role for this protein in host defense mechanisms.

A novel point mutation affecting the tyrosine kinase domain of the TRKA gene in a family with congenital insensitivity to pain with anhidrosis.

A nerve growth factor receptor encoded by the TRKA gene plays an important part in the formation of autonomic neurons and small sensory neurons in dorsal root ganglia and in signal transduction through its intracytoplasmic tyrosine kinase domain. Recently, three mutations in the tyrosine kinase domain of TRKA have been reported in patients with congenital insensitivity to pain with anhidrosis, which is an autosomal recessive disorder characterized by recurrent fever due to absence of sweating, no reaction to noxious stimuli, self-mutilating behavior, and mental retardation. We examined the TRKA gene in five generations of a large Japanese family with many consanguineous marriages who live in a small remote island of the southern part of Japan. We found a novel point mutation at nucleotide 1825 (A-->G transition) resulting in Met-581-Val in the tyrosine kinase domain. Two of the three affected patients were homozygous for this mutation; however, the third affected patient was heterozygous. Further analysis revealed that the third patient was a compound heterozygote with the Met-581-Val mutation in one allele and with a single base C deletion mutation at nucleotide 1726 in exon 14 in the other allele, resulting in a frameshift and premature termination codon.

T-cell receptor gene rearrangement in cells infiltrating skin eruptions specific to adult T-cell leukemia.

We examined T-cell receptor gene rearrangement in skin lesions and peripheral blood from 6 patients with adult T-cell leukemia (ATL) using the Southern blot method and a c beta 1 probe. A rearrangement signal common to skin lesions of all 6 patients was observed. One patient (Case 4) exhibited another rearrangement signal in the skin lesion and an identical signal was detected in the peripheral blood. This is the first report describing a specific pattern of T-cell receptor gene rearrangement in ATL. The signal obtained is assumed to represent receptors of T cells involved in surveillance of HTLV-I infected T cells.

Morphological, biochemical and molecular biological characteristics of a granulocyte colony-stimulating factor-producing human eccrine carcinoma cell line.

We describe here a newly established cell line from an eccrine carcinoma which produced an abundant amount of granulocyte colony-stimulating factor (G-CSF). An eccrine carcinoma of the scalp of a 69 year-old-Japanese female had metastasized to the pleura. Clinically, she had marked neutrophilia (up to 60,000/mm3), and a high level of G-CSF (38.7 x 10(3) pg/ml) was detected in the pleural effusion, as determined by enzyme-linked immunosorbent assay (ELISA). We established a cell line in vitro and maintained the cells in culture for 30 months in 90 subcultures. We investigated whether these tumor cells were able to produce G-CSF in culture and found that they were. We also found that the amount of G-CSF produced paralleled the rise in cell number (26.5 x 10(3) pg/ml at confluency). When culture media were administered to rabbits (25 ml/rabbit), the amount of circulating neutrophils increased until the number was equal to or greater than that resulting from injection of recombinant human G-CSF (rhG-CSF)(75 micrograms). This effect persisted for 7 days. When tumors were induced in SCID and nude mice by injecting cultured cells (1 x 10(7) cells/mouse), the number of circulating neutrophils also correlated well with tumor size in these mice (200,000/mm3, 3 cm tumor). After tumor removal, the neutrophil number returned to normal within 30 days. G-CSFmRNA in cultured, cells was detected by RT-PCR. Based on these results, it was confirmed that the marked neutrophilia observed in the patient was caused by the tumor-generated G-CSF. This is the first G-CSF-producing cell line developed from a cancer of the skin.

Crystalloid inclusion bodies of the endothelial cells in human fetal skin blood vessels and human umbilical cord vessels: a possible relationship to Weibel-Palade bodies?

Crystalloid inclusion bodies (CIB) of the endothelial cells (EC) were investigated in blood vessels of human fetal skin and the umbilical cord by electron- and immunoelectron microscopy. They were found in up to 15% of the investigated EC in various types of vessels. Their sizes ranged from 0.2 microns to 0.6 microns in the largest diameter. Most frequently we observed a laminated pattern of the crystalloid structure with a regular periodicity of dark and light bands. Additionally a honeycomblike pattern was also seen. Measurement of the CIB laminated structure revealed similar dimensions to Weibel-Palade bodies (WPB). In EC of all vessel types we found numerous WPB differing in electron density, shape and size from those of WPB found in adult blood vessels. WPB were found much less frequently in EC with CIB, suggesting that CIB is a precursor of WPB. After incubation with monoclonal antibody against von Willebrand factor (vWf) both WPB and large organelles were labeled. Because of their shape and size the labeled large organelles seemed to represent inadequately preserved CIB. After incubation with anti-lysozyme only the large organelles were labeled. A possible relationship of CIB to WPB is thus suggested. The presence of lysosomal enzymes such as lysozyme suggest that CIB are lysosomal organelle and participate in the uptake of vWf. The crystalloid pattern of CIB may represent an accumulation of a highly condensed form of vWf.

Differences in HTLV-I integration patterns between skin lesions and peripheral blood lymphocytes of HTLV-I seropositive patients with cutaneous lymphoproliferative disorders.

We examined HTLV-I integration patterns in nine cases of HTLV-I-seropositive patients with cutaneous lymphoproliferative disorders. The Southern blot on EcoRI digests of DNA revealed a discrete band of HTLV-I provirus (monoclonal integration) in either skin lesions or peripheral blood lymphocytes (PBL). Four cases showed the monoclonal integration of HTLV-I provirus only in skin lesions: one case showed only in PBL and two cases showed in both skin and PBL. The Southern blot on PstI digests of DNA revealed a 2.4 Kb band of the internal construct of HTLV-I provirus (polyclonal integration) in the PBL of EcoRI-negative samples. The difference in HTLV-I integration patterns between skin lesions and PBL in these cases suggests that the monoclonal outgrowth of HTLV-I-infected cells in the skin is causatively associated with the pathogenesis of cutaneous ATL.

Monoclonal anti-melanoma antibodies IKH-1 and IKH-2 which work on formalin-fixed, paraffin embedded tissues: characterization, clinical trials and comparative studies with HMB-45.

Mouse monoclonal antibodies (MoAbs), IKH-1 and IKH-2, were produced against cloned human melanoma cells, KHm-6, which were cultured with 12-O-tetradecanoylphorbol-13-acetate (TPA) and processed by formalin fixation and alcohol dehydration (FFAD). According to the biochemical analysis, antigenic substances which reacted with IKH-1 were 34.0-60.0 kDa glycoproteins, and those which reacted with IKH-2 were 33.5, 34.5 and 36.0 kDa glycoproteins. Immunoelectron microscopy revealed that reaction products of IKH-1 were seen in some membranous vesicles, premelanosomes and cell membrane of TPA-treated KHm-6 cells, while IKH-2 recognized only premelanosomal structures. Immunohistochemical tests revealed that IKH-1 and IKH-2 have a high sensitivity (94.0% and 85.0%, respectively) to formalin-fixed, paraffin-embedded (FFPE) tissue sections of malignant melanomas. IKH-1 had a high specificity and IKH-2 and 100% specificity to FFPE tissue sections of melanocytic lesions.

Desmoplakin I and II in acantholytic dermatoses: preservation in pemphigus vulgaris and pemphigus erythematosus and dissolution in Hailey-Hailey's disease and Darier's disease.

Desmoplakin I and II are important components of the attachment plaque of the desmosome which mediates cell to cell adhesion, in epithelial cells. In this study we used well-characterized antibody against desmoplakin I and II immunohistochemically and immunoelectron microscopically on two cases of pemphigus vulgaris and one case of pemphigus erythematosus and two cases each of Hailey-Hailey's disease and Darier's disease. In the normal human epidermis the desmosomes were demonstrated in a dotted pattern along cell periphery. In pemphigus vulgaris and pemphigus erythematosus acantholytic cells and the perilesional cells exhibited normal dotted pattern along the cell periphery. In Hailey-Hailey's disease and Darier's disease, the dotted pattern is lost in acantholysed and perilesional areas and anti-desmoplakin I + II positive proteins were observed diffusely in the cytoplasm. Immunoelectron microscopical findings correspond to these light microscopical observations. It is concluded that in autoimmune acantholytic disease such as pemphigus vulgaris and pemphigus erythematosus, desmoplakins are intact even in acantholytic cells, whereas in genodermatoses such as vulgaris and pemphigus erythematosus, desmoplakins are intact even in acantholytic cells, whereas in genodermatoses such as Hailey-Hailey's disease and Darier's disease primary or secondary abnormalities abnormalities of desmosomes may be involved in their pathogenesis.

Autologous bone marrow transplantation for patients with advanced chronic myelogenous leukemia.

We report on seven chronic myelogenous leukemia (CML) patients who received autologous bone marrow transplantation (ABMT) using bone marrow (BM) cells while at the chronic phase (CP) under the various treatments. Of the seven patients, four progressed to accelerated phase (AP) in 83-248 weeks after onset and three patients entered blastic crisis (BC) in 84-171 weeks after onset. All patients received high-dose chemoradiotherapy followed by infusion with 11.3 +/- 12.1 x 10(7) (average +/- S.D.) of bone marrow mononuclear cells (BM-MNCs)/kg IFN-alpha was resumed shortly after platelet recovery. Of the four patients in AP, one developed a recurrence of blastoma in 7 weeks, one progressed to second AP in 138 weeks after ABMT and two patients have survived the second CP for 159 and 330 weeks since ABMT, respectively. One of them achieved the complete disappearance of Ph1-positive metaphases for 33 weeks after ABMT. Of patients who received AMBT in BC, three relapsed within 8 weeks and died in 9, 17 and 58 weeks after ABMT, respectively. Hematological recovery was delayed in four patients. Therefore, we retrospectively re-evaluated the number of BM-MNCs collected through 50 procedures from 40 patients with CML-CP. The total MNCs obtained from 30 collections under IFN-alpha treatment was 27.4 +/- 30.9 x 10(8) cells (average +/- S.D.), being significantly lower than that obtained from 20 collections in pre-treatment state or with single chemotherapy other than IFN-alpha treatment (81.8 +/- 68.2 x 10(8) cells) (P < 0.005). The total number of MNCs correlated to white blood cell (WBC) count at BM collection (P < 0.01), which was also lower in the IFN-alpha(+) group than in the IFN-alpha(-) group (7.2 +/- 5.7 and 25.6 +/- 32.3 x 10(9)/l; P < 0.005). Our findings suggested that ABMT with the use of a sufficient number of progenitor cells might be helpful to CML patients in early AP and reach in extended periods of second CP. In addition, we suggest that BM collection is required before the start of IFN-alpha therapy because the total number of BM-MNCs correlated to the WBC count, which might be lower in IFN-alpha treatment.

Present features of gallstones in Japan. A collective review of 2,144 cases.

A collective review of 2,144 patients operated on for cholelithiasis during the last twenty-two months has shown that younger Japanese adults have predominantly cholesterol stones in their gallbladders and that elderly persons still frequently have bilirubin stones not only in their gallbladders but also in their common bile ducts. Recent westernization of dietary habits in Japan is considered to be the most probable factor causing the increased incidence of cholesterol stones. The decreased incidence of bilirubin stones is considered to be caused by the decreased incidence of biliary infection and increased intake of proteins in food.

Successful mapping of lymphorrhea using patent blue dye after lymph node dissection for malignant melanoma.

Patent blue is a dye that has been used for intraoperative lymphatic mapping. We used this mapping method on a patient with lymphorrhea after groin dissection. We easily detected the lymphatic channel causing lymphorrhea and successfully ligated it. This technique may have great merit for treating of lymphorrhea.

A case of lupus meningitis treated successfully with methylprednisolone pulse therapy.

A 46-year-old female had suffered from systemic lupus erythematosus (SLE) for 8 years. Headache, vomiting and stiff neck appeared in the active phase of SLE. Findings in the cerebrospinal fluid were consistent with those of lupus meningitis. No pathogenic microbes were detected by microbiological or immunological examinations. She was diagnosed as having lupus meningitis. The method discussed herein which elucidates the cause of fever in SLE using white blood cell count (WBC) and alpha-2 globulin appeared to be useful for examining this case of meningitis. Lupus meningitis seems to preferentially occur in SLE patients with positive anti-ribonucleoprotein (RNP) antibody. Pulse therapy with methylprednisolone appeared to work well in this lupus meningitis patient who had had a long course of corticosteroid therapy.

Immunolocalization of desmoglein I ("band 3" polypeptide) on acantholytic cells in pemphigus vulgaris, Darier's disease, and Hailey-Hailey's disease.

Acantholysis is defined as loss of coherence between epithelial cells and is histologically shown in several bullous diseases. It was postulated that desmoglein I, one of the major transmembrane glycoproteins of the desmosome, may adhere to the attachment plaque inside the cell and contribute to desmoglea outside the cell. In this study we used a well characterized antibody against desmoglein I for immunofluorescence and immunoelectron microscopic techniques on 2 cases each of pemphigus vulgaris and Darier's disease and one case of Hailey-Hailey's disease. In the normal epidermis desmosomes were demonstrated in dotted or rim-like patterns along cell periphery on immunofluorescence study. In pemphigus vulgaris dotted or rim-like patterns were still identified in many acantholytic cells, particularly in early phase of acantholysis. In Darier's disease and Hailey-Hailey's disease, dotted or rim-like patterns were already lost in early acantholysis and immunoreactive desmoglein I proteins were observed diffusely in the cytoplasm. Immunoelectron microscopy confirmed these immunofluorescence observations. It was suggested that in pemphigus vulgaris desmoglein I is unlikely to be the primary site of acantholysis because dotted or rim-like patterns of immunoreactive desmoglein I are relatively preserved on lesional cells, whereas in genodermatoses such as Darier's disease and Hailey-Hailey's disease primary abnormalities of desmosomes may be involved in their acantholysis.

Clinicopathologic analysis of 124 cases of adult T-cell leukemia/lymphoma with cutaneous manifestations: the smouldering type with skin manifestations has a poorer prognosis than previously thought.

Adult T-cell leukemia/lymphoma (ATLL) commonly involves the skin as well as peripheral blood and lymph nodes. During the last 15 years we have studied 124 cases of ATLL with specific skin manifestations. Twenty-one patients (16.9%) were classified as acute, 21 (16.9%) as chronic, 26 (21.0%) as lymphoma, and 56 (45.2%) as smouldering according to Shimoyama's classification. Many patients had nodules/tumors (34.7%), erythematous plaques (22.6%), and erythematous papules (19.4%) similar to those occurring with other cutaneous T-cell lymphomas. Some patients displayed characteristic skin manifestations resembling non-neoplastic cutaneous disorders. The median survival time (MST) of all patients was 12.0 months. The MSTs of individual clinical types were: acute type, 4 months; chronic type, 14 months; lymphoma type, 7 months; and smouldering type, 16 months. In the smouldering type, cases with a deeper infiltration pattern (MST, 14 months) had a more aggressive course than those with a superficial infiltration pattern (MST, 24 months) (p < 0.05). The results indicate that smouldering type ATLL with skin manifestations may have a worse prognosis than without skin manifestations. Moreover, some cases of the smouldering type with specific skin lesions should be classified into another group with a much poorer prognosis.

A case of intravascular malignant lymphomatosis (angiotropic large-cell lymphoma) presenting memory T cell phenotype and its expression of adhesion molecules.

A case of intravascular malignant lymphomatosis (angiotropic large cell lymphoma), T cell type was reported. The patient, a 59-year-old woman, had reddish or violaceous indurated macules scattered over the entire body surface. Neither lymphadenopathy nor hepatosplenomegaly was recognized. A chest Roentgenogram, whole body CT scan, and 67Ga-citrate scintigraphy yielded normal findings. Serum anti-HTLV-1 antibody was negative. Histopathologically, lesions showed intravascular large mononuclear cell proliferation associated with occasional fibrin thrombi formation in the dermis to subcutis. Immunohistochemically, the large mononuclear cell immuno-phenotype had a memory T cell character. Also, both lymphocyte function-associated antigen-1s, CD11a and CD18, and intercellular adhesion molecule-1 were demonstrated on the tumor cells and vascular walls in the lesions. To our knowledge, the present case is the fourth case of intravascular malignant lymphomatosis in the T cell lineage.

A case of adult T-cell leukemia/lymphoma (ATLL) with angiocentric and angiodestructive features.

This report describes a case of adult T-cell leukemia/lymphoma (ATLL) with angiocentric and angiodestructive features. The patient was a 66-year-old Japanese woman who began developing widespread skin lesions ten months prior to admission. The diagnosis of ATLL was made on the basis of her having an antibody to human T-cell lymphotropic virus type-1 (HTLV-1) and typical flower cells (ATLL cells) in peripheral blood smears. Once hospitalized, the course of her disease was very acute and severe, as is seen with angiocentric lymphoma. Based on histological features, this case was judged not to be angiocentric lymphoma; however, it may lie within the spectrum of angiocentric immunoproliferative lesions (AIL). The findings in this case strongly suggest that HTLV-1 can be a pathogenic factor in the expression of angiocentric and angiodestructive features in ATLL, as is Epstein-Barr virus (EBV) (1-4). To our knowledge the present case is the sixth reported in the literature of lymphoma in which these features are associated with HTLV-1 infection (5-7).

Squamous cell carcinoma of the skin associated with sarcoid reactions in the regional lymph nodes.

Sarcoid reactions in lymph nodes with or without metastasis from a primary malignant neoplasm are well-known. However, it is extremely rare to find these reactions associated with cutaneous solid tumors; only one such case has appeared in the literature. Here we describe a case of an 83-year-old man with cutaneous squamous cell carcinoma accompanied by sarcoid reactions and metastatic foci in the regional lymph nodes. The possibilities of systemic sarcoidosis and tuberculosis were excluded after extensive examinations specific for these diseases. Some authors regard the sarcoid reaction to be a sign of a good prognosis on the basis of studies of a few patients with solid tumors. In our case, however, the patient died of pulmonary metastasis with pleuritis carcinomatosa shortly after surgery. Systematic analysis of a sufficient number of cases should be carried out to evaluate the clinical significance of this type of reaction.

Junctional proteins of keratinocytes in Grover's disease, Hailey-Hailey's disease and Darier's disease.

Alterations of junctional structures in non-immune mediated acantholytic diseases (Grover's, Hailey-Hailey's and Darier's diseases) were examined using monoclonal antibodies against desmosomal attachment constituents (desmoplakin I & II and plakoglobin), desmosomal intercellular cement glycoprotein (desmoglein), protein of adherens junction (vinculin), and protein of gap junction (43Kd connexin). Universal cell surface (transmembrane) glycoprotein CD44 was also studied. In acantholytic foci of these diseases, attachment plaque proteins had dissolved and diffused into the acantholytic cells. The normal dotted linear pattern of immunostaining on the cell membrane was totally lost. In contrast, CD44 was well preserved on the cell membranes of acantholytic cells. Adherens junction and gap junction proteins were mostly preserved. Acantholytic cells of pemphigus vulgaris were similarly studied. In these cells, desmosomal attachment plaque proteins were very well preserved, while intercellular cement substance (desmoglein), adherens junctional proteins (vinculin), and gap junction protein (connexin) were totally absent, either on the cell membrane or in the cytoplasm. Electron microscopy confirmed an early dissolution of attachment plaque. Internalized desmosomal structures were seldom found in acantholytic cells of non-immune diseases. It was concluded that the primary event in acantholysis in these three diseases is the dissolution of desmosomal attachment plaque.