Regulation of neurokinin-1 receptor messenger RNA expression in synovial fibroblasts of patients with rheumatoid arthritis.

We examined whether soluble mediators regulate the expression of tachykinin receptor mRNAs in synovial fibroblasts of patients with rheumatoid arthritis (RA). mRNAs encoding long and short isomers of neurokinin 1 receptor (NK1R), and neurokinin 2 receptor (NK2R) were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Level of long, but not the short, of NK1R mRNA was increased by treatment with 10-100 ng/ml basic fibroblast growth factor (bFGF) or 20 ng/ml tumor necrosis factor-alpha (TNF-alpha), but not with 1ng/ml interleukin 1beta (IL-1beta). TNF-alpha upregulated NK2R mRNA as well as long NK1R mRNA whereas bFGF had no effect on NK2R mRNA. Expression of neurokinin 3 receptor (NK3R) mRNA was not observed in RA fibroblasts, and its expression was not induced by bFGF and TNF-alpha. The basal and increased levels of long NK1R mRNA were inhibited by treatment with 20 microM SU5402, an inhibitor of the tyrosine kinase activity of FGF receptor 1 (FGFR1), or 10 ng/ml transforming growth factor-beta1 (TGF-beta1). SU5402 and TGF-beta1 had no effect on the basal level of short NK1R mRNA. Immunocytochemistry revealed the enhancement by bFGF of immunoreactive NK1Rs in the cells at 24 h after treatment. These results suggest that bFGF, TGF-beta1, and TNF-alpha in synovial tissue and fluid play a role in the regulation of long NK1R expression in synovial fibroblasts of RA patients. It appears that the pathway of downregulation by TGF-beta1 is more dominant in the long NK1R mRNA expression than that of upregulation by bFGF or TNF-alpha. Furthermore, the regulation of short NK1R mRNA expression seems to be performed via a different pathway from that of long isomer mRNA.

A critical mutation in both WT1 alleles is not sufficient to cause Wilms' tumor.

The WT1 gene is a tumor suppressor gene for Wilms' tumor (WT). Inactivation of both alleles has been proposed as the cause of WT. We encountered a patient with Denys-Drash syndrome associated with WT whose WT1 gene had a homozygous point mutation not only in WT but also in renal tissue adjacent to the WT and in the germline. These findings indicate that factor(s) other than the loss of WT1 are required for WT to develop.

A point mutation found in the WT1 gene in a sporadic Wilms' tumor without genitourinary abnormalities is identical with the most frequent point mutation in Denys-Drash syndrome.

We have analyzed exon 9 of the WT1 gene of 18 non-familial/sporadic unilateral Wilms' tumors (WTs) from Japanese patients, by the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method. After screening these WTs, a nucleotide alternation, which was present on both alleles, was found in only one case. Furthermore, PCR-SSCP analysis of the constitutional DNA revealed that this patient carried the mutation on only one allele in the germline. Sequence analysis showed that the tumor carried a point mutation (C-1180 to T-1180) in WT1 exon 9 of both alleles, resulting in an Arg-394 to Trp-394 amino acid substitution within the third zinc finger domain of the WT1 product. Interestingly, this mutation is identical with the most frequent point mutation associated with the Denys-Drash syndrome. However, the classical triad of Denys-Drash syndrome does not apply to this patient. This is in the first report of the point mutation in the zinc finger domain of both WT1 alleles in a sporadic unilateral WT without genitourinary abnormalities, and the mutation suggests that some sporadic WTs carry the Denys-Drash WT1 mutations.

Gene gun-mediated oral mucosal transfer of interleukin 12 cDNA coupled with an irradiated melanoma vaccine in a hamster model: successful treatment of oral melanoma and distant skin lesion.

Malignant melanoma involving the oral cavity has a highly metastatic potential. Curative surgery is required to resect extensive oral tissues and often results in dysfunction as well as a severe cosmetic deformity in patients with the disease. An alternative technology for the local and sustained delivery of cytokines for cancer immunotherapy has been shown to induce tumor regression, suppression of metastasis, and development of systemic antitumor immunity. However, local immunization of the oral cavity has not previously been studied. In this study, we examined the efficacy of particle-mediated oral gene transfer on luciferase and green fluorescent protein production. The results showed that these proteins were more significantly expressed in oral mucosa than the skin, stomach, liver, and muscle. Using an established oral melanoma model in hamsters, particle-mediated oral gene gun therapy with interleukin (IL) 12 cDNA was then conducted. The results indicated that direct bombardment of mouse IL-12 cDNA suppressed tumor formation and improved the survival rate. The skin tumor model created by inoculation of melanoma cells was also significantly inhibited by the oral bombardment of IL-12 cDNA coupled with an irradiated melanoma vaccine administrated to the oral mucosa, compared to treatment with a percutaneous vaccine. IL-12 gene gun therapy, combined with an oral mucosal vaccine, induced interferon-gamma mRNA expression in the host spleen for a long time. These results suggest that immunization of oral mucosa may induce systemic antitumor immunity more efficiently than immunization of the skin and that oral mucosa may be one of the most suitable tissues for cancer gene therapy by means of particle-mediated gene transfer.

The distribution and production of cholesteryl ester transfer protein in the human aortic wall.

Cholesteryl ester transfer protein (CETP) has been considered to mediate the transfer of cholesteryl ester from arterial wall, however, the distribution and production of CETP in human arterial wall remains unclear. Present study histopathologically demonstrated the distribution of CETP and CETP mRNA in the human aortic wall by immunohistochemistry and in situ hybridization. While CETP was constantly distributed in the media, the protein was recognized within the intima with fibrocellular thickening and atherosclerotic intima. Double immunostaining methods demonstrated CETP expression in smooth muscle cells in the intima and media. CETP mRNA was detected not only in intimal cells but medial smooth muscle cells. Intimal cells expressing CETP mRNA were considered to be monocyte-derived macrophages and smooth muscle cells by immunohistochemistries using two antibodies against smooth muscle actin and human macrophage on the subserial sections. Our in vivo study provides that CETP is produced by smooth muscle cells in the intima and media of human aorta, and it is suggested that arterial smooth muscle cells positively participate in the removal of excessive cholesteryl ester from the arterial wall by CETP production.

Immunolocalization of apolipoproteins in aortic atherosclerosis in American youths and young adults: findings from the PDAY study.

The immunohistochemical distribution of apolipoproteins in the abdominal aortas of 142 men, 15-34 years of age, collected in a cooperative multicenter study group (Pathobiological Determinants of Atherosclerosis in Youth) was examined in relationship to serum VLDL+LDL+HDL cholesterol levels. ApoB deposits were limited to the intima of specimens with intimal fibro cellular thickening or atherosclerotic lesions. Apo A-I, E and J were observed in both the intima and media of the aortas with intimal lesions. The pattern of apoJ distribution was similar to that of apoA-I and E. The distribution patterns of these apolipoproteins in these young adults were very similar to those in adults and old men seen in an earlier study. The extent of apolipoprotein distribution in the intima and media increased with age and the stage of atherosclerosis development, but was not correlated significantly with serum VLDL+LDL or HDL cholesterol levels. The infiltration of lipoprotein particles into the aortic wall seems to be more strongly associated with the progression of intimal lesions rather than with serum cholesterol levels.

Pyrimidine nucleoside phosphorylase-activity and biological characteristics of breast-cancer.

We investigated whether Pyrimidine nucleoside phosphorylase (PyNPase) activity in breast cancer tissue correlated with biological characteristics of breast cancer. PyNPase activity, ER, PgR, EGFR, DNA ploidy pattern, PCNA positive cells and amplification of the c-erbB-2 gene were determined in specimens from 138 patients. PyNPase activity was significantly higher in ER negative than ER positive carcinomas (p<0.05), in PgR negative than PgR positive carcinomas (p<0.05) and significantly higher in tumors with c-erbB-2 gene amplification compared with tumors with no amplification (p<0.05). The results suggest that PyNPase activity in breast cancer tissue may be a new biological characteristic of breast cancer.

Different effects of pemphigus antibody and plasmin on the distribution of keratin intermediate filaments and desmoplakins between cultured oral and epidermal keratinocytes.

In order to clarify the molecular mechanism of blister formation in oral mucosa in pemphigus vulgaris (PV) comparing with that in epidermis, we analyzed the effects of PV serum on the distribution of keratin intermediate filaments (KIFs) and desmoplakins in oral as well as epidermal cultured keratinocytes by immunofluorescence microscopy using anti-keratin and anti-desmoplakin I/II monoclonal antibodies. After incubation with PV serum for 96 h at 37 degrees C, clusters of anti-keratin positive dots were formed around the nucleus in some of the keratinocytes from normal gingiva and soft palate but not in keratinocytes from tongue and skin, and desmoplakins also changed their distribution from linear arrangement at cell-cell contacts to clusters of dots around the nucleus in gingiva but not in epidermal keratinocytes. The dotted structures similar to those induced by pemphigus serum were formed also by incubation with human plasmin in gingival keratinocytes. However, no dot-formation of keratins was induced in these cells after incubation with trypsin. Furthermore, in epidermal keratinocytes, no keratin-dot formation was observed even after incubation with plasmin or trypsin. These results suggest that the dotted structures of KIFs caused by PV serum and plasmin might be a feature characteristic for the response of oral keratinocytes to PV serum and that there are some distinct differences in susceptibility to, and mode of, bulla formation between oral epithelium and epidermis.

Collagen alteration in vascular remodeling by hemodynamic factors.

The collagen alterations in the vascular wall remodeled by hemodynamic change were investigated by electron microscopy and immunohistochemistry. The left anterior descending coronary artery (LAD) without a myocardial bridge (MB) showed both lower matrix metalloproteinase-1 (MMP-1) expression and a smaller extent of spiraled collagen (SC) distribution than the LAD wall with MB, in which the intima was influenced by high shear stress. In the wall of the varicose great saphenous vein (GSV) the expression of MMP-1 was lower, while the expression of prolyl 4-hydroxylase was higher, than in the normal GSV. The extent of SC distribution in the intima and media of the varicose GSV was smaller than that in the normal GSV. An analogous difference in results was demonstrated between the portal vein (PV) of patients with liver cirrhosis and normal PV. However, the levels of expression of MMP-2, MMP-9 and tissue inhibitors of MMP (TIMPs) in these pathologic vessels were not different from those in the corresponding normal vessels. The results indicate that hemodynamic forces such as shear stress and increased intravascular blood pressure contribute to the collagen alterations in the vascular wall, which may lead to vascular wall remodeling.

Plant regeneration from hairy roots induced by infection with Agrobacterium rhizogenes in Crotalaria juncea L.

Hairy roots were induced from leaf segments of Crotalaria juncea, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without phytohormones. The adventitious shoots were induced from 30% of root segments 3 months after transfer onto medium containing 3 mg/l benzyl adenine. These shoots produced roots 1 month after transfer onto 1.2% agar-solidified 1/2MS medium without phytohormones. Regenerated plants were successfully grown under greenhouse conditions. The transgenic nature of the regenerated plants was confirmed by Southern-blot analysis.

[Structure and function of bone marrow environment].

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.

An ACTH-producing pituitary carcinoma developing Cushing's disease.

An autopsy case of an ACTH-producing pituitary carcinoma in a 59-year-old man who developed Cushing's disease is reported. The surgically removed pituitary tumor was diagnosed as chromophobe adenoma, however, pulmonary metastases appeared 2 years after the operation. Autopsy revealed a residual pituitary tumor in the sella turcica with systemic metastases to the lungs, liver, pulmonary lymph nodes, hypothalamus, dura mater, and the subarachnoid space of the midbrain and spinal cord. Immunohistochemistry revealed ACTH positivity in the tumor cells. Further immunohistochemical study showed positive high expression of Ki-67 in the tumor removed at surgery as well as in the autopsied tumor. Ki-67 labeling index provided valuable information about the invasive and proliferative potential compared to noninvasive benign pituitary adenoma.

Intratumoral pyrimidine nucleoside phosphorylase (PyNPase) activity predicts a selective effect of adjuvant 5'-deoxy-5-fluorouridine (5'DFUR) on breast cancer.

BACKGROUND: Pyrimidine nucleoside phosphorylase (PyNPase) is the enzyme that converts 5'-deoxy-5-fluorouracil (5'DFUR) to 5-fluorouracil (5FU). Its activity in cancer tissue may correlate with the selective antitumor activity of 5'DFUR in breast cancer. METHODS: Two hundred and sixteen T2 breast cancer patients were treated consecutively with surgery followed by 5'DFUR (600 mg/body/day) + tamoxifen (20 mg/body/day) for 2 years. PyNPase activity in breast cancer tissue, determined by high-performance liquid chromatography, ranged from 4.2-626.0 micrograms FU/mg protein/hr (mean +/- SD, 203.5 +/- 122.4), and the examined patients were divided into two groups: group A (high PyNPase group), cases with the PyNPase activity equal to or more than the mean value of 203.5 micrograms FU/mg protein/hr, and group B (low PyNPase group), cases with activity less than the mean value. RESULTS: Although there was no difference in relapse-free survival (RFS) between groups A and B, among node-positive patients (n = 83) those in group A tended to have a longer RFS. When divided into subgroups according to estrogen receptor (ER) status, among node-positive and ER-positive tumors (n = 49), the RFS was significantly better in group A than in group B (p < 0.05). CONCLUSION: Intratumoral PyNPase activity might be of use as a predictor of the effect of adjuvant 5'DFUR on breast cancer.

Primary embryonal carcinoma of the retroperitoneum.

A 32-year-old man with primary embryonal carcinoma of the retroperitoneum, a very rare condition, is reported. The disease took an acute course with rapid enlargement of cervical and inguinal lymph nodes, and high serum alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) values were observed. His testes were, however, intact. Remarkable tumor regression was achieved by systemic combination chemotherapy with cisplatin, vinblastine, and bleomycin (PVB therapy). This chemotherapy is beneficial even in advanced cases. In the two years since therapy, no recurrence has been observed.

Primary esophageal non-Hodgkin's lymphoma.

A 77-year-old woman with primary esophageal non-Hodgkin's lymphoma in clinical stage IEA (Ann Arbor Classification) developed pain and difficulty in swallowing. An upper gastrointestinal examination revealed a submucosal tumor from the upper to the middle portion of the esophagus. Histopathological examination at endoscopic biopsy with endoscopic partial incision showed non-Hodgkin's lymphoma (diffuse type--large cell). Immunohistological examination of tumor cells disclosed LCA (+), CD3(DAKO) (+), MT1 (+), UCHL1 (+), MB1 (+), MxPanB (-) and EMA (-) reactivity and showed T cell lymphoma. The clinical stage was determined to be IEA after further work-up. Improvement of swallowing difficulty and esophageal findings on upper gastrointestinal series were noted after modified CHOP therapy and radiotherapy (total 50 Gy).

The basic diagnostic approaches used in robotic still-image telepathology.

We investigated the basic diagnostic processes used in telepathology with our robotic still-image system, OLMICOS, by analysing the steps and patterns used in 20 consecutive tissue section diagnoses. Three basic approaches were recognized. One was magnifying a suspect finding in a low-powered microscopic image. This approach was used mostly for confirming or characterizing a tumour. The second approach was scanning over a low-powered image by magnifying square images to form a mesh. This was found to be useful to confirm the presence or absence of signs and was mostly used as the initial step in judging the surgical margin of malignant cases or diagnosing lesions. The third approach was a combination of these two and was used for delineating the surgical margin, confirming the absence of metastases or diagnosing difficult lesions. Recognition of these three basic diagnostic approaches is important in making a rapid and correct remote diagnosis.

[The early hemorrhage and development of a bacterial aneurysm after a cerebral ischemic attack caused by a septic embolism--a case report].

A 71-year-old female was admitted with the complaints of dysarthria and right hemiparesis. CT scan revealed subarachnoid hemorrhage in the left cerebral sulcus. The first angiography was performed 3 days after the onset and left carotid angiography revealed a small aneurysm arising from the left middle cerebral artery. After 3 weeks of antibiotic therapy, the second angiography showed the aneurysm to be clearly enlarged, so it was resected. The patient complained of marked dysarthria a day after the operation and CT scan revealed a new infarction in the right frontal lobe. The third angiography showed an aneurysm arising from the right middle cerebral artery and the fact that two peripheral arteries of the aneurysm had disappeared 3 weeks after the first operation. The second operation was performed and a bacterial aneurysm was resected. The patient left the hospital without any neurological deficits. Septic embolism is the most important complication of infective endocarditis and it is usually presented with subarachnoid hemorrhage and intracerebral hemorrhage caused by ruptured bacterial aneurysms. In this case the septic embolism occurred two times. At each time cerebral ischemic attacks were presented. The reason why this case presented with ischemic symptoms was suspected to be that embolisms occurred at the trifurcation of the distal middle cerebral arteries. We were able to detect a bacterial aneurysm angiographically 3 days after the ischemic attack and we suspected that a bacterial aneurysm had been able to develop within 3 days after the septic embolism.

[Multilocular chronic intracerebral hematoma due to metastatic brain tumor: a case report].

Chronic intracerebral hematoma is a fairly rare clinical entity. We report a case of a 76-year-old man who presented with left hemiparesis on admission. CT scan revealed a hematoma in the right basal ganglia. Under observation, the hyperdense lesion on CT scan changed to isodense 2 weeks after admission and then changed to low density 4 weeks after the first scan. These findings suggested hypertensive intracerebral hemorrhage but, 3 months after admission, hemiparesis grew worse and the appearance of the lesion changed from low density to isodensity again. When the patient presented disturbance of consciousness, CT scan revealed a new hemorrhage from the capsule of the hematoma and the new hematoma markedly expanded 2 weeks later. The contents of the hematoma was old uncoagulated hematoma resembling chronic subdural hematoma and drainage was carried out under local anesthesia. The patient died because of respiratory failure and autopsy was performed. Histopathologically cancer cells were shown in the hematoma cavity and the capsule of the hematoma was composed of two layers: a collagenous layer on the inside and a granulation layer with neovascular system on the outside. As the neovascular system existed on the outside of the capsule, the rebleeding occurred outside the capsule and the second hematoma appeared beside the initial one. We suspected that the initial bleeding occurred in the tumor tissue and the rebleeding occurred in the granulation layer of the capsule.

[Two case reports of retroperitoneal leiomyosarcoma].

Two cases of retroperitoneal leiomyosarcoma are presented. The first case was in a 67-year-old female, whose chief complaint of right upper abdominal mass and dull pain. The tumor, 13 x 12 x 8 cm in size, developing in the retroperitoneum was removed with the right kidney and vena cava. The pathological diagnosis was reported as leiomyosarcoma. The second case was in a 62-year-old male, whose complaint was left abdominal swelling, also with general fatigue. A large tumor mass, invading over all of the left flank organs, was palpable by physical examinations, from which the mass was far beyond surgical approach. Needle biopsy revealed the pathological finding of leiomyosarcoma. One month later, the patient died of cachexia. Review of the literatures for the retroperitoneal leiomyosarcoma, revealed only a few cases; 1.7%, of all leiomyosarcoma to date.

[A case of distal ureteral atresia].

A case of distal ureteral atresia with the chief complaint of a mass in the left lower quadrant is reported. On palpation of the abdominal region, a 3 X 4 cm mass, smooth-surfaced and movable, was palpable in the left lower quadrant, IVP failed to show the left kidney, while CT revealed an atrophic left kidney and dilated ureter. For the diagnosis of distal ureteral atresia, total left ureteronephrectomy was performed. The left kidney measured 4.5 X 3 X 3 cm and the ureter was dilated and ended blind. The pathohistological findings included dysplasia with normal nephrons here and there of the kidney. We examined the distal ureteral atresia from the renal tissue.