Milky spots as the implantation site for malignant cells in peritoneal dissemination in mice.

We examined the site-specific implantation of cancer cells in peritoneal tissues after an i.p. inoculation of 10(5) P388 leukemia cells. Twenty-four h after the inoculation, the number of viable cancer cells infiltrating into specific tissue sites of the peritoneum was estimated by an i.p. transfer method. A descending order of tissue implantation with cancer cells was established as omentum > gonadal fat > mesenterium > posterior abdominal wall > stomach, liver, intestine, anterior abdominal wall, and lung. A significant correlation was established between the logarithm of the number of infiltrating cancer cells and the logarithm of the number of milky spots. Next, the omentum was examined microscopically after i.p. inoculation with P388 leukemia cells labeled with bromodeoxyuridine or B-16 PC melanoma, which were differentiated from the other cells by an immunocytological method using anti-bromodeoxyuridine antibody or by the melanin of the B-16 PC melanoma cells. These cancer cells were found microscopically to be infiltrating only the milky spots, whereas none were seen at the other sites. These results suggest that cancer cells seeded i.p. specifically infiltrate the milky spots in the early stage of peritoneal metastases.

[Quality control (QC) of CT on rail system (FOCAL Unit) with a micro-multi leaf collimator (mMLC) using new GafChromic film for stereotactic radiotherapy].

PURPOSE: Recent years, CT on rail system was reported to be useful as a tool for image-guided radiotherapy (IGRT). This system was clinically developed with the aim of stereotactic irradiation (STI) for brain, lung, liver, prostate and other sites. Quality assurance and quality control (QC) is an important issue in CT on rail system to assure geometric accuracies. The purpose of this study is to estimate the geometric accuracies of our CT on rail system using a detachable micro-multi leaf collimator (mMLC) with new type radiochromic films. Carrying out our original QC program, translational errors, setup reproducibility, beam misalignment and beam characteristics were evaluated. METHODS AND MATERIALS: We have studied with CT on rail system (FOCAL unit, Toshiba Medical systems, Tokyo, Japan) and mMLC unit (Accuknife, Direx Inc., Tokyo, Japan). We have developed original alignment phantom and small steel markers (2 mm phi) were implanted on its surface at certain intervals. Firstly, we have evaluated the accuracy of self-moving CT gantry and CT resolutions for cranio-caudal directions by changing slice thickness. And then using the phantom, we have measured the accuracy and reproducibility of geometric isocenter of the linac side and the CT gantry side by scanning the phantom. We have also measured the geometric changes of the common treatment couch by weight-loaded test (up to 135 kgw). To estimate dosimetric and geometric accuracies with the mMLC unit, the misalignment of the beam axes (gantry, collimator and couch rotation axis), mMLC leaf positions, and dose distributions for the verification plan were measured with new type GafChromic films (GafChromic-RTQA, ISP Inc., USA) and cylindrical phantom. The dose characteristics of the GafChromic film were also evaluated. RESULTS: The reproducibility of the self-moving CT gantry have a good agreement within 1 mm. Weight-load test have shown a good reliability within 2 mm at the common treatment couch. The translational precision of the common treatment couch was 0.0 +/- 0.1 mm at linac side and -0.2 +/- 0.5 mm at CT gantry side. The misalignments of beam axes have been kept within 0.4 mm at maximum. Gap test have shown the accuracies of the mMLC leaf positions, which is needed to keep within 1 mm by a routine calibration. CONCLUSIONS: To practice quality control program for the FOCAL unit and the mMLC unit is essential for a regular interval to reduce systematic errors. New type radiochromic film would be useful for a verification tool as alternative to conventional film.

Identification and characterization of two novel mutations (Q421 K and R123P) in congenital factor XII deficiency.

The factor XII genes of two unrelated factor XII-deficient Japanese families were screened, and two novel mutations were identified. A heterozygous mutation (Q421K) was identified in the gene of a cross-reacting material (CRM)-negative patient with reduced FXII activity (entitled Case 1). No mutations were discovered in the other allele. Case 2 was a CRM-negative patient with severe FXII deficiency. In this case, a homozygous mutation (R123P) was discerned. Expression studies in Chinese Hamster Ovary (CHO) cells demonstrated accumulation of mutant Q421 K factor XII in the cell, and insufficient secretion, while the R123P mutant showed lower levels of accumulation than wild-type, and no evidence of secretion in culture supernatant. In the presence of proteasome inhibitor, all types of FXII (wild-type. Q421K, R123P) accumulated in the cells. Protease protection experiments using the microsomal fraction of these cell lines demonstrated that while 20% wild-type FXII (total wild-type:100%) and 10% R123P mutant (total R123P-type: 40%) were resistant to treatment with trypsin, 50% Q421K-type FXII (total Q421K-type:130%) remained resistant to digestion. From these results, we conclude that Q421K is less susceptible to proteasome degradation than wild-type, but is unable to exit the ER efficiently, resulting in insufficient secretion phenotype. In contrast, R123P is susceptible to proteasome degradation and is not secreted.

Evaluation of an artificial dermis full-thickness skin defect model in the rat.

An artificial dermis product was applied to full-thickness skin defects in rats and cell infiltration into the collagen matrix was investigated. Host fibroblasts and capillaries infiltrated as far as the upper end of the collagen matrix by day 14 after application. Determination of glycosaminoglycan levels in the matrix showed that hyaluronic acid was generated in a similar amount to that seen in the intact skin by day 14. An autologous thin split-thickness skin graft was placed onto the artificial dermis simultaneously or several days after its application to the defect. The take rate was 100% when a split-thickness skin graft was performed on day 14 after application of the artificial dermis. At 6 weeks after the skin defect was created, the wound area was 80% of the original area and the dermis at the grafted site was as thick as that of normal skin. These results suggested that the artificial dermis provides a good matrix for thin split-thickness skin graft and is useful for the reconstruction of full-thickness skin defects. This method is considered to be an alternative to the conventional procedure using thick skin grafts or skin flaps.

Histological evaluation of skin reconstruction using artificial dermis.

An artificial dermis, composed of a collagen matrix, was applied to a full-thickness skin defect prepared on the back of rats. Two weeks later, a thin split-thickness skin autograft was overlaid on the matrix at each recipient site. The dermal layer at the recipient sites was 1.02 mm thick with prior application of artificial dermis, as compared with the 0.46 mm thickness observed without such pretreatment. Histologically, the split-thickness skin graft normally lies with no gap on the artificial dermis, which looks like natural dermis. Six days after grafting, the epithelial basal cells in the grafts showed an active uptake of bromodeoxyuridine (a thymidine analogue), indicating high activity of cell proliferation. About 50 and 20% respectively of the artificial dermis remained at each recipient site at 12 and 20 weeks after its application (after the skin defect). This finding indicates that bovine collagen, which is a constituent of the artificial dermis, is gradually replaced by the host tissue.

Acquired Pelger-Huët anomaly in association with concomitant tacrolimus and fluconazole therapy following allogeneic bone marrow transplantation.

A 38-year-old Japanese woman with severe aplastic anemia received an allogeneic bone marrow transplant from her serologically HLA-identical father. Cyclosporine and methotrexate were administered to prevent graft-versus-host disease (GVHD). However, grade III acute GVHD developed on day 44, which was successfully treated with methylprednisolone and tacrolimus. Fluconazole therapy was started for oral candidiasis on day 112, but she complained of headache soon after. In addition to glycosuria and increased serum creatinine levels, Pelger-Huët anomaly of granulocytes was found in her blood, which disappeared after discontinuation of tacrolimus. Transient occurrence of Pelger-Huët cells may be associated with tacrolimus toxicity due to drug interaction with fluconazole.

Increased incidence of cytomegalovirus (CMV) infection and CMV-associated disease after allogeneic bone marrow transplantation from unrelated donors. The Fukuoka Bone Marrow Transplantation Group.

Cytomegalovirus (CMV) infection and CMV-associated disease were monitored using the CMV antigenemia assay in 72 patients who received allogeneic bone marrow transplantation (BMT), and their incidences were compared between related and unrelated donor transplant patients. The incidence of CMV infection after BMT was significantly higher in patients who received transplants from HLA-matched unrelated donors than from HLA-matched sibling donors (87% vs 53%, P < 0.05). CMV-associated disease developed in 73% of unrelated and in 14% of sibling donor transplant patients (P < 0.01). The peak levels of CMV antigenemia were significantly higher in unrelated donors than in sibling donor transplant patients (16 vs 1 CMV antigen-positive cells per 50000 WBCs, P < 0.01). The median number of CMV antigen-positive cells on first detection was also significantly higher in unrelated donor transplant patients (15 vs 1, P < 0.01). The detection of CMV antigen-positive cells preceded the development of CMV-associated disease in 18% of unrelated donor transplant patients, suggesting a lower predictive value of CMV antigenemia for subsequent CMV-associated disease in unrelated donor BMT. Careful monitoring and further studies are needed for the early diagnosis and prevention of CMV-associated disease in unrelated donor BMT.

Role of milky spots as selective implantation sites for malignant cells in peritoneal dissemination in mice.

We investigated the significance of milky spots for malignant cells in peritoneal dissemination using three mouse carcinomatous peritonitis models. P388 leukemia and Colon 26 cancer cells were labeled with bromodeoxyuridine (BrdU) and mice were inoculated intraperitoneally. After 24 h the greater omentum and the mesenterium were removed and stained immunohistochemically with anti-BrdU antibody. The labeled cells were found to have preferentially infiltrated into the milky spots in these specimens. Next, using B16 PC melanoma cells, which can be easily distinguished from the other cells by the intrinsic black melanin, the distribution of the melanoma cells was observed macro- and microscopically following intraperitoneal inoculation. The melanoma cells were similarly found to have selectively infiltrated into the milky spots in the omentum and mesenterium after 1 day. Moreover, the melanoma cells were growing and forming distinct metastic lesions within the milky spots 1 week later.

Gastrointestinal angiodysplasia in congenital platelet dysfunction.

We herein report three cases of repeated massive bleeding from the stomach and small bowel. One patient suffered from both thrombasthenia (type II) and von Willebrand disease (type 1) simultaneously. Two others had Bernard-Soulier's syndrome (BSS). One patient with BSS had bleeding from gastric angiodysplasia and was treated endoscopically by clipping. The other patients had massive bleeding from the small intestine, and had partial resection of the affected small intestine. Histologically, irregular dilatation and proliferation of the blood vessels were demonstrated in the submucosa in bleeding spots from a resected small intestine, and these findings were consistent with the features of acquired angiodysplasia. The development of gastrointestinal angiodysplasia may not only be associated with a dysfunction of von Willebrand factor but also with that of platelets.

Bronchiolar-alveolar carcinoma in a cow.

Bronchiolar-alveolar carcinoma was observed in the lung of an 8-year-old Holstein cow. Grossly, the lung contained multiple tumour masses, which were solid, yellowish-white in colour, and firm in consistency. These tumours also occurred in the liver, pancreas, uterus and lymph nodes in the thoracic, abdominal and pelvic cavities. Histologically, the masses were composed of abundant fibrous stroma and proliferating atypical cuboidal epithelial cells, occasionally forming glandular structures. Electron microscopy revealed that the neoplastic cells had microvilli and lamellar bodies in the cytoplasm, suggesting that they originated from immature respiratory epithelial cells differentiating towards either Clara cells or type II pneumocytes.

The effect of a collagenous implant on deep dermo-periosteal defects in the rabbit.

In plastic surgery, extensive wounds with exposed bone and loss of the periosteum (i.e., deep dermo-periosteal defects) are difficult to treat, even with split-thickness skin grafts, because such grafts rarely survive. Even when these grafts do survive, functional impairment often occurs subsequently. The application of a collagen sponge (Terudermis, Terumo, Tokyo) to such wounds has previously been reported to accelerate granulation tissue formation, resulting in would healing and graft survival. However, this previous report only presented data relating to gross morphological appearance. In this paper, we present histological evidence to demonstrate the beneficial effect of the collagen sponge on experimental dermo-periosteal scalp defects in rabbits. About two weeks after the application of collagen sponge to the experimental wounds, a well-vascularized granulation tissue was formed. Autologous split-thickness skin grafts applied to this new granulation tissue were found to be viable one week after grafting. The results confirm histologically that collagen sponge is effective for the treatment of deep dermo-periosteal defects which would not have regenerated skin cover with conventional therapies such as skin grafting or the temporary use of dermoprotective materials followed by skin grafting.

[A pertussis outbreak in a ward for severely retarded].

In June 2000, many cases with persistent cough were observed among inpatients and the staff of a ward for severely retarded. Some of them had symptoms suggestive of pertussis, such as whooping, post-tussive apnea. We performed a retrospective investigation to assess symptoms and serological findings suspicious of pertussis. There were a total of 14 cases of persistent cough over 3 weeks (4 to 9 weeks). 6 cases were inpatients and 8 were hospital staff. Of those, serological test for pertussis infection was performed in 10 cases and 6 cases were diagnosed as serologically confirmed pertussis. The other cases with persistent cough were also considered to be probable pertussis as they have had intensive contact with serologically confirmed cases. 12 cases were treated by antibiotics, but they all failed to respond. It was suggested that Bordetella pertussis must be considered as a causal organism of persistent cough even in adults. To prevent nosocomial transmission of pertussis, droplet precautions and macrolide treatment should be provided for patients with symptoms highly suggestive of pertussis.

[Non-destructive and rapid identification of bacteria using near-infrared spectroscopy].

Near-infrared (NIR) spectroscopy has recently come to be applied extensively in agricultural, food and chemical industries, and pharmaceutical science. We have been attempting to expand this method in the field of medical science. For example, we tried to use NIR spectroscopy for determination of bacteria. As the first step of this attempt, we differentiated between Escherichia coli and Staphylococcus aureus using NIR spectroscopy. This method could still further differentiate Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA). Using those results as reference, the true name of bacteria from unknown bacteria was given. Not only untreated bacteria, but also we differentiated untreated MSSA, MSSA cultured in sub MIC concentration of ABPC and heat-killed MSSA. This identification method is sensitive to the bacterial concentration. In the future, the some new idea of a new direction of research from the result of plots of weights from two different bacteria will appear.

[Therapy of carcinomatous peritonitis by the angiogenesis inhibitor TNP-470 in mice: analysis of timing and doses for intraperitoneal administration].

In order to perform therapy for carcinomatous peritonitis by a new angiogenesis inhibitor, TNP-470, we investigated the effective timing and the optimal doses for intraperitoneal administration using two mice models. In both carcinomatous peritonitis models caused by M 5076 tumor and B 16 melanoma, the early administration of TNP-470 within one week after tumor inoculation extended the survival times of the mice receiving the drugs, whereas the administration of TNP-470 one week or later after inoculation did not affect the survival time. However, there were significant differences in the effective therapeutic doses of TNP-470 between the two models. It is important to select the best timing and doses for intraperitoneal administration of TNP-470 based on the state of angiogenesis and the sensitivity of the tumor tissues to TNP-470.

[A case of Fabry's disease detected by renal biopsy findings].

A 39 year-old man was found to have mild proteinuria by urinary examination since one year ago. He was for the first time diagnosed as having Fabry's disease by histopathological and electronmicroscopic findings of the renal biopsy specimens, which showed the presence of numerous vacuolated cells and electron dense bodies inside the cells. The level of WBC alpha-galactosidase was significantly lower than normal level. The pedigree of this patient showed a familial history of various types of renal disease. One of the patient's brothers also showed decreased level of WBC alpha-galactosidase, who has been treated by maintenance hemodialysis for 2 years. It is concluded that early diagnosis of this disease through renal biopsy and WBC alpha-galactosidase level is important to manage the future course of patients with Fabry's disease.

[Graves' disease complicated with systemic lupus erythematosus (SLE): a case report].

A 25 year old man had been subjected to subtotal thyroidectomy under the diagnosis of Graves' disease. About a year later the patient developed systemic lupus erythematosus (SLE). Reports on cases of Graves' disease complicated with SLE have barely been observed so far. Consequently, the authors reckoned, with reference to the other literatures on the subject, our case worth reporting.