Ruxolitinib treatment for GvHD in patients with myelofibrosis.

Jak1/2 inhibitor ruxolitinib is a promising agent for treating steroid-refractory GvHD after allogeneic hematopoietic stem cell transplantation (SCT) to produce quick and durable responses. However, optimal dose and tapering schedule of ruxolitinib remain to be determined. Discontinuation of ruxolitinib in myelofibrosis often induces 'withdrawal syndrome' characterized by acute relapse of the disease, but this issue is not well addressed in the treatment of GvHD. Four patients with GvHD (one acute and three chronic) after SCT for myelofibrosis were treated with ruxolitinib. Low-dose ruxolitinib at 5 mg/day was safe and effective, but one of two patients treated at 10 mg/day of ruxolitinib was complicated with severe cytopenia. Withdrawal syndrome developed in one patient, who died of recurrence of GvHD shortly after discontinuation of ruxolitinib. Slow tapering or maintenance with low-dose ruxolitinib inhibited GvHD flare. Our experience calls attention that initiation at low-dose of ruxolitinib may be safe and careful tapering schedule is required to avoid withdrawal syndrome in patients with GvHD after SCT for myelofibrosis.

Three-Dimensional Study of the Terminal Portion in Sprague-Dawley Rat Ejaculatory Ducts.

In mammals, a pair of ejaculatory ducts exists in the urethra at the seminal colliculus. The detailed anatomical structures of the distal end of the ejaculatory ducts of Sprague-Dawley rats were investigated by the computer-assisted three-dimensional reconstruction analysis using light-microscopic serial sections. A three-dimensional reconstruction revealed that in adult rats, the ejaculatory sinus pair consists of two parts: the cranial section - a compartment region composed of a fusion of the ampullary gland duct and the seminal vesicle duct, and the caudal section - a grooved region composed of a long slitlike ejaculatory ostium that extends into the urethra on both sides of the seminal colliculus. But the sphincter structure was not observed. The long axis of the compartment region was approximately 58 µm in length, and that of the groove region was approximately 495 µm. Although many epithelial glands ducts were distributed throughout the ejaculatory sinuses, the prostate and coagulation gland ducts did not open in these sinuses. The urethra was composed of transitional epithelium, while the ejaculatory sinuses were composed of single to stratified cuboidal epithelium. The ejaculatory ducts continued to the ejaculatory ostium in male adult Sprague-Dawley rat were composed of the seminal vesicle ducts received the ampullary gland ducts.

Relationship between neutrophil influx and oxidative stress in alveolar space in lipopolysaccharide-induced lung injury.

We intratracheally administered lipopolysaccharide (LPS) to ICR mice and then collected BAL fluid and lung tissue to determine whether levels of neutrophils and/or myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid reflect lung tissue damage. Robust neutrophil accumulation into the alveolar space and lung tissue were almost completely abolished at seven days along with oxidative stress markers in the lung. However, lung injury scores and lung wet/dry ratios, as well as MPO and oxidative stress markers in BAL fluid were significantly increased at five and seven days after LPS administration. At later time points, BAL neutrophils generated more MPO activity and ROS than those harvested sooner after LPS administration. Although elevated neutrophil levels in BAL fluid reflected oxidative stress in the lungs, MPO might serve as a useful marker to evaluate damage sustained by epithelial cells over the long term.

Microvascular growth of 7,12-dimethylbenz(alpha)anthracene-induced adenocarcinomas in rats: histology and scanning electron microscopy of resin casts.

Vascular changes at various stages of growth of 7,12-dimethylbenz(alpha)anthracene (DMBA)-induced mammary adenocarcinomas in 22 female Sprague-Dawley rats were investigated using histology, immunohistochemistry and scanning electron microscopy (SEM) of corrosion casts. In the early stage of tumour growth, capillaries within the neoplasms were thin, with 8-10 microm diameter, and characterized by rows of vascular sprouts representing extensive neovascularization and formation of a high-density capillary plexus. In the intermediate stage of tumour growth, the growing tumour was multi-nodular and tumour cells were arranged in a tubulopapillary pattern. Capillaries formed spheroid vascular capsules and were characterized by dilation, to a diameter of 10-80 mum, and blind ends. In the late stage of tumour growth, a remarkable reduction in the number of vascular endothelial growth factor-positive cells and Ki-67-stained nuclei was demonstrated. The tunica media of nutritive arteries displayed a tendency to atrophy. Many arteriovenous anastomoses between the major arteries and the veins were found in regions just before their entry into the tumour. The central regions of the tumour were degenerative and necrotic, and vasculature was confined to surface regions of the tumour, forming a basket-like configuration around the avascular central regions. Resin leakages representing haemorrhage or oedema and distortions such as flattening, break-off and strangulations of capillaries were frequently observed, all of which are known as late tumour signs. We concluded that these microvascular alterations might change the homogeneity of tumour perfusion and contribute to necrosis in central portions of the tumour.

[Two cases of canine histoplasmosis in Japan].

Histoplasmosis is distributed in tropical, subtropical and temperate zones of the world. The disease is one of the imported mycoses in Japan. To date, although more than 30 human and one canine case of histoplasmosis have been reported in Japan, some including that of the canine might have been infected domestically, since the patients have no history of going abroad. The pathogen of histoplasmosis is thus believed to be present in our country. We examined skin biopsies from two dogs in Tokyo and Kumamoto, and found fungal elements 1-2 or 2-4 microEm in diameter in the macrophages. The homology of DNA sequences for the ITS rRNA gene were correspondent to Ajellomyces capsulatus at a rate of more than 97.4%. Therefore, the two dogs were diagnosed as having been infected with Histoplasma capsulatum which is the anamorph of A. capsulatus. Since the dogs had no history of having been outside Japan and had not been brought from an endemic area, they might have been infected domestically. Further epidemiological surveys on canine histoplasmosis may be able to estimate autochthonous human cases in Japan.

Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at -196 degrees C.

This study reports the development of a reliable method for cryopreservation of rat epididymal spermatozoa and the production of live young by artificial insemination using these cryopreserved spermatozoa. The motility and membrane integrity of rat spermatozoa were investigated after spermatozoa had been subjected to physical stress and frozen with various concentrations of glycerol (0, 3 and 6%) either in the presence or absence of Equex Stem as cryoprotective agents. The ability of cryopreserved spermatozoa to generate normal offspring by intrauterine insemination was also evaluated. Rat spermatozoa that had been centrifuged at 700 g for 5 min showed a significant decrease in motility compared with non-centrifuged spermatozoa. In addition, after centrifugation three times the percentage of membrane-intact spermatozoa decreased to approximately 0%. The percentage of membrane-intact spermatozoa was significantly higher (P < 0.01) in semen samples that had been frozen in medium without glycerol than in samples frozen in medium with 3% glycerol. Although the addition of 0.7% Equex Stem to medium without glycerol or with 3% glycerol did not influence rates of sperm motility after freezing and thawing, the percentage of membrane-intact spermatozoa was improved by the presence of 0.7% Equex (P < 0.05). Therefore, rat spermatozoa were handled gently to avoid physical stress and were frozen in medium containing 23% egg yolk, 8% lactose monohydrate and 0.7% Equex Stem, at pH 7.4 adjusted with 10% Tris(hydroxymethyl)aminomethane solution. Thirteen female rats were inseminated into the oviductal end of both uterine horns with frozen-thawed spermatozoa. Forty-one normal live offspring were obtained from nine of the inseminated females. These results indicate that frozen-thawed rat spermatozoa can generate normal offspring. To our knowledge, this procedure is the first successful production of offspring using spermatozoa cryopreserved in liquid nitrogen.

[High-dose rate Iridium-192 brachytherapy with flexible applicator--a trial toward decrease of stress during treatment and improvement of quality of life].

PURPOSE: We tried to improve the materials and methods of high-dose rate Iridium-192 brachytherapy for localized prostate cancer and evaluated the stress during the treatment in 20 patients with whom the therapy was performed. MATERIALS AND METHODS: Rigid applicators made of stainless steel of 1.6 mm in diameter were indwelt with a template as usual for 30 hours in 14 patients (group A). Flexible applicators made of polyoxymethylene rosin (POM) of 2.0 mm in diameter were indwelt without a template for 30 hours after the applicator insertion in 6 patients (group B). We made inquiries about lumbago, inconvenience and necessity of assistant help and sleep in the course of therapy, and urinary incontinence and erectile function after the course of therapy as the QOL. RESULTS: The stress during the course of therapy in the patients of group B was obviously less than that of group A. There were no significant differences in urinary incontinence and erectile function after the course of therapy between group A and B. CONCLUSION: In this study, our trial successfully reduced the stress during the course of therapy in the patients with localized prostate cancer in the course of high-dose rate Iridium-192 brachytherapy.

[High-dose rate iridium-192 brachytherapy combined with external beam radiotherapy for localized prostate cancer].

PURPOSE: We report our technique and also preliminary results in the cases with localized prostate cancer treated by the combination of high-dose rate Iridium-192 (HDR-Ir192) brachytherapy and external irradiation. MATERIALS AND METHODS: From June 1999 to August 2000, 17 patients were treated by the combination of HDR-Ir 192 and external beam. The mean age of patients was 72 years (range, 48-81 years). The clinical stage was B1 in 5, B2 in 7 and C (no cancer with seminal vesicle) in 5 cases. Of 10 patients without neoadjuvant hormonal therapy, the median initial pretreatment PSA was 15.3 ng/ml (6.93-222.32 ng/ml). The treatment was given by HDR-Ir 192 brachytherapy (6 Gy x 3 times/2 days) and external beam irradiation (40 or 45 Gy). The brachytherapy was given using TRUS guided percutaneously inserted temporary needles with a high dose rate remote afterloading control. Local control was evaluated by digital rectal examination. TRUS-guided biopsies and serum PSA evaluations. Follow-up ranged from 2 to 14 months, with a median of 8 months. RESULTS: In 4 (40.0%) of 10 patients without neoadjuvant hormonal therapy the level of serum PSA was decreased to less than 4.0 ng/ml within 3 months after the therapy. The effective grade in the biopsy specimens of 8 patients without neoadjuvant hormonal therapy was Grade Ob in 4, Grade 1 in 1, Grade 3 in 3 cases at 3 months after the therapy. No severe intra- or peri-operative complications occurred. CONCLUSION: The combined radiotherapy treatment is safe and effective for use in the patients with localized prostate cancer. However, more comprehensive studies involving long-term follow-up and great numbers of the cases with localized prostate cancer treated by the combination of HDR-Ir 192 brachytherapy and external irradiation will be necessary to determine whether this therapy contributes to better prognosis.

New antimetastatic hypoxic cell radiosensitizers: design, synthesis, and biological activities of 2-nitroimidazole-acetamide, TX-1877, and its analogues.

We designed, based on the molecular orbital (MO) calculation, synthesized, and evaluated the biological activities of the new antimetastatic hypoxic cell radiosensitizer, 2-nitroimidazole-acetamide, TX-1877, and its analogues. Each analogue has an electron-affinic imidazole group, an acetamide group and a certain hydrophilic group to control its biological effect, toxicity, and pharmacokinetics. In in vitro radiosensitization assay, most TX-1877 analogues, which have an electron affinity (EA) of more than 0.9 eV and partition coefficient (P) of more than 0.021, showed satisfactory enhancement ratios (ER > 1.60) at doses of I mM. On the other hand, imidazole analogues, such as TX-1908 (EA = 0.67 eV), TX-1910 (EA = -0.34 eV) and TX-1931 (EA = -0.37 eV), which have low electron affinities, had an ER of 1.31 or less. TX-1877 and KIN-806 effectively inhibited tumor regrowth when administered with irradiation in vivo at a dose of 0.4 mg/g. Tumor lung metastasis was inhibited by treatment with either TX-1877 or KIN-806 without irradiation at a dose of 0.4 mg/g. TX-1877 reduced markedly the mean number of metastatic lung nodules in comparison with KIN-806. Moreover, TX-1877 and KIN-806 enhanced macrophage and helper T lymphocyte infiltration for 3 weeks after drug treatment. TX-1877 shows a high EA value and has the C2 of HOMO localizing on N-methylamide and the C2 of LUMO localizing on 2-nitroimidazole group. The MO data might be useful for designing a bifunctional hypoxic cell radiosensitizer. TX-1877 and its analogues are potential antimetastatic hypoxic cell radiosensitizers, which would improve the efficiency of radiotherapy and quality of life in cancer treatment.

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs.

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.

Microvasculature of hydronephrotic kidneys in KK-A(Y) mice.

Spontaneous hydronephrosis in KK-A(Y) mice was studied using light and electron microscopy and scanning electron microscopy of resin casts to evaluate micro vascular changes in the kidney. The renal parenchyma was extremely thin as a result of tubular atrophy. Histologically, varying degrees of glomerulosclerosis were observed. Ultrastructurally, marked thickenings of the glomerular basal lamina, an increase in mesangial cells and matrix, and marked effacement of foot processes were observed. In resin casts, a marked reduction in number of glomeruli was evident. The capillaries were thin, strangulated and tom-off to varying degrees in severely affected glomeruli. In the medulla, the three-dimensional capillary network running along the tubules was lost and changed to a two-dimensional vascular bed. Despite severe hydronephrosis, the glomerular capillary network was relatively well preserved, being either slightly or moderately injured in approximately 60% of surviving glomeruli.

Toremifene-induced fatty liver and NASH in breast cancer patients with breast-conservation treatment.

We have described fatty liver, diagnosed by computed tomography scanning (CT) in more than 30% of patients with breast cancer who received tamoxifen. Therefore, it is urgent to elucidate the frequency and the degree of fatty liver induced by toremifene, an analogue of tamoxifen, which is also used in breast cancer. We enrolled 52 breast cancer patients who were treated with breast-conservation treatment and administered oral toremifene for 3-5 years as adjuvant endocrine therapy. We evaluated the degree of fatty liver by abdominal CT performed annually. CT demonstrated toremifene-induced fatty liver in four (7.7%) of 52 breast cancer patients. Toremifene-induced fatty liver did not correlate with abnormal levels of AST, ALT, GGT or total cholesterol. One patient who demonstrated moderate fatty liver by CT was histologically diagnosed as non-alcoholic steatohepatitis (NASH) by liver biopsy. The incidence of toremifene-induced fatty liver was significantly lower than that induced by tamoxifen. Accordingly, in terms of fatty liver and NASH, toremifene is considered to be more appropriate agent than tamoxifen. Though toremifene is less likely to induce fatty liver, the possibility remains that toremifene-induced steatohepatitis occurs. Because the diagnosis of fatty liver or NASH can be easily missed if only a blood test is performed, it is necessary to screen fatty liver by annual CT examination for patients who receive an antiestrogen agent.

Unrecognized hepatic steatosis and non-alcoholic steatohepatitis in adjuvant tamoxifen for breast cancer patients.

Adjuvant tamoxifen has become the treatment of choice against estrogen receptor-positive breast cancer. Adverse effects are rarely observed and since symptoms of hepatic steatosis, non-alcoholic steatohepatitis and cirrhosis are usually negligible, such effects are not well characterized despite large cohort studies of adjuvant tamoxifen. This issue remains to be systematically studied. The present study consisted of 136 breast cancer patients treated with or without tamoxifen. Patients had laboratory tests once each month and underwent abdominal computed tomography (CT) annually for 5 years. The extent of hepatic steatosis was assessed by CT as the liver/spleen ratio. While receiving adjuvant tamoxifen, 40 of 105 patients developed hepatic steatosis (liver/spleen ratio <0.9) without obvious changes in body mass index. Twenty-one had a liver spleen ratio of <0.5, whereas none of the 31 patients treated without tamoxifen had a ratio <0.9 or <0.5 (p<0.0001 and p<0.0001, respectively). Hepatic steatosis was recognized in 35 of the 40 patients within the first 2 years of receiving adjuvant tamoxifen and 21 of the 40 had increased transaminase levels. Liver biopsy revealed NASH in 6 of 7 patients among the 21 with a liver/spleen ratio of <0.5. A subset of individuals given adjuvant tamoxifen developed progressive hepatic steatosis without significant changes in the body mass index. We suggest a liver/spleen ratio of <0.5 as a criterion upon which liver biopsy should be recommended since NASH frequently occurred in such patients.

Breast-conservation treatment for patients with ductal carcinoma in situ.

Fourteen cases with symptomatic ductal carcinoma in situ (DCIS) were treated with breast-conservation treatment intensified with endocrine therapy. Nine of 14 patients with palpable mass had tumor detected on mammography. CT, ultrasonography, and MRI were able to detect linear and/or spotty lesion or enhancement suggesting DCIS. Whereas these findings were not particular to DCIS, the combination of these modalities would be useful in deciding the extent of resection for DCIS. There was no patient selection for breast-conservation treatment in our department. All patients received tangential and boost radiation, and were treated with endocrine therapy using anti-estrogen drugs. The reason that nine cases had close margins (<5 mm) might be on account of the treatment including lumpectomy with 1 cm of surgical margin. In spite of their margin status, no local or systemic failure was experienced, and the cosmetic results of most patients were rated as excellent or good. Therefore, our breast-conservation treatment intensified with systemic therapy is thought to be adequate for patients with symptomatic DCIS. Six of eight cases who received preoperative treatment containing endocrine therapy with or without CAF chemotherapy showed a decrease in tumor size. Preoperative treatment may effect the microinvasion and/or breast tissue surrounding a DCIS tumor.

Anti-CD2 monoclonal antibodies prevent the induction of experimental autoimmune myocarditis.

We investigated the effect of a monoclonal antibody against CD2 molecules (OX34) in preventing the induction of experimental autoimmune myocarditis (EAM) induced by immunizing Lewis rats with cardiac myosin. Administration of OX34 before immunization, on Days -6, -4, -2 and 0, completely prevented EAM. On the other hand, treatment with OX34 just before the appearance of myocardial lesions, on Days 9, 11, 13 and 15, had only a partial effect in preventing the disease. Flow cytometric analysis of lymph node cells showed that CD3+ T cells were immediately depleted with the administration of OX34 but had largely recovered on Day 21. Lymph node cells in OX34-treated rats had no proliferative responses to cardiac myosin-rod, but the proliferation was restored when recombinant IL-2 was added. Ultimate production of the anti-myosin antibody was not inhibited by the treatment with OX34. These results suggest that the prevention of EAM by administering the anti-CD2 monoclonal antibody OX34 resulted from T cell depletion during the induction phase, and might in addition result from T cell anergy of Th1, but not Th2 cells.

Soft dipole resonance in the neutron-skin nucleus 6He

A candidate for a soft dipole resonance, a dipole oscillation mode between a core cluster and a neutron skin, was observed at Ex = 4+/-1 MeV and with a width of 4+/-1 MeV in 6He via the 6Li( 7Li, 7Be) reaction at an incident energy of 65A MeV and forward scattering angles including 0 degrees. Its cross section is deduced to be sigma(0 degrees ) = 0.9+/-0.2 mb/sr. This value is comparable to that of the giant dipole resonance simultaneously measured.

Experimental autoimmune myocarditis and its pathomechanism.

The pathomechanisms of autoimmune myocarditis are quite different from viral infection. In this type of myocarditis, cardiac myosin fragments, proper dendritic cells and autoreactive T cells are the 3 major elements in initiating and promoting the inflammation. The causative epitope is locating on the S2 rod portion of the myosin heavy chain (MHC). Through our recombinant study, the peptide was found to be located on the latter half of MHC residues 1070 to 1165. Activity of antigenicity was not different between alpha and beta chain. The cardiac dendritic cell presents a unique structure with large mononuclear and interdigitating processes. This antigen presenter is quickly activated and suppressed by the antigen. The autoreactive T cell is closely linked with cytokine production. In the initial stage of myocarditis, IL-2 and IL-12 are increased. According to the progression of inflammatory changes, a great amount of IL-1b, INF-gamma and TNF-alpha is released around the diseased tissue. At the same time, NO is massively produced from infiltrating macrophages. Cytokines secreted from inflammatory cells accelerate T cell induction from Th0 to Th1. In the convalescent stage, production of TGF-beta 1 and IL-10 become dominant. They contribute to cell induction from Th0 to Th2.

Influence of castration on development of the thymus in neonatal male rats.

The influence of castration on the development of the thymus in neonatal rats was studied to elucidate when after birth the thymus comes under inhibitory regulation by the testis in rats. The relative and absolute weights of the thymus were measured five days after castration these cases. No excessive changes in the weights of the thymus with castration were observed by 31 days after birth. Significant changes in the thymus appeared in the relative weight at 36-day-castration. The absolute weight of the thymus was also significantly increased after 41-day-castration. These findings suggest that in rats the inhibitory regulation of the thymus by testis development does not appear before at least 31 days of age.

Conservation treatment intensified with an anti-estrogen agent and CAF chemotherapy for stage I and II breast cancer.

In order to improve both cosmetic results and survival rates, we performed breast-conservation treatment (BCT) intensified with tamoxifen and CAF chemotherapy to 218 out of 224 patients who visited our department with the desire of breast-conservation between August 1989 to December 1998. Of these patients, 68 presented with tumors of stage I, and another 122 stage II. All patients were administered tamoxifen (for pre-menopausal women) or tremifene (for post-menopausal women) orally: tamoxifen and tremifene administration was started just after confirmation of the breast cancer based on the findings of fine-needle aspiration cytology. All patients underwent lumpectomy with or without axillary dissection (level I and II). For patients with T2 tumors, the lumpectomy was performed following two to four times of CAF chemotherapy. Following conservative surgery, patients were treated with radiation therapy to the intact breast and ipsilateral axilla to a total dose of 4400 cGy with a conedown to a total median dose of 5300 cGy. At the end of March 1999, the mean follow-up time was 49.0 months. In spite of high-positivity (approximately 30%) of microscopically surgical margin, local recurrence rate is considerably low, and only 2 patients experienced local recurrence. Cause-specific survival rate for patients of stage I is 100%, and that of stage II is 91.7% at 5 years. The cosmetic results of therapy were also considered good.