Characterization of Spontaneous Mammary Tumors in Domestic Djungarian Hamsters (Phodopus sungorus).

Mammary tumors that spontaneously occurred in domestic Djungarian hamsters (Phodopus sungorus) were histologically examined. Forty-five mammary tumors included 14 adenomas, 18 adenocarcinomas, 1 lipid-rich carcinoma, 2 adenoacanthomas, 2 malignant adenomyoepitheliomas, 1 benign mixed tumor, and 7 "balloon cell" carcinosarcomas. The latter 4 types were newly recognized neoplasms in Djungarian hamsters. The relatively high incidence of spontaneous mammary carcinosarcomas in domestic Djungarian hamsters is intriguing. Carcinosarcomas exhibited anomalous histological features made up of a mixture of glandular cells, polygonal cells (including "balloon cells"), and sarcomatous spindle cells in varying proportions. Transitional features from glandular cells to polygonal cells and subsequently to sarcomatous spindle cells were observed. Using immunohistochemistry, we observed that glandular cells exhibited an epithelial phenotype (cytokeratin(+)/vimentin(-)), spindle cells exhibited a mesenchymal phenotype (cytokeratin(-)/vimentin(+)), and polygonal cells exhibited an intermediate phenotype (cytokeratin(+)/vimentin(+)). Reduction or loss of ß-catenin expression and gain of S100A4 expression were observed in polygonal and spindle cells. The polygonal cell population included a varying number of characteristic cells that were expanded by large intracytoplasmic vacuoles. Electron microscopy revealed that these "balloon cells" had large cytoplasmic lumens lined by microvilli. These observations suggest that epithelial-mesenchymal transition may account for the pathogenesis of mammary carcinosarcomas in Djungarian hamsters.

Motility of bile canaliculi in the living animal: implications for bile flow.

Modern fluorescence microscopic techniques were used to image the bile canalicular system in the intact rat liver, in vivo. By combining the use of sodium fluorescein secretion into bile, with digitally enhanced fluorescence microscopy and time-lapse video, it was possible to capture and record the canalicular motility events that accompany the secretion of bile in life. Active bile canalicular contractions were found predominantly in zone 1 (periportal) hepatocytes of the liver. The contractile movements were repetitive, forceful, and appeared unidirectional moving bile in a direction towards the portal bile ducts. Contractions were not seen in the network of canaliculi on the surface of the liver. Cytochalasin B administration resulted in reduced canalicular motility, progressive dilation of zone 1 canaliculi, and impairment of bile flow. Canalicular dilations invariably involved the branch points of the canalicular network. The findings add substantively to previous in vitro studies using couplets, and suggest that canalicular contractions contribute physiologically to bile flow in the liver.

Cardiovascular toxicity of cryopreserved cord blood cell infusion.

Although infusion of cryopreserved bone marrow or peripheral blood stem cell is associated with a variety of symptoms, there have been no reports detailing the data of infusion-related toxicities of cryopreserved cord blood (CB) units. We prospectively evaluated the incidence and significance of infusion-related toxicities in 34 adult patients undergoing unrelated CB transplantation. Cryopreserved CB units were thawed and immediately infused, unfiltered, through a central intravenous catheter without further manipulation. Heart rate, blood pressure, oxygen saturation and clinical symptoms were recorded during and after infusion. Twenty-four percent of patients experienced non-cardiovascular toxicities related to infusion. The incidence of systolic and diastolic hypertension and bradycardia was 58, 64 and 32%, respectively. Although three patients (9%) with severe systolic hypertension after the infusion required treatment with antihypertensive agents, no patients experienced life-threatening side effects or needed discontinuation of CB unit infusion. Patient or transplant characteristics had no effect on the hypertension and bradycardia related to the infusion of CB. These data suggest that infusion of cryopreserved CB without further manipulation after thawing is safe and well tolerated. However, cardiovascular toxicities including hypertension and bradycardia were frequently observed.

Preemptive therapy with ganciclovir 5 mg/kg once daily for cytomegalovirus infection after unrelated cord blood transplantation.

The efficacy and safety of preemptive therapy using ganciclovir (GCV) 5 mg/kg once daily for CMV infection after unrelated cord blood transplantation (CBT) were studied. The initial preemptive therapy with GCV 5 mg/kg once daily led to resolution of CMV antigenemia in 25 of 34 patients (74%). In the remaining 9 patients (26%), antigenemia resolved after dose-escalation of GCV or change to foscarnet therapy. Recurrence of antigenemia was seen in 18 patients (53%). A total of 12 patients received the second preemptive therapy with GCV 5 mg/kg once daily, which led to resolution of antigenemia in 11 of 12 patients (92%). The remaining 1 patient (8%) required change to foscarnet therapy. None of 34 patients developed CMV disease. Neutropenia with an absolute neutrophil number of less than 1 and 0.5 x 10(9) per liter after GCV therapy occurred in 12 (35%) and 1 (3%) patients, respectively, after the initial therapy, and in 2 (17%) and 0 (0%) patients, respectively, after the second therapy. No patients developed neutropenic fever or secondary graft failure after GCV therapy. There were no deaths directly attributable to GCV therapy. The present study suggests that antigenemia-based preemptive strategy using GCV 5 mg/kg once daily is feasible and effective for CBT recipients.

No occurrence of Pneumocystis jiroveci (carinii) pneumonia in 120 adults undergoing myeloablative unrelated cord blood transplantation.

The incidence of pneumonia caused by Pneumocystis carinii (PCP) (organism now renamed Pneumocystis jiroveci) during the early period after cord blood transplantation (CBT) was studied in 120 adults. Initially 89 patients (74%) received oral administration of 2 single-strength trimethoprim-sulfamethoxazole (TMP-SMZ) tablets twice daily from day -21. In 45 of 89 patients (51%), TMP-SMZ administration for a scheduled duration was completed. In the remaining 44 patients (49%), however, TMP-SMZ administration was discontinued prior to day -3 because of toxicity. Among these patients, 42 subsequently received aerosolized pentamidine (AP) on a median of day -13 (range, -20 to -6). Thirty-one patients (26%) received AP without TMP-SMZ administration on a median of day -14 (range, -21 to -9). None of the 120 patients were diagnosed with PCP within 100 days or 2 years after CBT; however, one patient who received AP before CBT but no prophylaxis after CBT developed cerebral toxoplasmosis on day +91. Pre-transplant prophylaxis against PCP did not significantly affect transplantation-related mortality or disease-free survival at 2 years after CBT. The results suggest that PCP during the early period after CBT can be effectively prevented by any pre-transplant prophylactic method.

Graft-versus-host Disease-free, Relapse-free Survival After HLA-identical Sibling Peripheral Blood Stem Cell Transplantation With Tacrolimus-based Graft-versus-host Disease Prophylaxis in Japanese Patients.

The ideal post-allogeneic hematopoietic cell transplantation recovery is not just the cure of hematologic malignancies but also freedom from ongoing morbidity. Recent studies have revealed that HLA-identical sibling peripheral blood stem cell transplantation (PBSCT) had been providing impaired graft-versus-host disease (GVHD)-free relapse-free survival (GRFS) due to a higher risk of GVHD. Study on GVHD prophylaxis bears clinical reliance when focused on Japanese population because risk of GVHD differs among races. We identified 15 consecutive Japanese patients who received tacrolimus-based GVHD prophylaxis after myeloablative HLA-identical sibling PBSCT. No episode of grade ≥ II acute GVHD and only one episode of grade III toxicity were documented, with the control of mean weekly blood tacrolimus concentrations during the first 4 weeks at 13 to 17 ng/mL. An estimated 46.7% (95% CI: 21.4% to 71.9%) of the patients enjoyed their GRFS at 3 years after transplantation, and failure in the treatment of chronic GVHD was not reported during the median follow-up period of 1059 days (range, 784 to 1778 days) after the development of chronic GVHD. The results suggest that the application of tacrolimus with the optimization of its blood concentrations may effectively prevent ongoing morbidities after HLA-identical sibling PBSCT.

Fibroblast-like synoviocytes support B-cell pseudoemperipolesis via a stromal cell-derived factor-1- and CD106 (VCAM-1)-dependent mechanism.

B-cell accumulation and formation of ectopic germinal centers are characteristic changes in the diseased joints of patients with rheumatoid arthritis (RA). Earlier studies suggested that interactions between B lymphocytes and specialized synovial "nurse-like" cells peculiar to the RA synovium may be responsible for the homing and sustained survival of B cells in the synovium. However, in this study, we found that B cells spontaneously migrate beneath ordinary fibroblast-like synoviocytes (FLSs) and then experience prolonged survival. FLSs isolated from joints of patients with osteoarthritis also supported this activity, termed B-cell pseudoemperipolesis. We found that FLSs constitutively expressed the chemokine stromal cell-derived factor-1 (SDF-1), and that pertussis toxin or antibodies to the SDF-1 receptor (CXCR4) could inhibit B-cell pseudoemperipolesis. However, expression of SDF-1 is not sufficient, as dermal fibroblasts also expressed this chemokine but were unable to support B-cell pseudoemperipolesis unless previously stimulated with IL-4 to express CD106 (VCAM-1), a ligand for the alpha(4)beta(1) integrin, very-late-antigen-4 (VLA-4 or CD49d). Furthermore, mAb's specific for CD49d and CD106, or the synthetic CS1 fibronectin peptide, could inhibit B-cell pseudoemperipolesis. We conclude that ordinary FLSs can support B-cell pseudoemperipolesis via a mechanism dependent upon fibroblast expression of SDF-1 and CD106.

Impact of ABO incompatibility on engraftment and transfusion requirement after unrelated cord blood transplantation: a single institute experience in Japan.

The impact of ABO incompatibility between donor and recipient on engraftment and transfusion requirement was studied in 95 adults who underwent unrelated cord blood transplantation (CBT). The patients included 27 ABO-identical, 29 minor, 21 major and 18 bidirectional ABO-incompatible recipients. Neutrophil engraftment did not differ between ABO-identical/minor ABO-incompatible and major/bidirectional ABO-incompatible recipients (hazard ratio (HR) 1.17, P=0.48). Cumulative incidence of platelet engraftment in ABO-identical/minor ABO-incompatible recipients was higher than in major/bidirectional ABO-incompatible recipients (HR 1.88, P=0.013). In addition, fewer platelet transfusions were required during the first 60 days after CBT in ABO-identical/minor ABO-incompatible recipients (HR 0.80, P=0.040). RBC engraftment did not differ between the two groups (HR 1.25, P=0.33). However, fewer RBC transfusions were required in ABO-identical/minor ABO-incompatible recipients than in major/bidirectional ABO-incompatible recipients (HR 0.74, P<0.005). No patients developed pure red-cell aplasia after CBT. These results indicate that ABO incompatibility affected platelet engraftment and transfusion requirement of RBC and platelet in CBT recipients. Further studies including larger patient numbers are required to elucidate the impact of ABO incompatibility on the clinical outcome of CBT.

Risk factors for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: retrospective analysis of 73 patients who received cyclosporin A.

Cyclosporin A (CsA) has been used most widely as an immunosuppressive agent for preventing graft-versus-host disease (GVHD). To explore the risk factors including CsA blood levels for grades II-IV acute GVHD, we retrospectively analyzed the data of patients who underwent allogeneic hematopoietic stem cell transplantation in our hospital between March 1989 and July 2001. Seventy-three patients (47 males and 26 females) received CsA and short-term methotrexate for GVHD prophylaxis. CsA 1.5 mg/kg was administered as a 3-h infusion twice daily from day 1 until the patient recovered from the toxic gastrointestinal complication. Methotrexate was given at a dose of 15 mg/m(2) on day 1 and 10 mg/m(2) on days 3, 6 and 11. Grades II-IV acute GVHD occurred in 18 patients (24.7%). Multivariate Cox regression analysis revealed that higher C(5) (the whole-blood CsA concentration at 5 h after the start of infusion) before the onset of acute GVHD reduced the onset of grades II-IV acute GVHD with a hazard ratio of 0.994 (95% confidence interval 0.989-0.999) for every increase of 1 ng/ml. Our data indicate that inadequate exposures of CsA can be a vital risk for developing acute GVHD. From our results, we consider that precise monitoring of CsA concentrations and adjustment of CsA dose using the concentration may be effective to prevent the onset of severe acute GVHD. To confirm this finding, further prospective study will be needed.

Successful Second Allogeneic Stem Cell Transplantation From a Sibling Donor for Relapse of Myelodysplastic Syndrome in a Recipient of a Renal Transplant From His Mother: Case Report.

There have been few reports on allogeneic stem cell transplantation in patients who have previously undergone solid organ transplantation. The clinical course of such patients is not yet well recognized. Therefore, appropriate immunosuppressive prophylaxis for the rejection of a solid organ graft or for graft-versus-host disease has not yet been established. We present the case of a successful allogeneic stem cell transplantation in a patient who relapsed after a first allogeneic stem cell transplantation for myelodysplastic syndrome and who had previously undergone renal transplantation. The prophylaxis in this case for graft-versus-host disease and rejection of the transplanted kidney was mycophenolate mofetil and tacrolimus. No hyperacute rejection of the transplanted kidney was observed. However, the patient's renal function deteriorated after the cessation of the mycophenolate mofetil and the reduction of the tacrolimus. This deterioration seemed to be due to rejection with humoral immunity of donor lymphocytes, and we were able to control it by resuming the mycophenolate mofetil and local graft irradiation.

The out-patient management of patients with acute mild-to-moderate colonic diverticulitis.

BACKGROUND: There are no management criteria for optimum out-patient care in mild-to-moderate acute colonic diverticulitis. AIM: To enable such patients to be managed in an out-patient setting, by establishing criteria and treatment protocols. METHODS: We conducted an open trial and follow-up study from 1997 to 2002. On the basis of ultrasonography, we defined and categorized mild-to-moderate acute colonic diverticulitis ranging from limited inflammation within diverticulum to an abscess < 2 cm in diameter. Subjects were treated as out-patients and followed a 10-day treatment protocol consisting of an oral antibiotic and a sports drink for the first 3 days. Physical examination and laboratory testing helped determine whether or not a patient could resume a liquid diet on day 4, and a regular diet on day 7. RESULTS: Of the 70 patients, 68 were successfully treated. Two patients required hospitalization. Of the 65 patients who were tracked over several months [median (intraquarter range) = 30.8 (11.9-44.2) months], 16 had one or more clinical recurrences. The medical cost per episode was 80% lower than in-patient treatment. CONCLUSIONS: Patients with mild-to-moderate acute colonic diverticulitis can be safely and successfully treated as out-patients using this protocol.

Fatal thromboembolism in acute promyelocytic leukemia during all-trans retinoic acid therapy combined with antifibrinolytic therapy for prophylaxis of hemorrhage.

In contrast to patients with disseminated intravascular coagulation (DIC) due to other causes, patients with acute promyelocytic leukemia (APL) receiving standard cytotoxic chemotherapy can be treated safely with antifibrinolytic drugs for prophylaxis of hemorrhage, without the occurrence of thromboembolic complications. However, such drugs should be used cautiously in APL patients who are receiving all-trans retinoic acid (ATRA) differentiation therapy. We report here a patient with APL who had fatal thromboembolism after receiving ATRA and tranexamic acid therapy.

Hematopoietic cytokine-dependent differentiation to eosinophils and neutrophils in a newly established acute promyelocytic leukemia cell line with t(15;17).

We recently established an acute promyelocytic leukemia (APL) cell line (HT93) that has the capacity to differentiate into neutrophils and eosinophils in response to all-trans retinoic acid (ATRA) and human hematopoietic cytokines. The cells had a myeloblastic morphology, were positive for surface CD33, CD34, and CD56, and showed the following karyotypes: 46, XY, t(1;12)(q25;p13), 2q+, t(4;6)(q12;q13), and t(15;17)(q22;q11). When the cells were cultured with ATRA, they showed nuclear segmentation and developed secondary granules consisting in part of neutrophils and eosinophils. In the presence of ATRA and granulocyte colony-stimulating factor (G-CSF), the cells showed polymorphonuclear neutrophil differentiation accompanied by expression of surface CD11b, CD15, CD10, positive activity for neutrophil alkaline phosphatase (NAP), and NAP mRNA expression. In cultures with ATRA and granulocyte-macrophage colony-stimulating factor (GM-CSF), IL (interleukin)-3, or IL-5, HT93 showed remarkable eosinophil maturation at day 8 as determined by luxol fast blue staining, in addition to expression of eosinophil peroxidase and major basic protein. These results indicate that HT93 is an APL cell line with the ability to differentiate into neutrophils and eosinophils, and that these lineages are dependent on the CSF added. HT 93 should prove to be a useful model in analyzing the effects of hematopoietic cytokines on proliferation, differentiation, and maturation of hematopoietic progenitors.

Profile of cell cycle in hematopoietic malignancy by DNA/RNA quantitation using 7AAD/PY.

Using 7-amino-actinomycin-D/pyronin Y (7AAD/PY), we analyzed the surface phenotypes and cell cycle of 22 hematopoietic cell lines based on their cellular DNA/RNA content. Populations of G1a, G1b, S, and G2M, the DNA index (DI), and the RNA index of S phase (SRI) were calculated by means of DNA/RNA dot plots. Two new parameters were extracted from the cell-cycle profiles: the nucleic acid index of S phase (NI) and the coefficient of variations in the RNA at S phase (SVC). DNA/RNA dot plots of cell lines revealed four characteristic profiles of the cell cycle, defined with the calculated NI and SCV. These were type 0 (small NI, large SCV), type I (small NI, small SCV), type II (large NI, small SCV), and type III (large NI, large SCV). Type O included four stem cell lines: one t(1;19) leukemia, two Ph1+ acute lymphocytic leukemia (ALL), and one biphenotypic crisis of chronic granulocytic leukemia (CGL). Type I included five ALL cell lines: three T-ALL and two common B-ALL. Type II contained 10 myeloid cell lines: five AML and five myeloid crisis of CGL. Type III contained three relatively immature lymphoma cell lines: two Burkitt's lymphoma and one follicular center lymphoma. Calculated NI/SCV (%) were as follows: type 0, 2.27 +/- 0.19/16.7 +/- 3.7; type I, 2.20 +/- 0.30/11.1 +/- 0.7; type II, 3.64 +/- 0.52/11.8 +/- 1.0; and type III, 3.60 +/- 0.53/17.5 +/- 1.9. Cell-cycle analysis of blasts using 7AAD/PY combined with surface phenotyping may yield important information for classifying hematopoietic malignancy within 2 hours of patient admission.

Increased levels of intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor receptor in the cerebrospinal fluid of patients with multiple sclerosis.

We investigated the presence of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble tumor necrosis factor receptor (sTNF-R) antigens in the CSF of patients with multiple sclerosis (MS) using a double-determinant ELISA. Patients with acute relapsing MS during an exacerbation (p < 0.001) and those with chronic progressive MS (p < 0.001) had significantly increased CSF levels of sICAM-1 compared with subjects with other neurologic diseases. CSF levels of sTNF-R were also significantly increased in patients with acute relapsing MS during an exacerbation (p < 0.001) and chronic progressive MS (p < 0.001) compared with subjects with other neurologic diseases. CSF levels of sICAM-1 and sTNF-R were positively correlated in patients with acute relapsing MS during an exacerbation (r = 0.81, p < 0.01) and chronic progressive MS (r = 0.86, p < 0.001). These results suggest that active immune reactions involving ICAM-1 and TNF-R production are present within the CNS and that both sICAM-1 and sTNF-R are important immunologic markers of the clinical activity of MS.

Soluble E-selectin in the serum and cerebrospinal fluid of patients with multiple sclerosis and human T-lymphotropic virus type 1-associated myelopathy.

To evaluate the activation of endothelial cells of the brain and the spinal cord, we investigated the presence of soluble endothelial leukocyte adhesion molecule-1 (sE-selectin) in the serum and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and those with human T-lymphotropic virus type 1-associated myelopathy (HAM). There were significantly higher levels of sE-selectin found in the serum of patients with relapsing-remitting MS during an exacerbation (p < 0.001) and those with chronic progressive MS (p < 0.01) compared with controls. Serum levels of sE-selectin in patients with HAM did not differ significantly from serum levels in controls or non-HAM carriers. We also found sE-selectin in the CSF of eight patients during an exacerbation of relapsing-remitting MS. These results suggest that an active immune reaction involving E-selectin production that is indicative of endothelial cell damage occurs in the CNS of patients during an exacerbation of relapsing-remitting MS. Thus, sE-selectin may be useful in monitoring disease activity in patients with relapsing-remitting MS.

Immunologic and immunohistochemical study of familial amyloid polyneuropathy.

Amyloid fibril protein was purified from postmortem organs of patients with familial amyloid polyneuropathy. In immunodiffusion tests, the protein reacted with antihuman prealbumin antibody but not with antihuman retinol-binding protein or antihuman immunoglobulin G (IgG). In immunoelectrophoresis, the amyloid fibril protein gave a single line with a slightly faster mobility than prealbumin. Immunohistochemical analysis, using fluorescent and peroxidase-antiperoxidase methods, showed that the amyloid deposits contained antigenic determinants of human retinol-binding protein and IgG but not prealbumin.

Chronic relapsing demyelinating polyneuropathy associated with hepatitis B infection.

We studied a patient with chronic relapsing polyneuropathy associated with immune complexes of hepatitis B virus. There were cycles of remission and exacerbation in parallel with liver dysfunction. Immunofluorescent deposits of hepatitis B antigen, immunoglobulin, and C3 component were detected in the vasa nervorum. Ultrastructural study revealed electron-dense deposits that might have been immune complexes composed of hepatitis B virus, both around the endoneural capillary and in the endoneurium. Immune complexes composed of hepatitis B virus may play a role in the pathogenesis of chronic relapsing polyneuropathy.

Thrombomodulin in the sera of patients with multiple sclerosis and human lymphotropic virus type-1-associated myelopathy.

Damage to the blood-brain barrier, which mainly consists of cerebral endothelial cells, has been demonstrated in multiple sclerosis (MS) clinically and histochemically. To investigate the endothelial cell damage, we evaluated the presence of soluble thrombomodulin in the sera of patients with MS and human T lymphotropic virus type-1-associated myelopathy (HAM) using an enzyme-linked immunosorbent assay. Serum thrombomodulin levels were significantly increased in patients with acute relapsing MS during an exacerbation and chronic progressive MS as compared with those of controls (P < 0.001, respectively). Patients with HAM also had higher serum levels of thrombomodulin than did controls (P < 0.001). There was significant difference between patients with HAM and seropositive non-HAM carriers (P < 0.01). These results suggest that the detection of serum thrombomodulin could be used as a marker of endothelial cell damage in inflammatory diseases such as MS and HAM.