Analysis of rotavirus antigenemia in hematopoietic stem cell transplant recipients.

Systemic rotavirus infection, such as rotavirus antigenemia, has been found in immunocompetent rotavirus gastroenteritis patients. However, the pathogenesis of rotavirus infection in immunocompromised transplant recipients remains unclear. Enzyme-linked immunosorbent assay was used to measure rotavirus antigen levels in serially collected serum samples obtained from 62 pediatric patients receiving allogeneic hematopoietic stem cell transplants (HSCT). Rotavirus antigen was detected in 43 (6.8%) of 633 serum samples (8 of 62 patients). The duration of rotavirus antigenemia ranged between 1 and 10 weeks, and diarrhea was concurrent with rotavirus antigenemia in Cases 3, 6, 7, and 8. The level of viral antigen in the transplant recipients (0.19 ± 0.20) was significantly lower than that observed in serum samples collected from immunocompetent patients on either day 1 (0.49 ± 0.18, P = 0.0011) or day 3 (0.63 ± 0.09, P = 0.0005). A patient who received a graft from a human leukocyte antigen (HLA)-mismatched donor was at significant risk for rotavirus antigenemia (P = 0.024; odds ratio = 9.44) in comparison to patients who received grafts from HLA-matched donors. Although the duration of antigenemia was clearly longer in HSCT patients than in immunocompetent rotavirus gastroenteritis patients, the levels of viral antigen were not as high. Therefore, mismatched HLA may be a risk factor for rotavirus antigenemia after HSCT.

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts.

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments.

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.

Prompt and durable hematopoietic reconstitution by unrelated cord blood transplantation in a child with Fanconi anemia.

We describe here the case of an 8-year-old girl with Fanconi anemia (FA) whose hematopoiesis was successfully restored by unrelated umbilical cord blood (UCB) transplantation. The patient became resistant to androgen therapy, and developed intracranial hemorrhage and dyserythropoiesis. Her hematopoietic recovery after the transplantation was excellent and a complete chimerism has been durably maintained. UCB should be considered as a stem cell source for transplantation when a patient with FA does not have an HLA-identical unaffected sibling donor.

Improvement in bronchiolitis obliterans organizing pneumonia in a child after allogeneic bone marrow transplantation by a combination of oral prednisolone and low dose erythromycin.

We report a 13-year-old boy who developed dyspnea at rest 1 year after the occurrence of cGVHD following an allogeneic bone marrow transplant (BMT). Pulmonary function data, imaging studies, lung biopsy, and bronchoalveolar lavage were consistent with the diagnosis of bronchiolitis obliterans organizing pneumonia (BOOP). Although reports suggest that oral methylprednisolone or methylprednisolone pulse therapies improve BOOP after BMT, we treated our patient with a combination of oral prednisolone (1 mg/kg) and low dose erythromycin (10 mg/kg) to avoid the side-effects of high-dose steroids. With this therapy, our patient showed clinical and radiological improvements within 1 week. The steroids were tapered off 12 months later and erythromycin was given for 14 months. We conclude that therapy consisting of a combination of oral prednisolone and low-dose erythromycin for BOOP after BMT may minimize the dose and duration of steroid use.

Massive pericardial and pleural effusion with anasarca following allogeneic bone marrow transplantation.

A 10-year-old girl presented with massive pericardial/pleural effusion with anasarca 216 days after an allogeneic bone marrow transplantation from her HLA-matched sibling for relapsed acute lymphoblastic leukemia. She did not show any other symptoms of chronic graft-versus-host disease (GVHD). The antinucleolar antibody was elevated in the blood and the pleural fluid. The lymphocytes in the fluid were mostly CD8+/HLA-DR+, and a majority of CD8+ cells in the blood expressed CD57. These data suggested that she had chronic GVHD. Immunosuppressive therapy including prednisolone, cyclosporin A, high-dose methylprednisolone, tacrolimus (FK506), and methotrexate had no effect, and the patient died of Aspergillus pneumonia 183 days after the presentation of the disease. Although it has not been described before, isolated serositis with edema should be recognized as a clinical feature of chronic GVHD.

A histological study of the exfoliation of human deciduous teeth.

For clarification of the histological details of the shedding of human deciduous teeth, exfoliated and extracted deciduous teeth were examined by light and electron microscopy. After the roots were completely resorbed, the dentogingival junction migrated along the inner resorbing surface and finally reached the pulpal surface of the crown. At the same time, the gingival epithelium also proliferated and migrated under the crown of the deciduous tooth in such a way that part of it lined the residue of the pulp and another part lined the surface overlying the erupting successional tooth. This phenomenon took place from various sides of the tooth surface. Therefore, just before exfoliation, the migrated gingival epithelium formed narrow necks of tissue, and the crown was only superficially attached to the gingiva by them. The final shedding of the tooth appeared to occur by a tearing of these narrow tissue regions. The results of the present study suggest that the dento-gingival junction as well as gingival epithelium play important roles in the process of exfoliation of human deciduous teeth.

Experimental development of a chitosan-bonded beta-tricalcium phosphate bone filling paste.

Bone filling substances are needed to meet several requirements including nontoxicity, setting time, changes in pH values, and amount of dissolved elements as well as mechanical properties. In this study, the bone-generating composites were prepared by employing the in vivo absorbable beta-tricalcium phosphate as a parent matrix kneaded with CaO, MgO, and ZnO as bone mineral additives with different compositions. The setting time, pH values, compressive strength were investigated as a function of the amount of these bone mineral additives. It was found that the setting time was shortened by increasing CaO, MgO, and ZnO contents. Increasing ZnO contents resulted in the pH value lower, while the pH values increased by increasing CaO and MgO contents. Increasing ZnO contents caused the compressive strength stronger, on the other hand, the compressive strength was weakened by increasing MgO contents. Furthermore, calcium appears to be selectively released from the hardened composite sample.

[Clinical trial of antithymocyte globulin for prophylaxis of acute graft-versus-host disease in pediatric recipients of bone marrow transplantation from unrelated donors].

We studied the effectiveness of antithymocyte globulin (ATG) in preventing acute graft-versus-host disease (a-GVHD) in children who received bone marrow transplants from unrelated HLA-matched donors at one institution. Of 39 patients who received transplants between 1993 and 1997, 23 were given ATG on the basis of informed consent. Either Thymoglobulin (Pasteur Merieux, 2.5 mg/kg/day) or Lymphoglobulin (15 mg/kg/day) was administered for 4 days. a-GVHD (> or = grade II) developed in 33% of the ATG group (n = 21) and in 44% of the non-ATG group (n = 16). Although a-GVHD (> or = grade II) appeared less frequent in the ATG group, the difference was not statistically significant. Among the subjects with hematological malignancies, no significant difference was observed in frequency of a-GVHD (> or = grade II) or 3-year survival rate for the ATG group (n = 10) and non-ATG group (n = 16). However, the incidence of cytomegalovirus infection was much higher (p < 0.01) in the ATG group (70%) than in the non-ATG group (19%). From this study, we were not able to confirm the benefits of ATG as described by other investigators.

Nocturnal blood glucose and IGFBP-1 changes in type 1 diabetes: Differences in the dawn phenomenon between insulin regimens.

OBJECTIVE: Insulin-like growth factor binding protein-1 (IGFBP-1) is known to regulate the bioavailability of insulin-like growth factor (IGF) and the levels of IGFBP-1 are increased in the morning in patients with type 1 diabetes mellitus. We investigated the nocturnal fluctuations of glucose, IGFBP-1, and free IGF-1 levels with three insulin regimens. RESEARCH DESIGN AND METHODS: Forty-eight type 1 diabetes patients were divided into three groups according to their basal insulin therapy (continuous subcutaneous insulin infusion [CSII], insulin glargine, NPH insulin). Blood samples were obtained every 2 h between 2 300 h and 0700 h to measure plasma glucose, IGFBP-1 and free IGF-1 levels. RESULTS: The dawn phenomenon was more frequent with NPH (62.1%) than with glargine (16.6%, p<0.05) and CSII (14.3%, p<0.05). In the NPH group, the serum IGFBP-1 levels were markedly increased from 21.0+/-3.6 ng/ml at 2 300 h to 200.3+/-21.8 ng/ml at 0700 h and free IGF-1 levels were inversely decreased; these changes were partially suppressed in the CSII and glargine groups. CONCLUSIONS: The use of insulin regimens that provide sufficient insulin levels in the early morning can suppress the dawn phenomenon, leading to improved glycemic control. The increase in circulating IGFBP-1 in the morning, as a result of waning of insulin action, lowers free IGF-1 levels and may cause insulin resistance.

Comparison of matched-sibling donor BMT and unrelated donor BMT in children and adolescent with acquired severe aplastic anemia.

From January 1991 to March 2007, 61 children and adolescent with acquired severe aplastic anemia received BMT in our institutions. We retrospectively compared the outcome of 30 cases of matched-sibling donor BMT (MSD-BMT) and 31 cases of unrelated donor BMT (URD-BMT). We observed one graft failure among MSD-BMT recipients and three graft failures among URD-BMT recipients, respectively. No patients in the MSD-BMT group developed grades II-IV acute GVHD compared with 11 of 30 patients (37%) in the URD-BMT group (P<0.001). One of 30 MSD-BMT recipients (3%) developed chronic GVHD compared with 8 of 30 URD-BMT recipients (27%) (P=0.013). The incidence of EBV and CMV reactivation was 11 of 20 URD-BMT recipients and 23 of 30, respectively. One patient in the URD-BMT group died of a motor accident 5.5 years after BMT. Ten-year OS was 100% in MSD-BMT recipients and 93.8% (95% CI, 81.9-100%) in URD-BMT recipients, respectively (P=0.252). Ten-year failure-free survival was 96.7% (95% CI, 90.2-100%) in the MSD-BMT group and 84.7% (95% CI, 70.2-99.2%) in the URD-BMT group, respectively (P=0.161).

Late-onset non-infectious pulmonary complications following allogeneic hematopoietic stem cell transplantation in children.

Late-onset non-infectious pulmonary complications (LONIPCs) that arise beyond 3 months after allogeneic hematopoietic SCT include bronchiolitis obliterans (BO), bronchiolitis obliterans with organizing pneumonia (BOOP) and idiopathic pneumonia syndrome (IPS). We retrospectively analyzed the incidence and outcome of LONIPCs among pediatric hematopoietic SCT recipients. We included 97 patients who survived for more than 3 months among the 114 who underwent allogeneic hematopoietic SCT between April 1997 and May 2007. Of the 97 enrolled patients, 10 (10.3%) developed LONIPCs at a median of 187 days after hematopoietic SCT (range, 123-826 days). Of the 10 patients with LONIPCs, eight had BO and two had IPS. Multivariate analysis showed that the onset of LONIPCs was associated with high-risk underlying disease and extensive chronic GVHD (hazard ratio, 5.42 (95% confidence interval, 1.36-21.7) and hazard ratio, 11.7 (95% confidence interval, 2.40-57.1), respectively). Only two patients responded to therapy with steroids and six of the 10 patients died. The 5-year OS rate was significantly lower among patients with, than without LONIPCs (28.0 vs 87.2%, P=0.000). Considering that we are lacking optimal therapies for LONIPCs, strategies aimed at the prevention of LONIPCs should be attempted.

Overexpression of anti-apoptotic Mcl-1 in testicular germ cell tumours.

AIMS: To determine the expression of Mcl-1 in testicular germ cell tumours in order to clarify the role of this anti-apoptotic factor in these tumours. Various members of the Bcl-2 family have been implicated in the apoptotic mechanisms regulating germ cell apoptosis. Mcl-1 is an anti-apoptotic Bcl-2 family member and has recently been reported to be related to the progression of malignancy; however, the involvement of Mcl-1 in the development of germ cell tumours is still unknown. METHODS AND RESULTS: Mcl-1 expression in testicular germ cell tumours was investigated by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). By immunohistochemistry, overexpression of Mcl-1 was present in all germ cell tumours that were studied, including embryonal carcinoma and yolk sac tumour, as well as choriocarcinoma and teratoma. In teratomas, Mcl-1 was widely distributed in the epithelial, myogenic, neural and mesenchymal components. RT-PCR analysis after microdissection revealed high levels of Mcl-1 mRNA in all tumour variants compared with non-neoplastic germ cells. CONCLUSION: Overexpression of anti-apoptotic Mcl-1 may function to enhance the viability of testicular germ cells, thereby leading to tumorigenesis.

Ultrastructural cytochemistry of carbohydrates in microfibrils associated with the amorphous elastin in the monkey aorta.

Two distinct ultrastructural components of elastic fibers can be identified--namely, the amorphous elastin and the microfibrils. We have examined the tunica adventitia of monkey aortas to demonstrate differential localization of carbohydrates in elastic fibers and collagen fibrils using Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of thin sections for vicinal-glycol-containing complex carbohydrates, en bloc concanavalin A (Con A) staining specific for alpha-D-mannosyl and alpha-D-glucosyl groups, and en bloc wheat germ agglutinin (WGA) staining specific for N-acetyl-D-glucosamine, N-acetylneuraminic acid, and N-acetyl-D-galactosamine. The PA-TCH-SP method moderately stained microfibrils and weakly stained collagen fibrils, but did not stain the amorphous elastin. Both Con A and WGA staining methods strongly stained microfibrils and moderately stained collagen fibrils, whereas the amorphous elastin lacked staining. Thus PA-TCH-SP, Con A, and WGA staining methods allow differential ultrastructural localization of carbohydrates in elastic fibers and collagen fibrils in monkey aortic adventitia and demonstrate the presence of more carbohydrate components in microfibrils than in collagen fibrils, whereas amorphous elastin lacks carbohydrate staining.

In vitro properties of a chitosan-bonded bone-filling paste: studies on solubility of calcium phosphate compounds.

The present study investigated properties of various mixtures of organic acids (malic and malonic) and calcium phosphate compounds (beta-tricalcium phosphate, ashed bovine bone, and synthetic hydroxyapatite) with the objective of determining the optimum combination of organic acid and calcium phosphate compound for components of a chitosan-bonded bone-filling paste. beta-tricalcium phosphate was decomposed by malic acid and malonic acid, but these two acids did not decompose synthetic hydroxyapatite and ashed bovine bone. Assessment of ion release from a set paste containing either synthetic hydroxyapatite or ashed bovine bone indicated that only calcium ions were appreciably released after storing and stirring the set paste in physiologic saline for 7 days.

Ultrastructural cytochemistry of oxytalan fibers in the periodontal ligament and microfibrils in the aorta with the periodic acid-thiocarbohydrazide-silver proteinate method.

Fullmer's oxytalan fibers are special connective tissue fibers in periodontal ligaments and some non-dental sites of certain animal species, and, ultrastructurally appear to resemble microfibrils related to elastogenesis. The present study has ultrastructurally examined the applicability of Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing complex carbohydrates to the study of oxytalan fibers in rat periodontal ligaments and microfibrils of the tunica adventitia in the rat aorta, where microfibrils are often associated with amorphous elastin and are thought to be oxytalan fibers. In the periodontal ligaments, the PA-TCH-SP method weakly to moderately stained collagen fibrils, moderately stained thin fibrils composing the oxytalan fibers, and intensely stained cytoplasmic granules of fibroblasts. In the aortic adventitia, the PA-TCH-SP method moderately stained collagen fibrils. Heavier staining was observed in microfibrils, whereas the amorphous elastin lacked staining. The most intense staining was seen in cytoplasmic granules and glycogen of mural cells. These studies demonstrate that oxytalan fibers in the periodontal ligament of rats and microfibrils in the aorta of rats contain vicinal glycol-containing glycoproteins and the PA-TCH-SP method is a useful tool in ultrastructural studies of oxytalan fibers and microfibrils of rats.

Ultrastructural distribution of sulfated complex carbohydrates in elastic cartilage of the young rabbit.

Sulfated glycosaminoglycans are an integral component of elastic cartilage. We have investigated the ultrastructural distribution of sulfated complex carbohydrates (CC) in the mature cartilage and the perichondrium of young rabbit auricles using the high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) and the tannic acid-ferric chloride (TA-Fe) methods. In the mature cartilage, HID-TCH-SP stained intracellular Golgi saccules of the mature face, secretory granules, and the extracellular matrix granules, but staining was not discernible in collagen fibrils and osmiophilic elastic fibers consisting of only amorphous elastin. The HID and TA-Fe staining were similarly observed in matrix granules, whereas the elastic fibers and collagen fibrils lacked the staining. The pericellular matrix granules had a diameter of 34 +/- 5 nm (mean +/- SD; n = 30). Thiéry's periodate-TCH-SP (PA-TCH-SP) method stained vicinal glycol-containing CC in collagen fibrils but failed to stain matrix granules and elastic fibers. In the perichondrium, HID-TCH-SP staining of the organelles was less intense in the flattened chondrocytes when compared with those in large mature chondrocytes. The extracellular HID and HID-TCH-SP staining were observed in the matrix granules. The diameter of pericellular matrix granules (19 +/- 4 nm, mean +/- SD; n = 30) was significantly smaller when compared to those in the mature cartilage (P less than 0.001). The HID-TCH-SP staining was closely associated with collagen fibrils. However, the staining was not seen in collagen fibrils and osmiophilic elastic fibers consisting of elastin and microfibrils. The PA-TCH-SP method stained collagen fibrils and microfibrils but did not stain the amorphous elastin. Thus these studies demonstrate that sulfated CC are packaged in chondrocyte secretory granules and are released into the extracellular matrix to form matrix granules, but are not incorporated into collagen fibrils and elastic fibers.

Effect of hydroxyapatite content on physical properties and connective tissue reactions to a chitosan-hydroxyapatite composite membrane.

The present study investigated the effect on certain physical properties of adding various amounts of hydroxyapatite (HAP) to chitosan sol. Also investigated were connective tissue reactions to a composite membrane that is being developed for possible use in guided tissue regeneration and for the limitation of HA particle migration at sites of implantation. The physical properties evaluated were shrinkage, tensile strength, hardness, calcium ion release, and morphology. Assessment of physical properties indicated that a ratio of HA to chitosan sol of 4/11 by weight is optimal in the preparation of the composite membrane. Subperiosteal implantation of the membranes over rat calvaria revealed that the membranes were well tolerated, with fibrous encapsulation and occasional areas of osteogenesis. Increasing the hydroxyapatite content seems to enhance membrane degradation.