Gonadal shielding to irradiation is effective in protecting testicular growth and function in long-term survivors of bone marrow transplantation during childhood or adolescence.

An increasing number of long-term surviving bone marrow transplantation (BMT) recipients have recovered from their primary disease but are at risk of developing failure of endocrine organs. We investigated 30 recipients who underwent allogeneic BMT during childhood or adolescence. Testicular growth and function were evaluated by serial measurement of testicular volume, basal luteinizing hormone (LH), basal follicle-stimulating hormone (FSH) and testosterone levels and by gonadotropin-releasing hormone (GnRH) provocative test. Puberty started spontaneously in all patients. However, all except four patients had normal testosterone levels with elevated LH, indicating partial Leydig cell dysfunction. Standard deviation scores of testicular volume at last evaluation were statistically lower in those who had received irradiation without gonadal shield compared to those with (-2.04+/-0.45 vs -0.30+/-1.17, respectively, P<0.005), suggesting damage of testicular germinal epithelium owing to gonadal irradiation. Serial measurement of testicular volume showed a tendency of growth to stop at 10 ml in those without gonadal shield. Among the 30 patients, only one patient has fathered a child after reaching spontaneous puberty. These results suggest that gonadal shield is effective to protect testicular growth and function, although the attainment of fertility is difficult to achieve.

Injection of CD4(+) and CD8(+) cells with donor or host accessory cells induces acute graft-vs-host disease in human skin in immunodeficient mice.

OBJECTIVE: We examined cell subsets with respect to cutaneous graft-vs-host disease by cell sorting selection of subsets of human mononuclear cells and injecting the subsets subcutaneously in a mouse model. MATERIALS AND METHODS: Cell suspensions containing cultured human epidermal cells and dermal fibroblasts from a single donor mixed with lymphoid cell subsets positively selected using the FACSVantage cell sorting instrument and/or MACS cell isolation kits from unrelated individuals were injected into immunodeficient mice. This model is known to generate human skin with histologic findings similar to human graft-vs-host disease. RESULTS: Donor T-cell subsets CD4(+) and CD8(+) plus either host or donor CD14(+) cells were necessary to cause acute cutaneous graft-vs-host disease. Although graft-vs-host disease can result from recognition of class I antigens expressed on human cutaneous cells by donor peripheral blood mononuclear cells, additional recognition of class II antigens expressed on host mononuclear cells resulted in more severe histologic manifestations. Dendritic cells that differentiated from donor and host monocytes also showed competent accessory cell function in this system. CONCLUSIONS: Based on this model, human cutaneous graft-vs-host disease was caused by donor CD4(+) cells and CD8(+) cells activated through recognition of host antigens, including class I and class II antigens presented by either donor or host CD14(+) cells or dendritic cells.

An in vivo model of human skin acute graft-versus-host disease: transplantation of cultured human epidermal cells and dermal fibroblasts with human lymphocytes into SCID mice.

The ability of mixed epidermal cell-lymphocyte reactions to detect allogeneic reactivities in an in vivo model was investigated by developing an in vivo model of acute graft-versus-host disease (GVHD), using SCID mice with a C.B-17 background in which human skin structures were generated by transplantation of cultured human epidermal cells (HEC) with dermal fibroblasts (HDFC). Suspensions containing cultured HEC and HDFC from a single donor were mixed with autologous peripheral blood mononuclear cells (PBMNC) or with PBMNC from unrelated individuals, and were injected into the flanks of C.B-17-SCID mice. Ten and 21 days after injection, subcutaneous nodules generated in the mice were examined histologically and immunohistochemically. Cystic structures developing after injection of HEC and HDFC without human PBMNC showed normal epidermislike tissue. Human skin generated in SCID mice injected with HEC and HDFC with auto-PBMNC showed no graft-versus-host reaction (GVHR) histologically, whereas those mice injected with PBMNC from siblings that shared an HLA haplotype showed mild GVHR. Human skin in SCID mice injected with HEC and HDFC with histoincompatible unrelated PBMNC showed moderate to severe GVHR. The severity of GVHR paralleled the dose of unrelated PBMNC, and GVHR was prevented by peroral treatment with cyclosporine A. Immunohistochemically, inflammatory cells infiltrating human cutaneous tissue formed in the SCID mice were stained by an anti-human CD45RO antibody that reacts with human T cells but not with murine lymphocytes, and most T cells were stained by an anti-human CD8 antibody recognizing HLA class I antigens. These findings are similar to those in clinical skin graft-versus host disease (GVHD) observed in patients undergoing allogeneic bone marrow transplantation. This experimental system should be useful as an in vivo model of human skin GVHD.

A Japanese boy with myalgia and cramps has a novel in-frame deletion of the dystrophin gene.

We report a Japanese Becker muscular dystrophy (BMD) patient with occasional myalgia and cramps during normal activity that developed at the age of 28 months. His family history was negative for neuromuscular diseases. Muscle biopsy analyses, including dystrophin immunostaining, disclosed no clinically relevant findings. The diagnosis of BMD was initially made at the age of 10 years, when indications of persistent high serum levels of CK prompted us to screen deletions in the dystrophin gene by amplification of 19 deletion-prone exons from the genomic DNA by the polymerase chain reaction (PCR). Among the exons examined, exons 13 and 17 were deleted. To clarify the size of the deletion, the dystrophin transcript was analyzed by reverse transcription PCR. The determined nucleotide sequence of the amplified product encompassing exons 10 to 20 disclosed that the entire segment corresponding to exons 13 to 18 (810 bp) was absent, a deletion that would be expected to cause the production of a dystrophin protein lacking 270 amino acids from the rod domain. This result indicates that occasional myalgia and cramps could be early clinical manifestations of mild BMD, especially in patients who have a deletion in the rod domain, and that deletion screening of the dystrophin gene might be the only reliable method to diagnose such cases.

Unique N-linked glycosylation of murine coronavirus MHV-2 membrane protein at the conserved O-linked glycosylation site.

The membrane (M) proteins of murine coronavirus (MHV) strains have been reported to contain only O-linked oligosaccharides. The predicted O-glycosylation site consisting of four amino acid residues of Ser-Ser-Thr-Thr is located immediately adjacent to the initiator Met and is well conserved among MHV strains investigated so far. We analyzed the nucleotide sequence of a highly virulent strain MHV-2 M-coding region and demonstrated that MHV-2 had a unique amino acid, Asn, at position 2 at the conserved O-glycosylation site. We also demonstrated that this substitution added N-linked glycans to MHV-2 M protein resulting in increment of molecular mass of MHV-2 M protein compared with JHM strain having only O-linked glycans.

Increased numbers of CD8+CD11+, CD8+CD11- and CD8+Leu7+ cells in patients with chronic graft-versus-host disease after allogeneic bone marrow transplantation.

Peripheral blood lymphocytes from 25 allogeneic bone marrow transplant recipients were studied serially using flow cytometry and two colour analysis. Fourteen patients were transplanted for haematologic malignancies, eight for aplastic anaemia, two for congenital immunodeficiencies and one for Morquio's disease. All patients were alive more than 100 days post-grafting; nine patients had chronic graft-versus-host disease (GVHD). Dual labelling with monoclonal antibodies, CD4/2H4, CD4/4B4, CD8/CD11, CD8/HLA-DR and CD8/Leu 7 was used to analyse the surface phenotypes of lymphocytes. The population of CD4+2H4+ cells was decreased, and CD8+CD11+, CD8+CD11- and CD8+Leu7+ cells were markedly increased in patients with chronic GVHD. The increase of CD8+CD11+, CD8+CD11- and CD8+Leu7+ cells closely correlated with clinical signs of chronic GVHD in each patient. These results suggest that CD8+ cells may play an important role in effector and/or suppressor mechanisms of chronic GVHD and could be used as an indicator of need for and response to treatment.

Transition of T cell receptor gamma/delta expressing double negative (CD4-/CD8-) lymphocytes after allogeneic bone marrow transplantation.

We studied peripheral blood CD4-CD8- gamma/delta T cells in recipients of allogeneic marrow grafts, using three-color immunofluorescence and flow cytometry, and investigated changes in their numbers in relation to the administration of hematopoietic growth factors or chronic graft-versus-host disease (GVHD). In the early post-bone marrow transplantation (BMT) period, the relative and absolute numbers of peripheral CD4-CD8- gamma/delta T cells in 22 allogeneic marrow graft recipients in relation to the use of hematopoietic growth factors were studied. During the first 4 weeks, increased numbers of CD4-CD8- gamma/delta T cells were observed in recipients of either recombinant human granulocyte colony-stimulating factor (rhG-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF). However, 4-8 weeks after BMT, the number of these cells was similar in both groups whether or not growth factors had been administered. At a later stage after BMT (3-12 months), peripheral CD4-CD8- gamma/delta T cells from 43 allogeneic BMT recipients were studied. These cells were markedly decreased in patients with chronic GVHD, and this decrease correlated closely with the clinical signs of chronic GVHD. These results suggest that CD4-CD8- gamma/delta T cells may play an important role in the recovery of neutrophils associated with growth factors during the very early post-BMT period, and in the immunodeficient state of chronic GVHD at a later stage after BMT.

Successful allogeneic bone marrow transplantation in a case with myelodysplastic syndrome which developed following Fanconi anemia.

We report the case of a 14-year-old boy with myelodysplastic syndrome (MDS/RAEB) which developed following Fanconi anemia. The patient received BMT from an HLA-identical sister. Based on the in vitro CY-sensitivity test, 100 mg/kg of CY was administered for conditioning combined with 6 Gy TBI. Mucosal symptoms such as stomatitis, diarrhea and hematuria were severe, but manageable, and engraftment was successful. The patient has maintained normal trilineage hematopoiesis with > 90% Karnofsky score for 30 months with disappearance of a clonal chromosomal abnormality (47,XY, +i(lq)) which was detected before BMT.

Role of interleukin-12 in the development of acute graft-versus-host disease in bone marrow transplant patients.

Interleukin-12 (IL-12) is a crucial cytokine regulating cell-mediated immunity, and may contribute to the development of graft-versus-host disease (GVHD). We investigated serum IL-12 concentrations, interferon gamma (IFN-gamma) production by peripheral blood lymphocytes (PBL) from allogeneic stem cell recipients after IL-12 plus anti-CD3 monoclonal antibody (mAb) stimulation. We also investigated IL-12 production by peripheral macrophages (Mphi) after lipopolysaccharide (LPS) stimulation from allogeneic stem cell recipients and patients receiving donor leukocyte transfusions (DLT) for treatment or prophylaxis of leukemia relapse and Epstein-Barr virus (EBV) lymphoproliferative disease (LPD). PBL from acute GVHD patients produced high IFN-gamma levels after IL-12 plus anti-CD3 mAb stimulation, whereas PBL from patients without acute GVHD produced low levels of IFN-gamma. However, serum IL-12 concentrations were low in both groups. Peripheral Mphi IL-12 production increased in patients who developed acute GVHD compared to patients without acute GVHD. Five patients receiving DLT for treatment or prophylaxis of leukemia relapse developed acute GVHD. IFN-gamma production by PBL stimulated by IL-12 plus anti-CD3 mAb increased, while IL-12 production by peripheral Mphi stimulated by LPS was very high after the development of acute GVHD. However, serum IL-12 concentration remained low. Three patients receiving DLT for EBV-LPD did not develop acute GVHD with no increase of IFN-gamma and IL-12 production. These results indicate that IL-12 may play an important role in the development of acute GVHD after allogeneic stem cell grafting or DLT, and increased IL-12 production by Mphi occurs with various stimuli, including LPS.

Bispecific antibody-mediated cytotoxicity by CD4+ and CD8(+)-activated T cells generated from leukemia patients after allogeneic bone marrow transplantation.

The F(ab')2 bispecific antibody (BSAb) was prepared from anti-CD3 moAb and anti-CD10 moAb. The BSAb could react with both CD3+ T cells and CD10+ leukemia cells and triggered T cell-mediated cytotoxicity. To apply the BSAb to prevention of leukemic relapse after BMT, we investigated the generation of both CD4+ and CD8+ anti-tumor effector T cells from patient's PBMC 14 days after BMT. Neither CD4+ T cells nor CD8+ T cells, which were activated with immobilized anti-CD3 moAb plus IL-2, could lyse CD10+ leukemia cells by themselves, but they showed augmented cytotoxicity against CD10+ leukemia cells by targeting with anti-CD3 x anti-CD10 BSAb. Moreover, the activated CD4+ T cells were demonstrated to produce IL-2 and IFN-gamma when they were cultured with CD10+ leukemia cells in the presence of the BSAb. The BSAb-mediated cytotoxicity of activated T cells was demonstrated not only against the recipient leukemia cells but also against third party leukemia cells. These results suggested that anti-CD3 x anti-CD10 BSAb might be a good tool to prevent relapse after BMT in combination with activated CD4+ T cells and CD8+ T cells.

Successful engraftment of allogeneic CD34-enriched marrow cell transplantation from HLA-mismatched parental donors.

The CD34 antigen is expressed on pluripotent stem cells and the CD34+ cell has been shown to be capable of hematopoietic reconstitution in animal and human autologous grafts. We asked if CD34+ cells could reconstitute hematopoiesis in human allogeneic transplantation from a HLA-mismatched donor. Three pediatric patients with advanced leukemia received allogeneic CD34-enriched marrow cell graft from HLA two (two patients) or three (one patient) loci-mismatched parental donors. CD34+ cell selection was performed with mouse anti-CD34 antibody 9C5 and magnetic beads coated sheep anti-mouse IgG1. 1.53 to 2.48 x 10(9) marrow cells were processed and 2.53 to 7.89 x 10(7) positively selected cells were recovered. The selected population showed 93.7 to 99.0% CD34+ cells and total recovery of CD34+ cells from the starting population was 54.6 to 62.3%. CD34+ cell selection resulted in more than 99.9% depletion of CD5+ cells from the bone marrow. The patients received 2.53 to 7.25 x 10(6) CD34-enriched cells/kg after myeloablative therapy. All patients achieved trilineage engraftment that was confirmed by various genetic markers. Acute graft-versus-host disease (GVHD) was grade 0 (two patients) or grade I (one patient), and hematological recovery was successfully achieved as follows; the days to reach granulocytes over 0.5 x 10(9)/I were 11 to 13 days, reticulocytes over 2% was 18 to 28 days, platelets over 50 x 10(9)/I was 33 to 58 days. One patient is surviving without relapse of leukemia and two patients died after either mixed hematopoietic chimerism or leukemia relapse was observed. These studies suggest that CD34+ marrow cells are capable of hematopoietic reconstitution from HLA two or three loci-mismatched donors even with the lowest dose of mature T cells.

Absence of a CD34- hematopoietic precursor population in recipients of CD34+ stem cell transplantation.

The purified CD34(+) cell fraction has been used for hematopoietic stem cell transplantation since they were demonstrated to have long-term reconstituting ability. Therefore, the potential effects of CD34(-) stem cells on the clinical course have been a major concern in recipients of CD34(+)-selected transplantation. To address this concern, we used an in vitro assay to determine whether transplant recipients have CD34(-)precursor population. Lin(-)CD34(-) cells were isolated from bone marrow cells in 11 transplant recipients including four CD34-selected transplantations, six standard bone marrow transplantations, and one T cell-depleted marrow transplantation. The frequency of the Lin(-)CD34(-) population in four CD34-enriched transplantation recipients was not different from those of normal donors or recipients of other modes of transplantation: 0.96 +/- 1.01% (mean +/- s.d., n = 4), 0.45 +/- 0.16% (n = 6), and 0.66 +/- 0.59% (n = 7), respectively. However, the Lin(-)CD34(-)population obtained from the recipients of CD34-enriched transplantation acquired neither CD34 expression nor colony-forming activity after 7 days of culture, whereas the cells from all the normal individuals and standard BMT recipients were able to differentiate into CD34(+) cells accompanied by the emergence of colony-forming activity.We conclude that recipients of CD34-enriched transplantation appear to have defects in their CD34(-) precursor population. The clinical significance of these defects will be determined in a life-long follow-up of these patients.

Successful hyperbaric oxygen treatment of life-threatening hemorrhagic cystitis after allogeneic bone marrow transplantation.

Hemorrhagic cystitis (HC) is a major cause of morbidity after allogeneic bone marrow transplantation (BMT). Many therapies have been investigated to prevent or treat HC, but effective treatment for HC is still limited. While the efficacy of hyperbaric oxygen therapy has been established for HC due to chemotherapy and/or radiation therapy, its role in HC occurring after allogeneic BMT has yet to be defined. We report two cases of life-threatening late-onset HC after allogeneic BMT in children, which resolved after treatment with hyperbaric oxygen.

Fatal interstitial pulmonary disease in a patient with dyskeratosis congenita after allogeneic bone marrow transplantation.

Chronic restrictive lung disease in a 9-year-old boy with dyskeratosis congenita (DC) 7 years after allogeneic bone marrow transplantation (BMT) is described. When he was 1 year and 10 months old, severe aplastic anemia developed. He received a marrow transplant from his HLA serologically identical, but HLA-DP mismatched brother. He developed grade II acute graft-versus-host disease (GVHD) and thereafter chronic GVHD of progressive type, and was treated with both prednisolone and azathioprine resulting in clinical improvement. Thereafter he complained of dyspnea, and bilateral noncircumscribed interstitial shadows on chest CT scan were present. His pulmonary function showed restrictive changes. Prednisolone was not effective and he died of respiratory failure. Post-mortem examination confirmed interstitial fibrosis, lymphocytic infiltration of the bronchioles and alveoli with luminal fibrosis. There was no evidence of chronic GVHD in the skin and the liver. These findings raise the possibility that this pulmonary complication was associated with DC itself.

Multivariate analysis of risk factors for hemorrhagic cystitis after hematopoietic stem cell transplantation.

To establish the most appropriate prophylactic therapy and risk factors for predicting hemorrhagic cystitis (HC) after stem cell transplantation (SCT), we retrospectively analyzed the clinical records of 450 transplant patients treated from 1982 to 2002. In all, 81 patients developed early- and/or late-onset HC (early=29, late=48, both=4). For the incidence of early-onset HC, administration of cyclophosphamide (CY) (p=0.0079, odds ratio (OD)=5.109, 95% confidence interval (CI)=1.533-17.030), busulfan (BU) (p=0.0015, OD=3.336, 95% CI=1.584-7.027), BU+CY (p=0.0001, OD=4.369, 95% CI=2.055-9.292), antithymocyte globulin (p=0.0009, OD=3.368, 95% CI=1.642-6.911), nonradiation (p=0.0163, OD=2.564, 95% CI=0.181-0.841), 2-mercaptoethane sodium sulfonate (Mesna) (p=0.0001, OD=7.519, 95% CI=2.847-19.858), and bladder irrigation (p=0.0001, OD=4.950, 95% CI=2.328-10.523) were risk factors. By Fisher's exact test, the combination of BU and Mesna was a more significant risk factor (P<0.001) than Mesna alone (p=0.008) compared to the administration of neither agent. By multivariate analysis, prophylactic administration of Mesna (p=0.0105, OD=5.301, 95% CI=1.477-19.026) and bladder irrigation (p=0.0001, OD=9.469, 95% CI=3.872-23.156) were significant risk factors of early-onset HC. We conclude that (i). high-dose BU as well as CY is a cause of HC, (ii). protective bladder irrigation has an opposite effect, and (iii). Mesna possibly has a toxic effect on bladder mucosa.

Use of total parenteral nutrition in pediatric bone marrow transplantation.

To evaluate the extent of the nutritional stress of pediatric bone marrow transplantation (BMT) and to evaluate the use of total parenteral nutrition (TPN), 35 consecutive pediatric patients who received BMT were studied retrospectively. Voluntary cessation of oral nutrition in almost all patients was observed, and significant decreases of serum albumin levels were seen after BMT. In 85% of these patients, TPN was necessary in response to severe wasting and fasting. No deaths were related to indwelling central venous catheters during the period of 2968 catheter-use days in these severely myelosuppressed patients. The mean of the total daily energy intake was 104% of basal energy expenditure (BEE), and 70% of patients lost their weight. Predicted energy requirement to maintain body weight after BMT would be 128% of BEE from a simple linear regression step in this study. Significant correlations were found between the marrow recovery time and the initial nutritional state, expressed as the percentage of ideal weight height ratio, as well as benign nature of the disease. The use of TPN did not show any beneficial effects on the time course of marrow recovery, although it showed favorable effects on the maintenance of body weight.

Differentiation of mouse hepatitis viruses in animal facilities in Japan by use of nucleotide analysis of the nucleocapsid gene.

The nucleotide sequences of the coding region of the nucleocapsid (N) gene of 12 mouse hepatitis virus (MHV) strains recently found in animal facilities in Japan were analyzed. Nucleotide sequencing was performed directly on polymerase chain reaction (PCR) products amplified by reverse transcription (RT) and polymerase chain reaction (RT-PCR) analysis from fecal samples or isolated viruses. Phylogenetic analysis of these MHV strains along with those reported previously indicated that sequence analysis of the N gene was a useful tool for differentiation of MHV strains,although most MHV strains in Japanese facilities were phylogenetically close. Results suggested that interchange of mice infected with MHV among facilities provided opportunities of introduction of MHV into otherwise MHV-free facilities and that the source of MHV infection could be traced by use of nucleotide analysis of the N gene.

Requirement of proteolytic cleavage of the murine coronavirus MHV-2 spike protein for fusion activity.

The spike (S) protein of a non-fusogenic murine coronavirus, MHV-2, was compared to that of a variant, MHV-2f, with fusion activity. Two amino acids differed between The S proteins of these viruses; one was located in the signal sequence (amino acid 12) and the other in the putative cleavage site (amino acid 757). To determine which one of these amino acid changes is important for the alteration of fusogenicity, chimeric S proteins between MHV-2 and -2f were constructed and expressed in DBT cells by a vaccinia virus expression system. The results revealed that one amino acid change (Ser to Arg) at position 757 is responsible for the acquisition of fusogenicity of the MHV-2f S protein. This change also altered the susceptibility to proteolytic cleavage of the MHV-2 S protein which was originally uncleavable. We concluded that the non-fusogenic activity of MHV-2 results from the lack of cleavage of its S protein.

Myeloproliferative disease in a calf.

A myeloproliferative disease was found in a 128-day-old Holstein female calf. The tumour consisted chiefly of primitive cells, and more mature cells, many of which were positive for haemoglobin, were admixed with them. Binucleated cells at various stages of maturation were occasionally seen, and siderosomes were confirmed in both primitive cells and more mature cells by electron microscopy. The primitive cells resembled proerythroblasts, and this tumour, thought to be an acute type neoplasm of the erythroid system, was distinguishable from chronic erythremic myelosis and polycythemia vera.

Sequence analysis of major structural proteins of newly isolated mouse hepatitis virus.

We have isolated the virus from a fecal pellet in the colon of a BALB/c mouse with X-linked immunodeficiency (xid) housed in a room in which there has recently been an epidemic due to mouse hepatitis virus (MHV) and designated it as the MHV-TY strain. Sequence analysis of the MHV-TY strain was performed on major structural, spike (S), membrane (M) and nucleocapsid (N), proteins directly from PCR products. The comparison of nucleotide sequences of MHV-TY with other strains investigated so far revealed that all three structural proteins of the TY strain had some unique amino acid sequences among MHV strains which can be used as markers of this strain.