The presence and longevity of peripherally expanded donor-derived TCRalphabeta+ mature T lymphocyte clones after allogeneic bone marrow transplantation for adult myeloid leukemias.

There are two major pathways for T-cell regeneration after allogeneic bone marrow transplantation; thymus-dependent T-cell differentiation of T-cell progenitors, and peripheral expansion of mature T cells in the graft. In order to learn to what extent the peripheral expansion of donor-derived mature T lymphocytes contributes to reconstitution of the TCRalphabeta+ T-cell repertoire after allogeneic bone marrow transplantation for adult myeloid leukemias, we pursued the fate of donor-derived T-cell clones using the amino-acid sequences of the complementarity-determining region 3 (CDR3) of the TCR-beta chain as a clonal marker. Clonal expansion of TCRalphabeta+ T lymphocytes with specific TCRBV subfamilies was identified in donor blood. Identical T-cell clones were not found in blood from recipients before transplantation. The donor-derived T-cell clones were identified in the circulating blood from recipients a few months after allogeneic bone marrow transplantation, and they remained in the blood for 18 months after transplant in two recipients, and for 56 months in one. These results suggest that the peripheral expansion of mature T lymphocytes in the graft makes a significant contribution to post-transplant T-cell regeneration during the early period of transplantation in humans, and that mature T cells can survive in recipients for several years. Further investigation will be required to explore which antigens drive the expansion of T-cell clones in donors and recipients, and the mechanisms of maintaining homeostatic balance between the thymus-dependent pathway and the peripheral expansion of mature T cells in post-transplant T-cell regeneration.

Distinct TCRAV and TCRBV repertoire and CDR3 sequence of T lymphocytes clonally expanded in blood and GVHD lesions after human allogeneic bone marrow transplantation.

Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the HLA-identical donor and recipient. If T lymphocytes infiltrating GVHD lesions recognize antigens expressed in these organs, T cell clones should expand in inflammatory tissues. We previously reported that recipients of allogeneic bone marrow grafts have clonally expanded TCRalphabeta(+) T lymphocytes soon after transplantation, which leads to a skew of TCR repertoires. To establish whether or not the same antigens cause clonal expansion of T lymphocytes in both blood and GVHD tissues, we examined the usage of TCR alpha and beta chain variable regions (TCRAV and TCRBV) and determined the complementarity-determining region 3 (CDR3) of T lymphocytes clonally expanded in circulating blood and GVHD lesions. We found that the repertoires and CDR3 diversity of TCRAV and TCRBV differed between the GVHD lesions and circulating blood, suggesting the selective recruitment of antigen-specific T cells into GVHD tissues. We also found that the usage of TCRAV and TCRBV by the clonally expanded T lymphocytes and their CDR3 sequences differed between the GVHD tissues and blood. These results suggest that the antigen specificity of TCRalphabeta(+) T lymphocytes clonally expanded in blood and GVHD lesions is different.

A clinicopathological study of 20 patients with T/natural killer (NK)-cell lymphoma-associated hemophagocytic syndrome with special reference to nasal and nasal-type NK/T-cell lymphoma.

We describe the clinicopathological features of 20 patients with T/natural killer (NK)-cell lymphoma-associated hemophagocytic syndrome (T/NK-LAHS). These patients were categorized into 2 groups according to the onset of hemophagocytic syndrome (HPS). Group 1 developed HPS during the clinical course, typically at the terminal phase of the disease. This group consisted of 7 patients with extranodal lymphoma arising in the nasal cavity, paranasal cavity, tonsils, or skin at presentation. In 5 of these patients, the preferred diagnosis was nasal and nasal-type NK/T-cell lymphoma, whereas the disease diagnoses in the remaining 2 patients were peripheral T-cell lymphoma of unspecified type and angioimmunoblastic T-cell lymphoma, respectively. Group 2 consisted of 13 patients whose disease corresponded to so-called malignant histiocytosis-like lymphoma, which is characterized by HPS at the initial presentation and the infiltration of the liver, spleen, and/or bone marrow without tumor formation. Nine of these 13 cases were found to have common histopathological features: CD56+, Epstein-Barr virus positivity, cytotoxic molecules, and nasal-type NK/T-cell lymphoma. The very poor prognosis of T/NK-LAHS may be partly explained by the finding that nasal and nasal-type NK/T-cell lymphoma, which is resistant to standard chemotherapy, made up the highest percentage (70%) of the cases.

A clinical analysis of 52 adult patients with hemophagocytic syndrome: the prognostic significance of the underlying diseases.

We retrospectively analyzed 52 adult patients with hemophagocytic syndrome (HPS). The underlying diseases were heterogeneous, including malignant lymphoma (lymphoma-associated hemophagocytic syndrome [LAHS]) in 26 patients, systemic lupus erythematosus in 3 patients, viral infections in 7 patients, and bacteria] or fungal infections in 6 patients. More than 83% of patients received prednisolone as an initial treatment. Multiple-agent chemotherapies (cyclophosphamide, doxorubicin, and vincristine) were administered to 96% of LAHS patients after a histopathological diagnosis of lymphoma. HPSs were controllable and remissions were achieved except for those patients with LAHS, fulminant Epstein-Barr virus-associated HPS, and an immunosuppressive state. Twenty-one (81%) of the LAHS patients had uncontrollable HPS and died of multiple organ failure and disseminated intravascular coagulation. The median survival time of LAHS patients was 83 days. In contrast, 3 (12%) of the other HPS patients died of multiple organ failure within 44 days.The clinical manifestations and the laboratory findings of LAHS and the other HPSs were too variable to establish the prognosis based only on the findings at the onset of HPS. The prognostic factors of adult HPS were found to be the underlying diseases, notably malignant lymphoma and infections, accompanied by the immunosuppressive state.

Identification of the T cell clones expanding within both CD8(+)CD28(+) and CD8(+)CD28(-) T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire.

We have previously reported that skewed repertoires of T cell receptor-beta chain variable region (TCRBV) and TCR-alpha chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3' terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the post-transplant skewing of TCR repertoires. Since the CD8(+)CD28(-)CD57(+) T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8(+)CD28(-) T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8(+)CD28(-) T cell and CD8(+)CD28(+) T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8(+)CD28(-) and CD8(+)CD28(+) T cell subsets in the same individuals. These results suggest that the clonally expanded CD8(+)CD28(-) T cells after allogeneic hematopoietic cell transplantation derive from the CD8(+)CD28(+) T cell subset, possibly by an antigen-driven mechanism, resulting in the skewed TCR repertoire.

Extensive clonal expansion of T lymphocytes causes contracted diversity of complementarity-determining region 3 and skewed T cell receptor repertoires after allogeneic hematopoietic cell transplantation.

We previously described skewed repertoires of the T cell receptor-beta chain variable region (TCRBV) and the TCR-alpha chain variable region (TCRAV) soon after allogeneic hematopoietic cell transplantation. To determine the characteristics of skewed TCRBV after transplantation, we examined the clonality of T lymphocytes carrying skewed TCRBV subfamilies and determined the CDR3 sequences of expanded T cell clones. In all 11 recipients examined, TCR repertoires were skewed, with an increase of certain TCRBV subfamilies that differed among individuals. In nine of 11 patients, clonal/oligoclonal T cell expansion was observed, although the expanded T cells were not necessarily oligoclonal. The extent of expansion after transplantation appeared to predict clonality. The arginine (R)-X-X-glycine (G) sequence was identified in clonally expanded T cells from four of five recipients examined, and glutamic acid (E), aspartic acid (D) and alanine (A) were frequently inserted between R and G. These results suggest that T lymphocyte expansion may result from the response to antigens widely existing in humans, and that the extensive clonal expansion of a limited number of T cells leads to contracted CDR3 diversity and post-transplant skewed TCR repertoires.

Pancreatic islet regeneration by ephedrine in mice with streptozotocin-induced diabetes.

In this experiment, we investigated the effects of crude Ephedrae herba, alkaloid extract of Ephedrae herba and 1-ephedrine, a major alkaloid component, on diabetic mice induced by streptozotocin (STZ). The alkaloid extract and 1-ephedrine showed suppression on the hyperglycemia. The suppression by Ephedrae herba of hyperglycemia may therefore be due to 1-ephedrine. Furthermore, we found that Ephedrae herba, alkaloid and 1-ephedrine promoted the regeneration of pancreas islets following atrophy induced by STZ. It is therefore suggested that Ephedrae herba may regenerate atrophied pancreatic islets, restore the secretion of insulin, and thus correct hyperglycemia.

Hemoperitoneum due to acute cytomegalovirus infection in a patient receiving peritoneal dialysis.

A 27-year-old man receiving continuous ambulatory peritoneal dialysis (CAPD) developed high-grade fever, dyspnea, and hemoperitoneum 32 months after the start of CAPD. A chest computed tomograph showed fine reticular shadows in the bilateral lower lung fields. Cytomegalovirus (CMV) antigenemia were detected, and immunoglobulin (Ig) M and IgG antibodies for CMV were also positive. The absolute counts of helper T cells (478/microL) and the ratio of helper T cells/suppressor T cells (0.25) decreased, despite no evidence of hematologic or immunologic diseases, including human immunodeficiency virus (HIV) or human T cell lymphoma virus-1 (HTLV-1) infection, or the use of immunosuppressive drugs. All symptoms, including hemoperitoneum and the ratio of helper T cells/suppressor T cells, improved gradually and spontaneously. Acute and primary cytomegalovirus (CMV) infection induced hemoperitoneum in a patient who was receiving CAPD.

Involvement of natural killer cells in patients with myelodysplastic syndrome carrying monosomy 7 revealed by the application of fluorescence in situ hybridization to cells collected by means of fluorescence-activated cell sorting.

Monosomy 7 is the most frequent chromosome abnormality among patients with secondary myelodysplastic syndrome (MDS). We used fluorescence in situ hybridization (FISH) and fluorescence-activated cell sorting (FACS) in order to clarify the lineage involvement. Four patients, three with de novo MDS and one with secondary MDS, were enrolled in this study. Monosomy 7 was observed in pluripotent stem cells (CD34(+)Thy-1(+)), and in B (CD34(+)CD19(+)) and T/natural killer (NK) progenitors (CD34(+)CD7(+)). The number of abnormal cells of B (CD19(+)) and T (CD3(+)) cells was below the cut-off value, but approximately 60% of the NK cells (CD3-CD56(+)) contained monosomy 7 in three of the patients.

Induction of tissue factor production but not the upregulation of adhesion molecule expression by ceramide in human vascular endothelial cells.

Binding of tumor necrosis factor-alpha (TNF-alpha) to p60 TNF-alpha receptor induces the activation of sphingomyelinase to generate ceramide, which in turn activates certain protein kinases and phosphatases, resulting in various TNF-alpha-mediated biological effects. We have investigated the role for the sphingomyelin/ceramide pathway in the TNF-alpha-induced upregulation of adhesion molecule expression and tissue factor production of human endothelial cells. TNF-alpha stimulated human umbilical vascular endothelial cells (HUVECs) to upregulate the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and HLA class I molecules in addition to the induction of procoagulant tissue factor production. C2-ceramide, a highly cell-permeable ceramide analog, was able to stimulate HUVECs to produce tissue factor activity as well as TNF-alpha. However, C2-ceramide did not stimulate HUVECs to upregulate the expression of VCAM-1, ICAM-1 and HLA class I molecules. These results suggest that there exist both the ceramide-dependent and -independent pathways in TNF-alpha signal transduction system in human vascular endothelial cells.

Endocapillary proliferative glomerulonephritis in a patient with parvovirus B19 infection.

A 45-year-old woman developed acute nephritic syndrome after erythema infectiosum. Laboratory data on admission showed decreased serum C3, C4, and CH50 levels and the presence of both immunoglobulin (Ig) M and IgG antibodies to human parvovirus B19 (HPV). A renal biopsy showed diffuse endocapillary proliferative glomerulonephritis. Immunofluorescence microscopy indicated 2+ granular staining for IgG, IgM, and C3 over the mesangial area and along glomerular capillary walls. HPV antigen was also detected in glomeruli by immunohistochemistry. Electron microscopy showed electron-dense deposits in the subendothelial space and the paramesangial area. These findings suggest that immune complex-type glomerulonephritis is caused by glomerular deposition of HPV antigen-antibody complexes in some patients with HPV infection.

Hematopoietic cell transplantation-related nephropathy in Japan.

To clarify the incidence and characteristics of hematopoietic cell transplantation (HCT)-related nephropathy (HCT-N) in Japan, we sent questionnaire letters to 188 hematologic divisions of 91 hospitals and analyzed the responses. Of 2,136 Japanese hematopoietic cell transplant recipients, 51 patients (2.4%) had HCT-N. The early-onset (180 days after HCT) groups included 20, 16, and 15 patients, respectively. The early-onset group mainly consisted of patients with acute renal failure (ARF) and hemolytic uremic syndrome and/or thrombotic thrombocytopenic purpura. ARF was the dominant type in the middle-onset group. The main phenotype of the late-onset group was nephrotic syndrome, which correlated with chronic graft-versus-host disease (P=0.008). The total amounts of irradiation for patients with chronic renal failure and urinary abnormality were significantly greater than those for patients with ARF (P=0.004). The survival rate of the early-onset and middle-onset groups was 47.2%, whereas 87% of patients in the late-onset group survived (P=0.002). HCT-N is expected to become a serious and important problem in Japan because of the increasing number of HCTs from unrelated donors.

Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation.

We analysed T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6-7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0.05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.

Reconstitution of gammadelta T cell repertoire diversity after human allogeneic hematopoietic cell transplantation and the role of peripheral expansion of mature T cell population in the graft.

We have examined the reconstitution of gammadelta T cell repertoire diversity after human allogeneic hematopoietic cell transplantation using a polymerase chain reaction (PCR)-based complementarity-determining region (CDR) 3 size spectratyping and DNA sequencing. The CDR3 complexity in the variable region of the T cell receptor (TCR)-delta chain was different amongst the individuals studied. Furthermore, CDR3 size distribution patterns of allogeneic hematopoietic cell transplant recipients were almost completely recovered by a few months after transplantation. In some patients, clonal predominance of the TCRDV1+ T cells became evident during the period after transplantation. In one particular donor/recipient pair, clonal predominance of TCRDV1+ T cells was already present in blood lymphocytes of the donor, and was also observed in the recipient after transplantation. Using this donor/recipient pair, we have questioned whether gammadelta T cell regeneration occurs via the peripheral expansion of mature T cells in the graft. In the donor lymphocytes, two expanding gammadelta T cell clones, which were demonstrated by CDR3 sequences of the TCR-delta chain, were recognized. These two clones were identified in the T cells from the recipient post transplant, but not before transplantation. One of the two clones was still detectable 1(1/2) years after the transplant procedure. These results strongly suggest that peripheral expansion of mature T cells in the graft is the principal pathway of gammadelta T cell regeneration after allogeneic hematopoietic cell transplantation in adults.

A PML/RARA chimeric gene on chromosome 2 in a patient with acute promyelocytic leukemia (M3) associated with a new variant translocation: t(2;15;17)(q21;q22;q21).

We describe a patient with acute promyelocytic leukemia (APL) carrying a new complex variant translocation of t(2;15;17)(q21;q22;q21). The karyotypic interpretation was confirmed by fluorescence in situ hybridization (FISH) with the use of painting probes of chromosomes 2, 15, and 17 and a PML/RARA dual color DNA probe. FISH showed a PML/RARA fusion gene on the der(2) instead of the der(15). These results suggest that the critical event in the development of APL is the formation of a PML/RARA chimeric gene, regardless of its locus in the genome.

Delayed recovery of CDR3 complexity of the T-cell receptor-beta chain in recipients of allogeneic bone marrow transplants who had virus-associated interstitial pneumonia: monitor of T-cell function by CDR3 spectratyping.

BACKGROUND: In the T-cell receptor (TCR)-beta chain, complementary-determining region 3 (CDR3) contains specific peptide sequences essential for recognition. Diversity of this region is considered to contribute to immunocompetence in humans. OBJECTIVE: The purpose of this study was to define the process of reconstitution of CDR3 complexity of the TCR-beta chain after allogeneic bone marrow transplantation and to investigate the association between host immunocompetence and CDR3 complexity. METHODS: Diversity of the CDR3 region of the TCR-beta chain was examined by CDR3 size distribution analysis with the use of an automated DNA sequencer. RESULTS: Reconstitution of the alphabeta T-cell repertoire and CDR3 diversity was incomplete for at least 2 months after bone marrow transplantation. Delayed reconstitution of T-cell diversity was more marked in immunocompromised hosts. Unlike the situation in patients who received allogeneic bone marrow grafts, the recovery of CDR3 complexity was almost perfect by 2 months after transplantation in patients who received allogeneic blood stem cells. Clonal expansion of alphabeta T cells after allogeneic bone marrow transplantation was readily detected by CDR3 size spectratyping analysis. CONCLUSION: PCR-based CDR3 size spectratyping may be a useful tool for clinically monitoring immune reconstitution after allogeneic bone marrow transplantation.

[Pulmonary complications after allogeneic hematopoietic stem cell transplantation].

We investigated the occurrence of pulmonary complications in patients who underwent allogeneic hematopoietic stem cell transplantation at our institution. Pulmonary complications were observed in 12 out of 60 patients. Interstitial pneumonia developed in 12 cases: 7 idiopathic, 2 cytomegalovirus-associated, 1 P. carinii, 1 HSV, and 1 HHV-6-associated. HSV- and HHV-6-associated pneumonias were exhibited 100 days after transplantation. PCR analysis was diagnostically useful for detection of viral DNA in bronchial alveolar lavage fluid. Respiratory disease with airway obstruction was observed in 4 patients with chronic graft-versus-host disease, and all 4 had a history of interstitial pneumonia. Three patients died of respiratory failure. Mycobacicrium avium complex was detected in 2. Exacerbation of respiratory failure may be associated with mycobacterial infection.

[High molecular-weight Bence Jones protein deposits in the kidney of a patient with plasma cell dyscrasia].

A 70-year-old man was admitted to our hospital because of edema of the face and legs, proteinuria, and hypoproteinemia. We made a diagnosis of nephrotic syndrome. Immunoelectrophoresis of the patient's serum and urine disclosed a small amount of kappa type Bence-Jones protein (BJP) in the fast-gamma area. A renal biopsy showed nodular expansion of mesangium with deposition of space kappa-light chain in the mesangial area. Light chain deposition disease was diagnosed. Western blotting analysis disclosed that the patient's BJP molecules had a molecular mass of 66 KDa, and were composed of two kappa chains of 33 KDa. Bone marrow aspiration revealed dysplastic plasma cells, and Western blotting analysis of an extract of these cells detected BJP-kappa with a molecular mass of 33 Kda. This BJP was larger than normal, indicating the possibility of abnormal structure. Structural abnormalities may be responsible for tissue precipitation of the kappa light chain.