Shin Hayashi

Involvement of interferon-γ and macrophage colony-stimulating factor in pathogenesis of haemophagocytic lymphohistiocytosis in adults

Summary We investigated the role of monocyte/macrophage‐activating cytokines in pathogenesis of haemophagocytic lymphohistiocytosis (HLH) in 21 adult patients. Sera from patients with active HLH contained extremely high levels of macrophage colony‐stimulating factor (M‐CSF) and of interferon‐γ (IFN‐γ). These levels returned to almost normal during remission. Neither interleukin‐4 nor granulocyte/macrophage colony‐stimulating factor could be detected. Active HLH sera also contained high concentrations of inflammatory monokines, such as interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Serum concentrations of soluble CD8 and soluble interleukin‐2 receptor were extremely high during active HLH, and returned to virtually normal levels during remission. Circulating CD2+ T‐cells obtained from patients with active HLH spontaneously secreted M‐CSF and IFN‐γin vitro, whereas circulating monocytes did not produce detectable levels of both M‐CSF and IFN‐γ, but produced high levels of IL‐6 and TNF‐α. These findings suggest that IFN‐γ and M‐CSF at least partly from T‐cells, such as CD8+ T‐cells, might contribute to activation of monocytes or histiocytes, resulting in the up‐regulated monokine production and haemophago‐cytosis in HLH.

Cytomegalovirus (CMV) antigenaemia for rapid diagnosis and monitoring of CMV‐associated disease after bone marrow transplantation

A technique for the rapid detection of cytomegalovirus (CMV) antigen‐positive blood leucocytes (CMV antigenaemia) was evaluated in 15 marrow transplant patients as a means of diagnosis and for monitoring CMV‐associated disease. CMV antigenaemia was determined by direct immunoperoxidase staining of leucocytes with a peroxidase‐labelled monoclonal antibody, HRP‐C7, which binds an immediate‐early antigen of human CMV.

Monitoring of human herpesviruses after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation

Herpesviruses frequently cause serious complications after allogeneic bone marrow transplantation (allo‐BMT). Recent studies have shown more rapid immune reconstitution after allogeneic peripheral blood stem cell transplantation (allo‐PBSCT) compared with allo‐BMT. However, it has not been clarified whether the improved immune reconstitution after allo‐PBSCT is associated with a lower incidence of herpesvirus infections. We monitored the emergence of Epstein‐Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV‐6) and HHV‐7 DNA by a nested‐double polymerase chain reaction in peripheral blood leucocytes from 22 allo‐BMT and 16 allo‐PBSCT patients. Each virus had an unique temporal profile of detection. HHV‐6 DNA was detected most frequently at 3 weeks after transplantation, whereas CMV and EBV DNA were detected later (2–3 months). Detection rates of HHV‐6 DNA at 3 and 4 weeks after allo‐BMT were significantly higher than those after allo‐PBSCT (9/16 v 2/13 at 3 weeks, P < 0.01; 10/21 v 1/15 at 4 weeks, P < 0.01). Detection rates of the other three herpesviruses after the two types of allogeneic transplantation were not significantly different throughout observation period. Furthermore, detection of HHV‐6 DNA within the first 4 weeks was associated with delayed platelet engraftment after both allo‐BMT and allo‐PBSCT (P < 0.01). These results suggest an advantage for allo‐PBSCT over allo‐BMT in terms of suppression of HHV‐6 reactivation and prevention of subsequent complications.

Constitutive production of granulocyte colony-stimulating factor and interleukin-6 by a human lung cancer cell line, KSNY: Gene amplification and increased mRNA stability

Abstract: A human lung squamous cell carcinoma cell line designated KSNY was established from a patient suffering from marked neutrophilia and polyclonal hyper‐γ‐globulinemia. In our previous report, we demonstrated colony‐stimulating activities in the culture supernatant of this cell line. To determine the exact molecules for the activities, we investigated the gene expression of various cytokines in KSNY cells and showed the mRNA expression of both granulocyte colony‐stimulating factor (G‐CSF) and interleukin‐6 (IL‐6). We also detected substantial amounts of G‐CSF and IL‐6 in the culture supernatant with sensitive enzyme‐linked immunosorbent assays (ELISA). The amplification of the gene locus for G‐CSF, but not for IL‐6 was shown by Southern blot analysis. Furthermore, we also showed that the mRNAs for G‐CSF and IL‐6 were relatively stable in KSNY cells. These findings are thought to be related to the constitutive production of the cytokines in KSNY cells.

Influence of transplanted dose of CD56+ cells on development of graft-versus-host disease in patients receiving G-CSF-mobilized peripheral blood progenitor cells from HLA-identical sibling donors.

Summary:We investigated effects of variations in the cellular composition of G-CSF-mobilized peripheral blood progenitor cell (G-PBPC) allografts on clinical outcomes of allogeneic PBPC transplantation. We retrospectively analyzed transplanted doses of various immunocompetent cells from 27 HLA-identical sibling donors in relation to engraftment, incidence of graft-versus-host disease (GVHD), and survival. Significant variability was documented in both absolute numbers and relative proportions of CD34+, CD2+, CD3+, CD4high+, CD4+25+, CD8high+, CD19+, CD56+, and CD56+16+ cells contained in these allografts. Stepwise Cox regression analysis revealed that the CD56+ cell dose was significantly inversely correlated with the incidence of GVHD. Thus, there was a significantly higher incidence of grade II acute GVHD in patients receiving a lower CD56+16+ cell dose (hazard ratio (HR) 0.0090; 95% confidence interval (CI), <0.00001–3.38; P=0.031), a higher incidence of chronic GVHD in those receiving allografts with a lower CD56+16+ to CD34+ ratio (HR <0.00001; 95% CI <0.00001–0.0007; P=0.0035), and a higher incidence of extensive chronic GVHD in those receiving allografts with a lower CD56+ to CD34+ ratio (HR <0.00001; 95% CI <0.00001–0.053; P=0.0083). These results suggest that CD56+ cells in G-PBPC allografts from HLA-identical sibling donors may play an important role in preventing the development of GVHD.

A comparison of glandular involvement between chronic graft-versus-host disease and Sjögren's syndrome.

Patients with chronic graft-versus-host disease (cGVHD) occasionally suffer from symptoms of xerostomia and xerophthalmia, which are also features of Sjógrens syndrome (SS). To identify differences in the glandular involvement between cGVHD and SS, we measured the proportions of infiltrating lymphocyte subsets and the expression of HLA-DR antigen and cell adhesion molecules in labial salivary glands (LSG). In cGVHD, more than 90% of the infiltrating lymphocytes were T cells with a slight predominance of CD8+ over CD4+ cells. In SS, CD4+ cells were predominant, and B cells accounted for 10-30% of the infiltrating lymphocytes. Ductal epithelial cell associated with lymphocytic infiltration expressed HLA-DR antigen in both cGVHD and SS. In SS alone, HLA-DR antigen expression also occurred without associated lymphocytic infiltration. The expression of adhesion molecules on ductal epithelial cells, especially vascular cell adhesion molecule 1, was more intense in SS than in cGVHD, while that on endothelial cell was similar in cGVHD and SS. These data suggest that the pathogenesis of glandular involvement of cGVHD is different from that of SS.

Multiple autoimmune haemopoietic disorders and insidious clonal proliferation of large granular lymphocytes.

We report a patient with clonal proliferation of CD3+8+TCRαβ+ large granular lymphocytes (LGL) presenting multiple episodes of autoimmune cytopenia, including autoimmune neutropenia, idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia, and pure red cell aplasia. Each disorder appeared separately or as a combination during an 11‐year clinical course. The increase of blood CD3+8+TCRαβ+ LGL was detected 6 years after the initial diagnosis of cytopenia, but the absolute number of LGL cells was always < 1.0 × 109/l. LGL cells were of monoclonal origin and had a chromosomal abnormality. LGL cells transiently responded to cyclosporine A therapy, which was also effective on all of these autoimmune cytopenias. Accordingly, an undetectable level of proliferation of a clonal LGL population could cause various autoimmune haemopoietic disorders.

FLT3 mutations in normal karyotype acute myeloid leukemia in first complete remission treated with autologous peripheral blood stem cell transplantation.

Summary:We retrospectively analysed the significance of FLT3 mutations in patients with acute myeloid leukemia (AML) having a normal karyotype, who were treated with high-dose chemotherapy and autologous peripheral blood stem cell transplantation (auto-PBSCT). In all, 34 patients with normal karyotype AML in first complete remission receiving high-dose chemotherapy and auto-PBSCT were analysed based on the presence or absence of FLT3/ITDs and FLT3/D835. They were 16 males and 18 females and with a median age of 41.5 years. FLT3/ITDs were detected in eight of 34 patients (23.5 %), and FLT3 D835 mutations in two of 34 patients (5.9%). White blood cell count (P=0.0087), serum concentration of lactate dehydrogenase (P=0.005), and percentages of peripheral blood (P=0.0131) and bone marrow (BM) blasts (P=0.0312) were significantly higher in patients showing the FLT3 mutations. Overall survival (OS) and disease-free survival (DFS) were similar between patients with or without FLT3 mutations (5 year DFS, 67.5 vs 68.55%, P=0.819; 5 year OS, 64.81 vs 78.88%, P=0.4457, by the log-rank test). FLT3 mutations demonstrate no further prognostic impact in patients with normal karyotype AML in first CR treated with high-dose chemotherapy and auto-PBSCT. Myeloablative chemotherapy supported by auto-PBSCT may overcome any poor prognostic implications of FLT3 mutations.

Rituximab does not compromise the mobilization and engraftment of autologous peripheral blood stem cells in diffuse-large B-cell lymphoma

To investigate effects of the preautografting administration of rituximab on the mobilization and engraftment of peripheral blood stem cells (PBSC), we retrospectively analyzed the outcomes of 43 newly diagnosed diffuse-large B-cell lymphoma patients who received CHOP chemotherapy with or without rituximab as a first-line treatment before autologous PBSC transplantation (PBSCT). There was no difference in the number of CD34+ cells among PBSC between the non-rituximab and the rituximab groups. Although B-cells were completely depleted from PBSC in the rituximab group, we found no difference in the expression of CXCR-4, VLA-4 and c-Kit on PBSC, indicating that rituximab did not affect the expression of these adhesion molecules, which might be involved in the mechanism of mobilization. There was no significant difference in the recovery of neutrophils and platelets, transplant-related toxicity and post-transplant complications between the two groups. Despite the short follow-up, there was no significant difference in progression-free survival between the two groups. These results indicated no adverse effect of rituximab on the mobilization and engraftment of PBSC. Larger studies are required to determine the impact of rituximab on the mobilization and function of PBSC as well as whether a survival advantage exists in patients who undergo auto-PBSCT with rituximab.

Donor lymphocyte transfusion for the treatment of Epstein-Barr virus-associated lymphoproliferative disorder of the brain.

EBV-associated lymphoproliferative disorder (LPD) is a rare but serious complication in marrow transplant recipients. A 31-year-old Japanese woman in the second chronic phase of CML received an allogeneic BMT from her HLA 2-locus-incompatible 62-year-old father. Around day +200, she developed EBV-LPD of the right parieto-temporal lobe which caused slowly progressive left hemiparesis. Two courses of donor lymphocyte transfusions (DLT) of 106 CD3+ T cells/kg of body weight failed to suppress her central nervous system (CNS) EBV-LPD. The patient died of recurrent blastic crisis of CML. This case suggests that DLT may be ineffective for the treatment of CNS EBV-LPD.