Decrease of blood dendritic cells and increase of tissue-infiltrating dendritic cells are involved in the induction of Sjögren's syndrome but not in the maintenance.

We have demonstrated previously that, in primary Sjögren's syndrome (SS), immature myeloid dendritic cells (DCs) are decreased in blood and mature myeloid DCs are accumulated in salivary glands, suggesting recruitment of the myeloid DCs from blood to salivary glands. To verify whether this finding is universal in patients of not only primary SS but also secondary SS, in this study we analysed the blood DCs of secondary SS patients. We examined 24 secondary SS and 29 primary SS patients. A direct correlation between the decreased number of myeloid DCs and the duration of Sicca syndrome in primary and secondary SS was observed; namely, the reduction of myeloid DCs in blood was restored spontaneously with duration time of Sicca syndrome. We also examined the immunohistochemical staining of salivary glands of SS patients with monoclonal antibodies against fascin, CD11c and human leucocyte antigen DR (HLA-DR). Fascin(+) or CD11c(+)/HLA-DR(+) mononuclear cells were present in the salivary glands of secondary SS patients, as in primary SS. However, fascin(+) mononuclear cells were barely detected in the salivary glands of a chronic phase of SS patients. We also found a negative correlation between the frequency of blood myeloid DCs and salivary gland-infiltrating DCs in secondary SS patients, as well as primary SS. Our results suggest that the reduction of blood myeloid DCs and preferential trafficking of myeloid DCs into salivary glands is a common event in the early stage of SS. Myeloid DCs may play essential roles in the pathogenesis of Sicca syndrome of SS by initiating T helper cell immune responses.

Step contrast reversal in LEEM during Pb deposition on W(110).

Anomalous step contrast in low energy electron microscopy (LEEM) images observed during Pb deposition on a W(110) surface is discussed. The steps are dark on the clean surface, and become bright by Pb deposition at about 200 °C. The contrast reversal is related to the presence of a two-dimensional (2D) Pb gas on the surface and its atomic density distribution. Upon further deposition the steps become dark again and show an anomalous intensity profile. This change is attributed to the 2D crystallization process.

Principal component analysis uncovers cytomegalovirus-associated NK cell activation in Ph+ leukemia patients treated with dasatinib.

Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) in a proportion of Ph+ leukemia patients, which associates with a better prognosis. The lymphocytosis is predominantly observed in cytomegalovirus (CMV)-seropositive patients, yet detectable CMV reactivation exists only in a small fraction of patients. Thus, etiology of the lymphocytosis still remains unclear. Here, we identified NK cells as the dominant LGLs expanding in dasatinib-treated patients, and applied principal component analysis (PCA) to an extensive panel of NK cell markers to explore underlying factors in NK cell activation. PCA displayed phenotypic divergence of NK cells that reflects CMV-associated differentiation and genetic differences, and the divergence was markedly augmented in CMV-seropositive dasatinib-treated patients. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis, and was further enhanced after starting dasatinib in virtually all CMV-seropositive patients. Thus, the extensive characterization of NK cells by PCA strongly suggests that CMV is an essential factor in the NK cell activation, which progresses stepwise during leukemia and subsequent dasatinib treatment most likely by subclinical CMV reactivation. This study provides a rationale for the exploitation of CMV-associated NK cell activation for treatment of leukemias.

Amplified and rearranged bcl-2 gene in two lymphoma cell lines, FL-218 and FL-318, carrying a 14;18 translocation.

Two human B-cell lines carrying a 14;18 chromosome translocation [t(14;18)(q32;q21)], designated FL-218 and FL-318, were established from effusion cells of two Japanese patients manifesting the transformed histology of follicular lymphoma. The FL-218 and FL-318 cell lines were composed of cells in the hyperdiploid range, which had two and three or four 18q- chromosomes, respectively. These 18q- chromosomes were not distinguishable from an 18q- chromosome derived from t(14;18) since they exhibited the same banding pattern. Southern blot analysis revealed that in both cell lines, breakage of the bcl-2 gene occurred within the major breakpoint cluster region and the truncated gene juxtaposed to an immunoglobulin heavy chain gene locus. The autoradiographic intensity of the retained fragment each on 18q- chromosome was more enhanced than that of the translocated fragment on 14q+ chromosome. These findings suggest that the extra 18q- chromosome found in t(14;18)-positive cancer does not arise from de novo independent t(14;18) but from duplication of a preexisting 18q- chromosome.

IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD.

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60-80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-gamma-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3-8.2%) than in patients without cGVHD (mean 1.2%; range 0.8-1.7%) and normal control subjects (mean 1.1%; range 0.1-1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4(+) T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8(+) T cells may be an immunological marker of cGVHD.

Immunoglobulin heavy chain class switching, mu to gamma, in a human lymphoma cell line FL-318 carrying a t(14;18)(q32;q21) chromosomal translocation.

Previously, we reported the establishment of a human lymphoma cell line, FL-318, carrying a t(14;18)(q32;q21) chromosomal translocation. FL-318 cells had mu-heavy chain on the cell surface, while they expressed 'sterile' germ-line gamma transcripts, suggesting that the chromatin structure of the immunoglobulin heavy chain (IGH) locus was 'accessible' to class switch recombination. After several months of in vitro cell culture, we found a small population of FL-318 cells expressing the gamma-chain. Using a limiting dilution method, a mu-producing cell clone FL-318M, and gamma-producing FL-318G were isolated. Hybridization studies with various DNA probes from the IGH locus as well as the BCL2 gene demonstrated that the mu-constant gene was deleted on the functional IGH allele of FL-318G cells, and that the cells produced abundant productive gamma-chain messages. These studies indicated that FL-318 cells underwent spontaneous class switching during in vitro cell culture, unrelated to T cell interaction or antigenic stimulation.

Sensitivity to dexamethasone and absence of bcl-2 protein in Burkitt's lymphoma cell line (Black93) derived from a patient with acute tumor lysis syndrome: comparative study with other BL and non-BL lines.

An Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell line, designated Black93, was established in culture from a patient who developed acute tumor lysis syndrome (ATLS). Growth inhibition in vitro by dexamethasone (DXM) and the expression of bcl-2 protein (Bcl-2) were investigated in Black93 and 17 other cell lines derived from EBV-negative or -positive BL, pre-B acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), and EBV-positive lymphoblastoid cell lines of normal B cell origin (B-LCL), assuming an inherent susceptibility of Black93 to cell death. The most marked growth inhibition by DXM was observed in Black93, two other BL, two pre-B-ALL and two FL lines. The other cell lines were less sensitive or were resistant. DNA extracted from the Black93 cells treated with DXM showed a ladder of oligo-nucleosomal DNA on electrophoresis. On testing of fixed smears by indirect immunofluorescence, bcl-2 protein (Bcl-2) was undetectable in Black93 and three BL lines but was detected in all the other cell lines at varying intensity. Western blot analysis showed mostly the same results. In the BL lines, the most DXM-sensitive cell lines lacked Bcl-2 expression, and the DXM-resistant cell lines always expressed Bcl-2. While none of the DXM-resistant cell lines lacked Bcl-2 expression, several pre-B or FL lines that expressed [correction of expessed] Bcl-2 were sensitive to DXM. Black93 is the first reported cell line established from a patient with ATLS. The positive sensitivity to DXM and the lack of Bcl-2 expression observed in Black93 are a major characteristic exhibited frequently by BL lines and, probably, by fresh BL cells. These properties may contribute to the precipitation of ATLS.

Intra-arterial steroid-injection therapy for steroid-refractory acute graft-versus-host disease with the evaluation of angiography.

We treated three patients with steroid-refractory acute graft-versus-host disease (aGVHD) with intra-arterial steroid-injection therapy (IAST). Two patients with gut aGVHD received IAST into both superior and inferior mesenteric arteries, while one patient with liver aGVHD received IAST into the proper hepatic artery. The volume of stools and the bilirubin level improved soon after IAST. Angiography of the superior and inferior mesenteric arteries was performed in the two patients with steroid-refractory gut aGVHD, and identical abnormal findings were obtained. IAST might be an earlier option for steroid-refractory aGVHD.

Blood dendritic cells are decreased in acute graft-versus-host disease.

The recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) often develop acute graft-versus-host disease (aGVHD), which is closely related to morbidity and mortality. However, the essential part of the immune responses elicited in aGVHD remains largely unknown. We attempt to determine if peripheral blood dendritic cells (PBDCs) are altered in aGVHD, and find that the number of PBDCs (both myeloid and lymphoid DCs) is significantly decreased. Immunohistochemical staining of the biopsied skin from patients with aGVHD demonstrates that a number of fascin(+) cells with dendritic projections infiltrate the dermis of the skin. Based on these findings, we hypothesize that the PBDCs are recruited to the affected tissues and may thus play important roles in immune responses elicited in aGVHD.

t(9;14)(p13;q32) involving the PAX-5 gene: a unique subtype of 14q32 translocation in B cell non-Hodgkin's lymphoma.

Newly identified t(9;14)(p13;q32) is a subtype of the well-defined 14q32 translocation and is closely associated with lymphoplasmacytic lymphoma (Revised European-American Classification of Lymphoid Neoplasms). The analysis of the breakpoint of t(9;14) unraveled its molecular structure as being the recombination of the PAX-5 gene on 9p13 with IgH locus located on 14q32. The molecular event does not seem to cause structural alteration of the protein product of PAX-5 and, instead, its deregulation is most likely the essential outcome of this translocation. In vitro experiments have shown that the overexpression of PAX-5 resulted in enhanced proliferation of B cells, implicating its potential capacity for lymphomagenesis. PAX-5 is crucial during most developmental stages of B cells mainly through regulating the expression of a variety of genes. Therefore, elucidating the nature of the altered expression of these downstream genes as well as the PAX-5 gene itself would be indispensable in clarifying the precise mechanism of lymphomagenesis caused by t(9;14).

Class switch recombination of the immunoglobulin heavy chain gene frequently occurs in B-cell lymphomas associated with rearrangement of the BCL2 gene.

B-cell lymphomas, mainly follicular lymphomas, carrying a t(14;18) chromosomal translocation associated with rearrangement of the BCL2 gene and the immunoglobulin heavy chain (IGH) gene, share many similarities with germinal center B cells in the secondary lymphoid follicle. In the germinal center, antigen-stimulated B cells proliferate and differentiate while undergoing isotype class switching of their immunoglobulin heavy chains. To examine whether BCL2-positive lymphoma cells show class switch recombination similar to that in the germinal center B cells, we studied the genomic configurations of the IGH gene loci in 38 patients with B-cell lymphomas. Sixteen (80%) out of 20 patients with BCL2-positive lymphomas showed class switch recombination on translocated and/or productive IGH gene loci. Lymphoma cells from 7 of the 16 patients expressed the gamma-heavy chain on their surfaces, indicating functional class switching to the gamma-constant gene on the productive allele. By contrast, 6 (33%) of the 18 patients lacking the BCL2 rearrangements exhibited class switch recombination. Statistical analysis revealed that the BCL2-positive lymphomas underwent switch recombination on either allele at a significantly higher frequency than the BCL2-negative lymphomas (P = 0.0099). This indicates that follicular lymphoma, not only morphologically but also functionally, recapitulates the germinal center. We propose that the up-regulated BCL2 expression itself is capable of playing an important role in immunoglobulin class switching.

Adult T-cell leukemia successfully treated with allogeneic bone marrow transplantation.

Adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 (HTLV-1) and is known to be a refractory disease of highly poor prognosis. We describe a case of ATL treated with allogeneic bone marrow transplantation (allo-BMT). The allo-BMT successfully induced complete remission in the patient. Currently, at 24 months post BMT, there has been no evidence of minimal residual disease (MRD) detected by polymerase chain reaction (PCR) assay for the T-cell receptor gamma chain gene. By contrast, PCR analysis demonstrated the reappearance of the cells harboring the integrations of the HTLV-1 proviral DNA 9 months after the BMT. These findings may imply a reversion to the carrier state rather than the recurrence of the leukemia from the MRD. The clinical consequence of our case illustrates that allo-BMT is an effective therapy, at least for achieving longer disease-free survival in ATL.

A Hodgkin's disease cell line, KM-H2, shows biphenotypic features of dendritic cells and B cells.

The origin of Reed-Sternberg (RS) cells, the neoplastic cells of Hodgkin's disease, has long remained controversial. Dendritic cells (DCs) are highly specialized antigen-presenting cells that have the unique capacity to prime naive T cells, and they may be progenitors of RS cells in a population of Hodgkin's disease cells. In this study, the KM-H2 cell line, previously established from a patient with Hodgkin's disease of mixed cellularity, was reevaluated for its cellular derivation, particularly in terms of DCs. KM-H2 cells were demonstrated to carry the newly proposed DC-associated molecules fascin, CD83, and DEC-205, as well as costimulatory molecules such as CD40, CD80, and CD86. In addition, KM-H2 cells were shown to be able to potently stimulate peripheral blood T cells and to have the strong endocytotic activity of fluorescein isothiocyanate-dextran. On the other hand, KM-H2 cells were shown to have variable-diversity-joining recombination of the immunoglobulin H gene, although they did not express any subclasses of immunoglobulin and they were negative for CD79a and CD79b. In addition, KM-H2 cells produced the messenger RNA of the Pax-5 gene. These findings lead to a hypothesis that KM-H2 cells originated from the cells that had differentiated through the possible common DC-B-cell progenitors along the newly proposed pathway.

Geographical aspects of bcl-2 gene involvement in Japanese patients with non-Hodgkin's B-cell lymphomas.

Using three separate bcl-2 probes, we examined involvement of the bcl-2 gene in Japanese patients with non-Hodgkin's B-cell lymphomas. Of 52 patients with follicular lymphoma (FL), 24 had rearrangements. In a group of 50 patients with diffuse lymphoma, three of 32 patients with diffuse large cell lymphoma had rearrangements. The frequency of rearrangements in each of these groups, as detected by both major and minor breakpoint cluster region probes, was compatible with that found in other Far Eastern studies. However, the difference in frequency between the groups studied in the Far East and the West was significant, and these two geographically distinct populations also displayed a difference in the breakpoint distribution. In the immunophenotype study of 33 patients with FL, the expression of CD10 antigen correlated with bcl-2 involvement, whereas none of the other B markers emerged as parameters to distinguish between the two lymphoma groups; those with, and without, the rearrangements.

A case of primary splenic large cell lymphoma with a t(9;14)(p13;q32).

t(9;14)(p13;q32), a subtype of 14q32 translocation, plays an essential role in the development of lymphoplasmacytoid lymphoma. t(9;14)(p13;q32) Causes juxtaposition of the PAX-5 gene on 9p13 and the IgH gene on 14q32, leading to the deregulation of the PAX-5 gene. We report a case of primary splenic lymphoma with a t(9;14). The histological diagnosis was diffuse large B-cell lymphoma without plasmacytoid differentiation. The lymphoma cells showed a complex karyotype including a t(9;14). Southern blot analysis localized the breakpoint of the PAX-5 gene within a couple of kb regions upstream of the exon 1A, although the involvement of the PAX-5 gene with the immunoglobulin heavy chain gene could not be confirmed.

A case of T-lineage lymphoblastic lymphoma/leukemia with t(4;11)(q21;p15) that switched to myelomonocytic leukemia at relapse.

A 51-year-old Japanese woman, initially diagnosed with T-lineage (CD2+, CD7+, CD3-, CD4-, CD8-) lymphoblastic lymphoma with t(4;11)(q21;p15), relapsed with acute myelomonocytic leukemia with the identical chromosomal abnormality. Southern-blot analysis revealed clonal rearrangements of an immunoglobulin heavy chain gene (JH) and T-cell receptor genes (J delta 1, J gamma 1, C beta 1) at first presentation, but germ line configurations of these genes at relapse. Leukemias with t(4;11)(q21;p15) may involve a hematopoietic progenitor capable of multilineage differentiation.

Involvement of c-myc oncogene in lymphoma cell lines with no detectable chromosome rearrangement of band 8q24.

Two lymphoma cell lines of B-cell type were established from Japanese patients with diffuse small noncleaved cell lymphoma. Cytogenetic analysis revealed a 14q+ marker chromosome in both cell lines, and a t(8;14)(q24.1;q32.3) seemed most likely to have occurred. The chromosome 8 pair, however, had no abnormalities. Molecular analysis demonstrated c-myc amplification lacking gross rearrangement in one cell line and genetic rearrangement of c-myc at the first intron as well as aberrant sizes of c-myc mRNA in the other cell line. In the latter case, it is possible that a t(8;14)(q24.1;q32.3) was buried in an unrecognized complex translocation. A combination of cytogenetic and molecular studies to determine the precise nature of the 14q32 translocation is discussed.

[bcl-2 gene involved in Japanese follicular lymphoma].

Using three chromosome 18-specific DNAs, rearrangements of a bcl-2 gene were detected in 20 (44%) of 45 Japanese patients (pts) with follicular lymphoma (FL) and 3 (8%) of 36 pts with diffuse large cell lymphoma. The 21 pts had t (IgH; bcl-2), and of the remaining two who did not display it, one had chromosome t(14; 18). Compared with the findings in American pts, the lower frequency of t(14; 18)-positive lymphoma could reflect a difference in the incidence of overall nodal B lymphoma between Japan and the United States. 11 of 18 pts with t(14; 18)-positive FL expressed CD10 antigen on the cell surface and all 12 pts with t(14; 18)-negative FL did not, and the difference is statistically significant, indicating that t(14; 18)-positive Japanese FL is the same disease entity as most of American FL. Extra 18q- chromosome found in the advanced grade diseases of t(14; 18)-positive lymphoma results in amplification of the rearranged bcl-2 gene on 18q-, suggesting that the change is closely associated with transformation of FL carrying t(14; 18). It is thus possible of international realization to institute the prognostic factors and the treatment strategy for conquering t(14; 18)-positive lymphoma.

[Bone marrow transplantation-associated thrombotic microangiopathy manifested by visual disturbance].

In October 1996, a 26-year-old woman was given a diagnosis of acute myeloblastic leukemia, FAB subtype M1. Treatment with combined chemotherapy achieved a complete remission (CR). In May 1997, the patient received an allogenic bone marrow transplant (BMT) from an HLA-identical sibling donor. Cyclosporine (CsA) and short-term methotrexate were given for graft-versus-host disease (GVHD) prophylaxis. Successful engraftment was obtained and signs of acute or chronic GVHD never developed. Five months after BMT, the patient experienced low-grade fever and blurred vision. Retinal examination demonstrated intraretinal hemorrhages, cotton-wool spots, and retinal detachments, which were presumably attributable to multiple thrombosis of retinal microvessels. The patient also exhibited hemolytic anemia with red cell fragmentation, thrombocytopenia, elevated lactate dehydrogenase, and renal impairment, and was thus given a diagnosis of BMT-associated thrombotic microangiopathy (BMT-TM). Discontinuation of CsA and administration of ticlopidine and prednisolone induced successful recovery from BMT-TM. Three months after the onset of BMT-TM, however, the patient experienced generalized clonic-tonic seizures with consciousness loss. Single-photon-emission computed tomography revealed blood-flow disturbances in the brain, suggesting the recurrence of microthrombosis. Accordingly, multiple transfusions of fresh frozen plasma were administered together with dipyridamole and aspirin. The patient gradually recovered and remained asymptomatic through the following 13 months. Currently, early diagnosis of BMT-TM is considered to be difficult. We suggest that careful examination of the ocular base may be useful for the early detection of BMT-TM.

[Antithymocyte globulin treatment in 11 patients with aplastic anemia].

We report the response to, and toxicity of antithymocyte globulin (ATG) treatment in 11 consecutive patients (7 men and 4 women; median age 46 years) with aplastic anemia (AA). Six of the patients had severe disease and 5 had moderate disease; all were treated within one year from diagnosis. The ATG regimen was the initial treatment for 6 patients, but a sequential treatment for the other 5. Cyclosporin A was administered orally 1-3 weeks after the ATG treatment. All patients were assessed for over 6 months (median, 20.4 months); 8 showed a good response, 1 a minimal response, and 2 no response. Disease severity had no influence on the response. In 1 patient, ATG treatment had to be discontinued because of hepatic toxicity. However, adverse reactions were not severe in the other 10 patients. These findings suggest that ATG treatment is a safe and effective therapy for AA.