Masatoshi Wakui

Dry eye after haematopoietic stem cell transplantation

AIMS To determine the incidence, natural course, and severity of dry eye occurring or worsening after haematopoietic stem cell transplantation (SCT). METHODS At a tertiary care hospital, 53 patients undergoing allogeneic or autologous SCT followed by at least 180 days of follow up were studied prospectively. Examination included grading of symptoms of dry eye, evaluation of ocular surface, tear break up time, and Schirmer tests with and without nasal stimulation. Meibomian gland secretion was also examined using a slit lamp while applying steady digital pressure. RESULTS Of the 53 patients, 44 received allografts. Half of these patients (22) developed dry eye or their pre-existing dry eye worsened after SCT, while none of nine autograft recipients did. Onset of dry eye was 171 (SD 59) days after SCT. Two types of dry eye occurred. One (n=10) was severe with ocular surface findings resembling Sjögren’s syndrome and reduction of reflex tearing soon after onset. A mild type (n=12) had unimpaired reflex tearing. Meibomian gland dysfunction (MGD) was more frequent and severe in patients with dry eye and chronic graft versus host disease (GVHD), and overall severity of dry eye was greater in patients with MGD and chronic GVHD. CONCLUSIONS Dry eye after SCT occurred only in allograft recipients, and was not evident in autograft recipients. The severe form of dry eye had a tendency to develop rapidly. Further study on the prediction and treatment of severe dry eye after SCT is necessary.

Visualization of volatile substances in different organelles with an atmospheric-pressure mass microscope

We have developed a mass microscope (mass spectrometry imager with spatial resolution higher than the naked eye) equipped with an atmospheric pressure ion-source chamber for laser desorption/ionization (AP-LDI) and a quadrupole ion trap time-of-flight (QIT-TOF) analyzer. The optical microscope combined with the mass spectrometer permitted us to precisely determine the relevant tissue region prior to performing imaging mass spectrometry (IMS). An ultraviolet laser tightly focused with a triplet lens was used to achieve high spatial resolution. An atmospheric pressure ion-source chamber enables us to analyze fresh samples with minimal loss of intrinsic water or volatile compounds. Mass-microscopic AP-LDI imaging of freshly cut ginger rhizome sections revealed that 6-gingerol ([M + K](+)at m/z 333.15, positive mode; [M - H](-) at m/z 293.17, negative mode) and the monoterpene ([M + K](+) at m/z 191.09), which are the compounds related to pungency and flavor, respectively, were localized in oil drop-containing organelles. AP-LDI-tandem MS/MS analyses were applied to compare authentic signals from freshly cut ginger directly with the standard reagent. Thus, our atmosphere-imaging mass spectrometer enabled us to monitor a quality of plants at the organelle level.

Risk-adapted pre-emptive therapy for cytomegalovirus disease in patients undergoing allogeneic bone marrow transplantation.

We prospectively evaluated a risk-adapted pre-emptive treatment with ganciclovir for CMV diseases in patients undergoing allogeneic bone marrow transplantation (BMT). High-level CMV antigenemia (10 or more positive cells on two slides) or CMV antigenemia at any level in patients with grade II–IV acute graft-versus-host disease (aGVHD) were chosen as risk factors. We also retrospectively evaluated virus reactivation in plasma using quantitative real-time polymerase chain reaction (PCR). Fifty patients were evaluable. None of the 27 patients with or without grade I aGVHD developed high-level CMV antigenemia or CMV disease. Among the 23 patients with grade II–IV aGVHD, 12 patients (52%) developed CMV antigenemia and were treated pre-emptively, of whom two developed CMV gastroenteritis or retinitis in spite of therapy. Six of the remaining 11 patients developed CMV gastroenteritis before CMV antigenemia was detectable. All of the eight patients with CMV diseases were successfully treated with ganciclovir and no deaths directly related to CMV disease occurred. In four of the seven evaluable patients with CMV gastroenteritis, real-time PCR was able to detect virus reactivation earlier than CMV antigenemia. Although our risk-adapted pre-emptive therapy effectively reduced CMV-related mortality, further refinements of this approach, particularly in the prevention of CMV gastroenteritis, may be achieved by incorporating real-time PCR. Bone Marrow Transplantation (2000) 25, 765–769.

Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice

Severely immunodeficient NOD/Shi-scid IL2Rg(null) (NOG) mice are used as recipients for human tissue transplantation, which produces chimeric mice with various types of human tissue. NOG mice expressing transgenic urokinase-type plasminogen activator in the liver (uPA-NOG) were produced. Human hepatocytes injected into uPA-NOG mice repopulated the recipient livers with human cells. The uPA-NOG model has several advantages over previously produced chimeric mouse models of human liver: (1) the severely immunodeficient NOG background enables higher xenogeneic cell engraftment; (2) the absence of neonatal lethality enables mating of homozygotes, which increased the efficacy of homozygote production; and (3) donor xenogeneic human hepatocytes could be readily transplanted into young uPA-NOG mice, which provide easier surgical manipulation and improved recipient survival.

Tumor necrosis factor α 5′‐flanking region, tumor necrosis factor receptor II, and HLA–DRB1 polymorphisms in Japanese patients with rheumatoid arthritis

Objective New polymorphisms affecting transcriptional activity were recently reported within the 5′-flanking region of the tumor necrosis factor α gene (TNFA). In addition, genome-wide linkage screening indicated 1p36 as one of the candidate chromosomal regions where the TNF receptor II gene (TNFR2) is located. In the present study, HLA–DRB1, TNFA promoter, and TNFR2 genotypes were determined to examine whether these polymorphisms are associated with rheumatoid arthritis (RA), either independently or in combination. Methods Genotypes of HLA–DRB1, TNFA upstream promoter, and TNFR2 codon 196 were determined in 545 Japanese patients with RA and 265 healthy controls. Association of these genes with susceptibility to RA was analyzed both independently and after stratification by one of the genotypes. Results As expected, the HLA–DRB1 shared epitope was strongly associated with RA. In addition, a significant negative association of DRB1*1405 and 1302 was observed. Furthermore, DRB1*1405 was suggested to possess a protective role for the development of RA in DRB1*0405-positive individuals. A significant increase in TNFA-U02 in RA was detected, which was not independent of DRB1*0405. A significant association was not observed between TNFR2-196M/R polymorphism and RA. Conclusion Among the 3 genes examined in this study, HLA–DRB1 was considered to be most strongly associated with RA.

Semi-quantitative analyses of metabolic systems of human colon cancer metastatic xenografts in livers of superimmunodeficient NOG mice

Analyses of energy metabolism in human cancer have been difficult because of rapid turnover of the metabolites and difficulties in reducing time for collecting clinical samples under surgical procedures. Utilization of xenograft transplantation of human-derived colon cancer HCT116 cells in spleens of superimmunodeficient NOD/SCID/IL-2Rγnull (NOG) mice led us to establish an experimental model of hepatic micrometastasis of the solid tumor, whereby analyses of the tissue sections collected by snap-frozen procedures through newly developed microscopic imaging mass spectrometry (MIMS) revealed distinct spatial distribution of a variety of metabolites. To perform intergroup comparison of the signal intensities of metabolites among different tissue sections collected from mice in fed states, we combined matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI–TOF-IMS) and capillary electrophoresis–mass spectrometry (CE–MS), to determine the apparent contents of individual metabolites in serial tissue sections. The results indicated significant elevation of ATP and energy charge in both metastases and the parenchyma of the tumor-bearing livers. To note were significant increases in UDP-N-acetyl hexosamines, and reduced and oxidized forms of glutathione in the metastatic foci versus the liver parenchyma. These findings thus provided a potentially important method for characterizing the properties of metabolic systems of human-derived cancer and the host tissues in vivo.

The influence of donor age on liver regeneration and hepatic progenitor cell populations.

BACKGROUND Recent reports suggest that donor age might have a major impact on recipient outcome in adult living donor liver transplantation (LDLT), but the reasons underlying this effect remain unclear. The aims of this study were to compare liver regeneration between young and aged living donors and to evaluate the number of Thy-1+ cells, which have been reported to be human hepatic progenitor cells. METHODS LDLT donors were divided into 2 groups (Group O, donor age ≥ 50 years, n = 6 and Group Y, donor age ≤ 30 years, n = 9). The remnant liver regeneration rates were calculated on the basis of computed tomography volumetry on postoperative days 7 and 30. Liver tissue samples were obtained from donors undergoing routine liver biopsy or patients undergoing partial hepatectomy for metastatic liver tumors. Thy-1+ cells were isolated and counted using immunomagnetic activated cell sorting (MACS) technique. RESULTS Donor liver regeneration rates were significantly higher in young donors compared to old donors (P = .042) on postoperative day 7. Regeneration rates were significantly higher after right lobe resection compared to rates after left lobe resection. The MACS findings showed that the number of Thy-1+ cells in the human liver consistently tended to decline with age. CONCLUSION Our study revealed that liver regeneration is impaired with age after donor hepatectomy, especially after right lobe resection. The declining hepatic progenitor cell population might be one of the reasons for impaired liver regeneration in aged donors.

Treatment of advanced myelodysplastic syndrome with a regimen including recombinant human granulocyte colony‐stimulating factor preceding allogeneic bone marrow transplantation

We treated 13 patients with morphologically advanced myelodysplastic syndrome using cytosine arabinoside and total body irradiation, followed by allogeneic marrow transplantation from HLA‐identical sibling donors. Granulocyte colony‐stimulating factor (G‐CSF) was added to the preparative regimen to selectively increase chemosensitivity of leukaemic cells and to improve transplant outcome. No regimen‐related deaths occurred, and no side‐effects related to the addition of G‐CSF were observed except for transient mild bone pain. At a median follow‐up time of 39 months the projected 5‐year disease‐free survival and 5‐year overall survival were 67.7% and 75.5%, respectively, with only one case showing cytogenetic relapse. The preparative regimen including G‐CSF is feasible, and preliminary results seem to be encouraging. However, a larger trial is clearly warranted to evaluate its efficacy.

Genetic dissection of lupus pathogenesis: Sle3/5 impacts IgH CDR3 sequences, somatic mutations, and receptor editing

Sle3/5 is a lupus susceptibility locus identified on mouse chromosome 7 of the New Zealand Black/New Zealand White (NZB/NZW)-derived NZM2410 strain. Based on previous observations, this locus appears to contribute to lupus pathogenesis through its impact on diversification of immune responses. To understand how Sle3/5 affects somatic diversification of humoral responses, we analyzed IgH rearrangements preferentially encoding hapten-reactive IgG1 repertoires after immunization and assessed peripheral IgH VDJ recombination activities in C57BL/6 (B6) mice congenic for Sle3/5 (B6.Sle3/5). In addition to altered somatic VH mutation profiles, sequences from B6.Sle3/5 mice exhibited atypical IgH CDR3 structures characteristic of autoreactive B cells and consistent with peripheral B cells bearing putatively edited receptors. Significant expression of Rag genes and circular VHD gene excision products were detected in splenic mature B cells of B6.Sle3/5 but not B6 mice, showing that peripheral IgH rearrangements occurred beyond allelic exclusion. Taken together, on the nonautoimmune background, Sle3/5 affected VHDJH junctional diversity and VH mutational diversity and led to recombinational activation of allelically excluded IgH genes in the periphery. Such impact on somatic IgH diversification may contribute to the development of autoreactive B cell repertoires. This is the first report to present evidence for significant association of a lupus susceptibility locus, which has been mapped to a chromosomal region in which no Ig genes have been identified, with somatic IgH sequence diversity and peripheral H chain receptor editing or revision without relying upon Ig transgene strategies.

Trichomonas foetus meningoencephalitis after allogeneic peripheral blood stem cell transplantation.

A 34-year-old man with refractory acute myelogenous leukemia underwent allogeneic peripheral blood stem cell transplantation (PBSCT) from his HLA-matched sibling. Engraftment was prompt and no acute GVHD developed. However, high fever persisted even after engraftment, and the patient developed headache, diplopia, vertigo and nuchal rigidity on day 20 post- transplant. Cerebrospinal fluid (CSF) showed pleocytosis with no detectable microorganisms. Despite therapy with broad-spectrum antibiotics, antifungal agents and antituberculous drugs, he developed rapid mental deterioration with seizures and died on day 40. Just prior to his death, trichomonads were isolated from both CSF and urine. Scanning electron microscopic examination identified the trichomonad as Trichomonas foetus. At autopsy, trichomonads were detected histopathologically in an area involving meningoencephalitis. To our knowledge, this is the first case of T. foetus meningoencephalitis in a recipient of allogeneic PBSCT and, more importantly, the first human case of T. foetus infection.