Abstract Background : Restoration of virus-specific immunity by virus specific T cells (VSTs) offers an attractive alternative to conventional drugs, and can be highly effective in immunocompromised ...patients, including hematopoietic stem cell transplant (HSCT) recipients. However, conventional VSTs manufacture requires preparation of specialized antigen-presenting cells (APCs), prolonged ex vivo culture in serum-containing medium and antigen re-stimulation with viruses or viral vectors to provide viral antigens for presentation on APCs. Methods : To simplify this complex process, we developed a method to generate multiple VSTs by direct stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries in serum-free medium. Results : We generated VSTs that targeted seven viruses (cytomegalovirus CMV, Epstein-Barr virus EBV, adenovirus AdV, human herpesvirus 6 HHV-6, BK virus BKV, JC virus JCV and Varicella Zoster virus VZV) in a single line. The phenotype, growth and specificity of multiple VSTs produced in serum-free medium were equivalent to those generated in conventional serum-containing medium. Discussion : The use of serum-free medium allows this approach to be readily introduced to clinical practice with lower cost, greater reproducibility due to the absence of batch-to-batch variability in serum and without concerns for infectious agents in the serum used. This simplified approach will now be tested in recipients of Human Leukocyte Antigen (HLA)–matched sibling HSCT.
h-Caldesmon is considered a novel specific marker for tumors with smooth muscle differentiation. To reassess its diagnostic use, the authors evaluated the immunohistochemical expression of ...h-caldesmon and other myogenic markers (calponin, alpha-smooth muscle actin, HHF35, and desmin) in 30 leiomyosarcomas (external soft tissues 15, retroperitoneum 8, uterus 5, other sites 2), 26 myofibroblastic lesions, and 26 fibrohistiocytic tumors of varying biologic potential and histology. In contrast with previous data, h-caldesmon was expressed only in 11 (36%) of the 30 leiomyosarcomas analyzed, whereas they consistently expressed actins and frequently expressed calponin (86%) and desmin (76%). Leiomyosarcomas with the expression of h-caldesmon were well or moderately differentiated and primarily confined to the retroperitoneum or uterus. All but one leiomyosarcomas in the external soft tissues examined were negative for h-caldesmon, and the h-caldesmon-negative tumors showed moderately to poorly differentiated morphology. All myofibroblastic lesions examined were negative for h-caldesmon despite their constant expressions of at least one of the other markers. h-Caldesmon was not expressed in fibrohistiocytic tumors either, although focal positivity for the other markers was seen in subsets of the tumors. Thus, h-caldesmon can be regarded as a specific myogenic marker. However, one should be aware that the expression of h-caldesmon in leiomyosarcomas can be more variable according to their locations and/or extent of smooth muscle differentiation than considered previously.
Chronic active Epstein-Barr virus infection (CAEBV) is a high-mortality and high-morbidity disease. To clarify the prognostic factors, a national survey was performed in Japan, and data for 82 ...patients who met the criteria for CAEBV were analyzed. Of these 82 patients, 47 were alive and 35 had already died. Multivariate analysis revealed that thromobocytopenia and age at disease onset were correlated with mortality. The probability of 5-year survival was 0.45 for older patients (onset age, ⩾8 years), 0.94 for younger patients (P<.001), 0.38 for patients with thrombocytopenia (platelet count <12×10 4 platelets/μL at diagnosis), and 0.76 for patients without thrombocytopenia (P=.01). Furthermore, patients with T cell infection by EBV had shorter survival times than patients with natural killer cell infection (probability of 5-year survival, 0.59 vs. 0.87; P<.009). Patients with CAEBV with late onset of disease, thrombocytopenia, and T cell infection had significantly poorer outcomes
CD40 is a 45-kDa glycoprotein member of the tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carcinoma cells. The unique capacity ...of CD40 to trigger immunoglobulin isotype switching is dependent on the activation of protein-tyrosine kinases, yet CD40 possesses no kinase domain and no known consensus sequences for binding to protein-tyrosine kinases. Recently, an intracellular protein (CD40bp/LAP-1/CRAF-1) which belongs to the family of TNFR-associated proteins was reported to associate with CD40. We describe a 23-kDa cell surface protein (p23) which is specifically associated with CD40 on B cells and on urinary bladder transitional carcinoma cells. Protein microsequencing revealed that p23 shows no homology to any known protein. A rabbit antibody raised against a peptide derived from p23 recognized a 23-kDa protein in CD40 immunoprecipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was not associated with TNFR p80 (CD120b). These findings suggest that p23 is a novel member of the CD40 receptor complex.
Apoptosis linked to oxidative stress has been implicated in pancreatitis. We investigated whether NADPH oxidase mediates apoptosis in cerulein-stimulated pancreatic acinar AR42J cells. We report here ...that cerulein treatment resulted in the activation of NADPH oxidase, as determined by ROS production, translocation of cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and interaction between NADPH oxidase subunits. Cerulein induced Ca(2+) oscillation, the expression of apoptotic genes p53 and bax, and apoptotic indices (DNA fragmentation, TUNEL staining, caspase 3 activity, decrease in cell viability) in AR42J cells. Treatment with a Ca(2+) chelator, BAPTA-AM, or transfection with antisense oligonucleotides for NADPH oxidase subunits p22(phox) and p 47(phox) inhibited cerulein-induced ROS production, translocation of NADPH oxidase cytosolic subunits p 47(phox) and p 67(phox) to the membrane, and the expression of apoptotic genes and apoptotic indices, as compared to the cells without treatment and those transfected with the corresponding sense oligonucleotides. These results indicate that NADPH oxidase may mediate ROS-induced apoptosis in pancreatic acinar cells in a Ca(2+)-dependent manner.
Summary
Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection ...revealed several clusters of co‐regulated genes, an enrichment of preferentially up‐ or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co‐incubation with Legionella hackeliae and L. pneumophilaΔdotA. One hundred and thirty‐one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host‐pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.
Autosomal recessive form of hyper-IgM syndrome type 2 (AR-HIGM2) is secondary to mutations affecting both alleles of
AICDA gene encoding activation-induced cytidine deaminase, characterized by ...defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in most of the patients. We herein report the immunological phenotype of seven patients carrying a single heterozygous R190X mutation in
AICDA. Variable defect in in vivo CSR inherited as an autosomal dominant (AD) trait strongly suggests that this heterozygous
AICDA mutation causes HIGM (AD-HIGM2). In AD-HIGM2 B cells, CSR was consistently found impaired in vitro. However, in contrast to AR-HIGM2, the CSR-induced double-stranded DNA breaks in the switch region of IgM heavy chain gene were detected. The SHM frequency in V regions of IgM heavy chain gene in B cells was normal in all (but one patient). The characteristics of the AD-HIGM2 phenotype indicate that the AID C-terminal region may be involved in DNA repair machinery required for CSR.
Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been ...hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on β-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin α and importin β reflects possible modification of nuclear import of Ku proteins. The importin β level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.
Abstract
Adoptive immunotherapies have been developed for antiviral agent‐refractory cytomegalovirus (
CMV
) disease after stem cell transplantation (
SCT
). However, the application of such ...strategies is limited, particularly in terms of need for donor cooperation regarding blood sampling and inaccessibility in the setting of cord blood transplantation. Herein, we describe the first successful treatment of antiviral agent‐refractory
CMV
enteritis after allogeneic
SCT
by the infusion of
ex vivo
‐expanded donor‐derived
CD
4
+
lymphocytes obtained from the recipient's peripheral blood.
In this study, we describe the cytological and cytogenetic features of six Epstein‐Barr virus (EBV)‐infected natural killer (NK) cell clones. Three cell clones, SNK‐1, ‐3 and ‐6, were derived from ...patients with nasal T/NK‐cell lymphomas; two cell clones, SNK‐5 and ‐10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK‐11, was from a patient with hydroa vacciniforme (HV)‐like eruptions. An analysis of the number of EBV‐terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV‐infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK‐cell lymphomas but also those isolated from CAEBV and HV‐like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T‐cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV‐positive NK cells, which proliferated in the diseased individuals. The results showed that EBV‐positive NK cells in malignant and non‐malignant lymphoproliferative diseases shared common cytological and cytogenetic features.
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