Yasser Osman

Activation of hepatic NKT cells and subsequent liver injury following administration of α‐galactosylceramide

It has been established that α‐galactosylceramide (α‐GalCer), a glycolipid, is recognized by natural killer T (NKT) cells together with the monomorphic MHC‐like antigen, CD1d, in mice and humans. In this study, we examined how NKT cells are modulated by in vivo administration of α‐GalCer in mice. When 2 μg (or more)/mouse of α‐GalCer was injected i.p., the majority of NKT cells disappeared in the liver and spleen, possibly undergoing apoptosis, on day 1. At this time, NKT cytotoxicity seen in liver lymphocytes also disappeared. In parallel with this numerical and functional change of NKT cells, there was always concomitant hepatocyte damage, as shown by histology and elevated levels of transaminases. Subsequently, the number and function of NKT cells continued to increase from day 3 to day 7. The response seen in hepatic (and splenic) NKT cells did not occur in thymic NKT cells. All these phenomena induced in the liver did not appear in NKT‐deficient mice such as β2‐microglobulin–/– and CD1d–/– mice. These results shed further light on the in vivo interaction between NKT cells and α‐GalCer in mice.

Amyopathic dermatomyositis associated with transformed malignant lymphoma

Amyopathic dermatomyositis is a disease of unknown origin characterized by the specific skin lesions of dermatomyositis but without clinical or laboratory evidence of myopathy. During the past 15 years, a great controversy between the different reports concerning a possible association of dermatomyositis with malignancy has been noted. In this report, the authors describe a patient with amyopathic dermatomyositis who presented first with a benign hyperplasia of the lymph node, which finally transformed into frank malignant lymphoma. In addition to follow-up care, screening tests to search for occult malignancy in patients with amyopathic dermatomyositis (or dermatomyositis) are recommended.

Generation of Ag-specific cytotoxic T lymphocytes by DC transfected with in vitro transcribed influenza virus matrix protein (M1) mRNA

BACKGROUND Application of DC transfected with tumor Ag RNA is promising for DC-based tumor immunotherapy. In this study, Ag-specific cytotoxic T lymphocytes (CTL) were generated by priming lymphocytes with DC transfected with in vitro transcribed (IVT) influenza virus matrix protein M1 (M1) mRNA. METHODS Human UC blood-CD34+ cell-derived DC were transfected with IVT mRNA encoding either the enhanced green fluorescence protein (EGFP), or M1 by square-wave electroporation. DC were confirmed to have typical morphology and phenotype. DC transfected with IVT EGFP mRNA were analyzed with the FACScan flow cytometer, to confirm the efficiency of this transfection method. On Days 7, 14, 21 and 28 after the start of DC culture, DC were harvested and electroporated with M1 mRNA. The transfected DC were co-cultured with autologous UC blood CD34- cells. One week after the fourth priming of autologous CD34 negative cells with M1 mRNA electroporated DC, Ag-specific CTL activity was evaluated. To prepare target cells, M1 mRNA was added to autologous DC 48 h prior to CTL assays. RESULTS Our CTL assays results indicate that UC blood CD34+ cell-derived DC transfected with M1 mRNA by electroporation stimulated Ag-specific CTL responses that are capable of recognizing and lysing autologous DC loaded with M1 mRNA. M1 mRNA transfected DC-primed CTL showed a significant cytotoxic activity against M1 mRNA loaded autologous DC, while nearly baseline cytotoxic activity was recorded for the M1 mRNA unloaded DC. DISCUSSION Our results showed that mRNA-transfected DC are potent stimulators of T-cell immunity in vitro. In addition, mRNA-loaded DC can function as targets in CTL cytotoxicity assays, which offer a practical substitute for tumor cells in assays to test the immunological effects of specific Ags.

A comparative study of the JAM test and 51Cr-release assay to assess the cytotoxicity of dendritic cells on hematopoietic tumor cells

Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti‐tumor cytotoxic ability. Nevertheless, the mechanism of anti‐tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [3H] thymidine (3H‐TdR) uptake by tumor cells; 2) to induce cytolysis on 51Cr‐labeled tumor cells; 3) and to induce DNA fragmentation on 3H‐TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of 3H‐TdR. However no cytolysis was verified by 51Cr‐release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between 51Cr‐release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released 51Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4‐h and the 10‐h 51Cr‐release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.

Expression of coxsackievirus and adenovirus receptor in neointima of the rat carotid artery

Our previous study revealed that the coxsackievirus and adenovirus receptor (CAR) is a homophilic cell adhesion molecule and may function as a sensor of cell-cell interactions in the brain and damaged heart. In this study, we investigated if CAR expression is involved in the formation of neointimal hyperplasia using a balloon injury model of rat carotid artery. Cultured vascular smooth muscle cells (SMCs) from rat aorta were also studied. CAR antigen was constitutively detected in the endothelial cells (ECs) but not in SMCs before injury. On Day 5 after balloon injury, CAR was expressed strongly in the first layer of medial SMCs. Neointimal hyperplasia was observed on Day 7, and strong expressions of CAR concomitantly with proliferating cell nuclear antigen (PCNA) were obvious in the neointimal SMCs, while CAR in medial SMCs disappeared. The expression of CAR mRNA reached a peak on Day 7 and declined gradually to the basal levels. When the ECs regenerated on Day 14, CAR antigen was observed in the ECs but disappeared in the neointima. CAR together with PCNA was expressed abundantly in the proliferating SMCs in vitro and diminished in cells grown to a confluent state. The abundant expression of CAR in the neointima may facilitate an adenoviral gene therapy.

Effect of prostaglandin E2, lipopolysaccharide, IFN-γ and cytokines on the generation and function of fast-DC

BACKGROUND Recent reports have described a new strategy for differentiation and maturation of monocyte-derived DC within only 48 h of in vitro culture (fast-DC). We compared the ability of various maturation stimuli with the generation of Ag-specific T-cell responses and generation of functional fast-DC. METHODS CD14+ cells were treated with GM-CSF and IL-4 for 1 day to generate immature DC, and were then matured with either inflammatory cytokines or a combination of lipopolysaccharide (LPS) and INF-gamma. Mature DC were then used to study the effect of prostaglandin E2 (PGE2) on the stimulatory function of fast-DC. RESULTS fast-DC were CD14- and expressed mature DC surface markers, and maintained this phenotype after withdrawing the cytokine from culture. Treatment of fast-DC with a combination of LPS and INF-gamma promoted the maturation of highly uniform fast-DC. The T-cell proliferative response to DC was enhanced by inclusion of PGE2 in the MCM-mimic (TNF-a, IL-1 a, IL-6, PGE2) cocktail. DISCUSSION fast-DC are very effective; they not only reduce the labor, cost and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.