Expression of rck/p54, a DEAD-box RNA helicase, in gametogenesis and early embryogenesis of mice.

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals.     

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of inflammatory bowel disease

4-1BB (CDw 137), a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB monoclonal antibody (mAb) enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of anti-4-1BB mAb reduced the incidence and severity of inflammatory bowel disease. In this study, we investigated the effects of anti-4-1BB mAb in a murine intestinal inflammation model, which induced by the hapten reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) and mimics immunologic characteristics of human Crohn's disease (CD). Colitis was induced by rectal administration of 2mg of TNBS in 35% ethanol using a vinyl catheter positioned 4cm from the anus. All mice were sacrificed 3 and 10 days after the TNBS administration. The disease activity index (DAI), histological changes of the colon and production of cytokines (IL-2, IL-4, IL-10 and IFN-gamma) were evaluated. The surface molecules of T cells in peripheral blood, spleen and mesenteric lymph nodes were analyzed by flow cytometry. When mice were treated with anti-4-1BB mAb, improvement in both wasting and histopathologic signs of colonic inflammation was observed. The increase a number of splenic CD4(+)CD25(+) T cells and decreased synthesis of the Th1 cytokine IL-2 also occurred. Interestingly, increased production of Th1 cytokine IFN-gamma and proportion of CD8(+) T cells were observed in mice treated with anti-4-1BB mAb in comparison to the colitic mice. These studies show, for the first time, that agonistic anti-4-1BB mAb can improve experimental colitis by reduction of IL-2 and augmentation of CD4(+)CD25(+) regulatory T cells. TNBS colitis is Th1-mediated and has similar histologic features and distribution of inflammation to CD. This study suggests that anti-4-1BB mAb therapy could be effective in the treatment of patients with CD.     

Morphometric evaluation of PGP9.5 and NCAM expressing nerve fibers in colonic muscle of patients with Hirschsprung's disease

A quantitative assessment of the density of the protein gene product 9.5 (PGP9.5), the neural cell adhesion molecule (NCAM), and the low-affinity nerve growth factor receptor (NGFR) expressing nerve fibers in the circular muscle layer in the colon was carried out by morphometric analyses from 13 patients with Hirschsprung's disease (HD). The difference in the nerve fiber density between the ganglionic and aganglionic segments was compared by calculating the ratio of the sum of the areas occupied by positively stained nerve fibers per unit area of the muscle after immunohistochemical staining on paraffin embedded tissue sections using computer software. There was an obvious difference in the density of the PGP9.5 stained nerve fibers between the ganglionic (0.0380 +/- 0.0171) and aganglionic segments (0.0143 +/- 0.01661). The NCAM-positive nerve fibers were fewer in number than those of both the PGP9.5-positive fibers and NCAM-positive fibers, which were also markedly lower in number in the aganglionic segment (0.0066 +/- 0.0076) than in the ganglionic segment (0.0230 +/- 0.0195). Immunostaining for low-affinity NGFR revealed much fainter staining in the ganglionic and aganglionic segment without a statistically significant difference in their density. Considering the fact that PGP9.5 is a very sensitive marker for nerve fibers, the results of this study reaffirm the innervation failure of the proper muscle in HD. The decreased NCAM expression level in the aganglionic segment appears to be caused not by the selective down-regulation of NCAM expression among the nerve fibers but by a markedly reduced number of nerve fibers.     

Cellular immuno-PCR. Detection of a carbohydrate tumor marker.

Carbohydrate tumor-antigens are important tumor markers for diagnosis and functional characteristics of human cancer cells. Detection of these carbohydrate tumor antigens on metastatic cancer cells in blood is a difficult task. We developed a highly sensitive method to detect a cell surface carbohydrate antigen using a hybrid technology referred to as cellular immuno-PCR. This technique uses the human monoclonal antibody (HumAb) L612, specific to a tumor-related antigen (ganglioside) GM3 that is expressed on the cell surface of human tumor cells and not normal cells. L612 coupled to a DNA oligonucleotide for exponential amplification by DNA polymerase chain reaction (PCR) can be used to enhance the detection signal. The DNA-HumAb conjugate was assessed for detection of a small number of human cancer cells after PCR amplification and Southern blot analysis. To assess the assay specificity human melanoma and other cancer cell lines, as well as healthy donor and melanoma patients, bloods were assessed. Cellular immuno-PCR requires < 1 ng/ml DNA-HumAb complex and was shown to have a detection level of < 10 cells in titration studies in which melanoma cells were diluted in 2 million healthy donor peripheral blood lymphocytes. The assay was shown to be very sensitive and could detect low levels of GM3 antigen expression by tumor cells. This novel approach for detecting a carbohydrate tumor antigen on tumor cells in blood provides a potential useful clinicopathological assay.     

Recognition of Pneumocystis carinii antigen on its surface by immunohistochemistry and immunoelectron microscopy.

The aim of this study was to detect the surface antigens in different stages of experimental induced Pneumocystis carinii in Sprague-Dawley rats. Immunohistochemical staining with monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies demonstrated that the P. carinii organisms were mostly in the alveolar lumina. The binding sites of the monoclonal (900, 902 and 904) and polyclonal (SP-D) antibodies developed against P. carinii were examined at the ultrastructural level by using a post-embedding immunogold labeling. The gold particles were observed evenly on the surface of precyst and cyst stages of the P. carinii. In the trophozoite stage, scattered gold particles were seen on the pellicles and tubular expansions. The monoclonal antibodies reacted mainly with pellicles of P. carinii, whereas SP-D labeled pellicles, intracystic bodies, cytoplasms of alveolar macrophages, free floating surfactant material in the alveolar spaces, and adjacent type II epithelial cells. In the immunogold labeling, basically no significant differences were found in the precyst, cyst, and ruptured cyst stages. These results indicate that the gold particles were observed adhering to every stage of P. carinii, mostly concentrated on the pellicles, and more concentrated in the precyst or cyst stage than trophozoite stage which may be due to an increase in antigen accumulation during development from the trophozoite to the cyst.     

Upregulation of VEGF and FGF2 in normal rat brain after experimental intraoperative radiation therapy

The expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)2 in the irradiated brain was examined to test how a single high dose radiation, similar to that used for intraoperative radiation therapy given to the normal cerebrum, can affect the vascular endothelium. After a burr hole trephination in the rat skull, the cerebral hemisphere was exposed to a single 10 Gy dose of gamma rays, and the radiation effect was assessed at 1, 2, 4, 6, and 8 weeks after irradiation. Histological changes, such as reactive gliosis, inflammation, vascular proliferation and necrosis, were correlated with the duration after irradiation. Significant VEGF and FGF2 expression in the 2- and 8-week were detected by enzyme-linked immunosorbent assay quantification in the radiation group. Immunohistochemical study for VEGF was done and the number of positive cells gradually increased over time, compared with the sham operation group. In conclusion, the radiation injuries consisted of radiation necrosis associated with the expression of VEGF and FGF2. These findings indicate that VEGF and FGF2 may play a role in the radiation injuries after intraoperative single high-dose irradiation.     

A pilot study of bortezomib in Korean patients with relapsed or refractory myeloma

Recent clinical trials showed that bortezomib, a novel proteasome inhibitor, had therapeutic activity in multiple myeloma. However, there was no data about the feasibility of bortezomib in Korean patients. We performed a pilot study of bortezomib in patients with relapsed or refractory myeloma (1.3 mg/m2 twice weekly for 2 week in a 3-week cycle). Seven patients were enrolled. The median age of patients was 59 yr. All patients previously received VAD (vincristine, doxorubicin and dexamethasone) and thalidomide chemotherapy. Three patients previously received alkylator-containing chemotherapy and 4 patients, autologous stem cell transplantation. Bortezomib monotherapy resulted in 3 partial remissions (43%), 3 no changes (43%) and 1 progressive disease (14%). One patient who had no response to bortezomib monotherapy experienced partial remission after addition of dexamethasone to bortezomib. The most common serious toxicity was thrombocytopenia (grade 3/4, 10 of 20 cycles (50%)) and grade 3 peripheral neuropathy was developed in 2 of 20 cycles (10%). Drug-related adverse event led to discontinuation of bortezomib in 1 patient. There was no treatment related mortality. Overall, bortezomib seems to be effective and feasible. Conduction of larger clinical studies on Korean patients is necessary to characterize clinical efficacy and safety of bortezomib more precisely.     

BK polyomavirus interstitial nephritis in a renal allograft recipient

Human polyomavirus (PV) interstitial nephritis has recently been recognized as a cause of severe renal allograft dysfunction. It occurs in immunosuppressed patients after reactivation of the latent virus PV type BK (BK virus) in the renal epithelium. BK disease is defined as a morphologically manifest renal infection with cytopathic signs accompanied by varying degrees of interstitial inflammatory cell infiltrates and functional impairment. It is also identified by the presence of cells containing viral inclusion bodies (decoy cells) in the urine. The authors report a case of BK PV interstitial nephritis in a 36-year-old renal allograft recipient. Under light microscopy the chief diagnostic indicator was detection of intranuclear viral inclusions, which were found exclusively in tubular epithelial cells. Cells with viral changes were often enlarged with nuclear atypia and chromatin basophilia. Widespread interstitial plasma cell infiltrates associated with tubulitis were present. Intranuclear paracrystalline arrays of virus particles 35-38 nm in diameter were present as characteristic ultrastructural indicators. Urine samples revealed decoy cells with ground-glass-type intranuclear inclusions positive for BK virus by electron microcopy.     

A case of multiple schwannomas of the trigeminal nerves, acoustic nerves, lower cranial nerves, brachial plexuses and spinal canal: schwannomatosis or neurofibromatosis?

In most cases, while schwannoma is sporadically manifested as a single benign neoplasm, the presence of multiple schwannomas in one patient is usually indicative of neurofibromatosis 2. However, several recent reports have suggested that schwannomatosis itself may also be a distinct clinical entity. This study examines an extremely rare case of probable schwannomatosis associated with intracranial, intraspinal and peripheral involvements. A 63-year-old woman presented with a seven-year history of palpable lumps on both sides of the supraclavicular area and hearing impairment in both ears. On physical examination, no skin manifestations were evident. Facial sensory change, deafness in the left ear and decreased gag reflex were revealed by neurological examination. Magnetic resonance imaging revealed multiple lesions of the trigeminal nerves, acoustic nerves, lower cranial nerves, spinal accessory nerve, brachial plexuses, and spinal nerves. Pathological examination of tumors from the bilateral brachial plexuses, the spinal nerve in the T8 spinal position and the neck mass revealed benign schwannomas. Following is this patient case report of multiple schwannomas presenting with no skin manifestations of neurofibromatosis.