Microendoscopic resection of lumbar discal cysts.

INTRODUCTION: A lumbar discal cyst is a relatively rare cystic lesion that communicates with lumbar intervertebral discs. Surgical resection of the cyst is the reported treatment of choice. In this study, the authors report the minimally invasive surgical resection of lumbar discal cysts using a microendoscopy. PATIENTS AND METHODS: Seven male patients with lumbar discal cysts underwent microendoscopic resections (mean age: 25.1+/-3.2 years and the mean follow-up period: 27.9 months). During the surgeries, the cysts were subtotally resected in a piecemeal fashion, and the fistulas forming the communications between the cysts and the corresponding intervertebral discs were coagulated using a bipolar coagulator. RESULTS: All the patients obtained relief from their pain after surgery, and no recurrences occurred during a mean follow-up period of 28 months. The mean operation time was 72.6+/-20.2 min, and the mean blood loss was 44.4+/-13.7 grams. No intra- or peri-operative complications were noted in any of the patients. CONCLUSIONS: Microendoscopic resection appears to be a minimally invasive and feasible surgical option for the treatment of lumbar discal cysts.

GRIP1 enhances estrogen receptor alpha-dependent extracellular matrix gene expression in chondrogenic cells.

OBJECTIVE: The role of postmenopause on the pathogenesis of cartilage degeneration has been an open question. We assessed cartilage degeneration in estrogen receptor (ER)alpha null mice and examined the role of glucocorticoid receptor-interacting protein 1 (GRIP1) in the ERalpha-dependent transcription of a type II collagen gene (col2a1) with special reference to a crosstalk with the transforming growth factor (TGF)-beta signaling pathway. METHODS: The vertebral cartilaginous endplate from female ERalpha null mice was subjected to histological analyses. Col2a1 expression of primary chondrocytes (PCs) obtained from ERalpha null mice after 17beta-estradiol (E(2)) and TGF-beta1 stimulation was examined by reverse transcription polymerase chain reaction (RT-PCR). Estrogen response element (ERE) or col2a1 promoter-enhancer luciferase reporter system was used to investigate the crosstalk among ERalpha, GRIP1, and MKK6. Col2a1 expression and glycosaminoglycan (GAG) content were measured in ATDC5 cells treated with GRIP1 small interfering RNA (siRNA). RESULTS: ERalpha deficiency clearly accelerated impairment of the vertebral cartilaginous endplate. E(2) and TGF-beta1 stimulation increased col2a1 expression in PC from wild-type mice, but not that from ERalpha null mice. The same stimulation increased the col2a1 promoter-enhancer reporter activity, and the elevated activity was decreased by dominant-negative ERalpha and p38 mitogen-activated protein kinase (MAPK) inhibitor. GRIP1 increased the E(2)-dependent ERE activation in the presence of ERalpha and constitutive-active MKK6. GRIP1 siRNA repressed col2a1 expression and GAG production in ATDC5 cells. CONCLUSIONS: Crosstalks between ERalpha/GRIP1 and TGF-beta/MKK6/p38 MAPK pathway have protective roles on cartilage metabolism via regulating the extracellular matrices expression. The finding may lead to the development of a novel therapeutic approach for cartilage degeneration.

Surgical treatment of intramedullary spinal cord tumors: prognosis and complications.

STUDY DESIGN: Retrospective case series. OBJECTIVE: To evaluate our recent treatment strategy for intramedullary spinal cord tumors. SETTING: Department of Orthopaedic Surgery, Keio University, Japan. METHODS: We reviewed 68 cases of intramedullary tumors (ependymoma, 33; astrocytoma, 23; hemangioblastoma, 12), treated surgically between 1994 and 2003. There were 42 males and 26 females whose mean age at the time of surgery was 43 years. The mean follow-up period was 6.2 years. The tumor malignancy grade according to the WHO classification was astrocytoma grade I, 3; grade II, 8 (low-grade: 11 cases); grade III, 10; grade IV, 2 (high-grade: 12 cases). All ependymomas were grade II. Three of the 12 hemangioblastomas were associated with von Hippel-Lindau disease. RESULTS: Total excision was achieved in 90% of the ependymomas and functional improvement was obtained when the preoperative neurological deficit was mild. Approximately 50% of low-grade astrocytomas could be totally excised with favorable survival outcomes, suggesting that total excision should be attempted for low-grade astrocytomas. However, total excision of high-grade tumors was difficult and the functional outcomes were poor. Cordotomy should be considered in patients with a thoracic high-grade astrocytoma. Total resection was possible in 92% of hemangioblastoma, and the functional outcomes were good, however, more attention should be paid for tumors with feeding arteries on the ventral side and for those associated with von Hippel-Lindau disease. CONCLUSIONS: Predictors of good surgical outcome for intramedullary spinal cord tumors were histological grades of the tumors, surgical margins, and neurological status of the patient before surgery.

Microendoscopic discectomy for lumbar disc herniation with bony fragment due to apophyseal separation.

The purpose of this study was to elucidate the feasibility of microendoscopic discectomy (MED) for the treatment of lumbar disc herniation with a bony fragment due to apophyseal separation. Eighteen patients with low back pain and unilateral sciatic pain due to lumbar disc herniation with a bony fragment were treated by MED using the unilateral approach (15 males and three females; mean age, of 28.9 years; mean follow-up period, 21.1 months); 18 age-and sex-matched patients with lumbar disc herniation without a bony fragment treated by MED served as the control group. The clinical outcomes were evaluated using the Japanese Orthopedic Association Score for Low Back Pain (JOA scores; maximum score, 29 points). Evaluation of the results revealed that good surgical outcomes equivalent to those in the control group were obtained in the subjects of LDH with a bony fragment (JOA scores; 14.1+/-3.5 in the patient group vs.15.4+/-2.6 in the control group before surgery; 26.3+/-1.8 in the patient group vs. 26.9+/-1.3 at follow-up after the surgery). Although the mean surgical time was significantly longer in the patient group, there were no intra- or postoperative complications in either group. We conclude that MED using the unilateral approach is a feasible minimally invasive surgical option for patients of lumbar disc herniation with an apophyseal bony fragment.

Surgical outcomes of spinal cord astrocytomas.

STUDY DESIGN: Retrospective case series. OBJECTIVES: To analyze prognostic factors for patients with spinal cord astrocytomas. SETTING: Department of Orthopaedic Surgery, Keio University, Japan. METHODS: Seven patients received total excisions (group T), eight underwent partial excisions (group P), and 15 had excisional biopsies (group B). Impacts of the tumor histological grade, the level of the tumor, the types of surgical interventions, and the use of adjuvant radiotherapies on the survival and functional outcomes of 30 patients (18 in low-grade and 12 high-grade malignancy tumors) were analyzed. RESULTS: The survival rate of the low-grade malignancy group was significantly higher than that of the high-grade group. The survival rate of the patients with thoracic astrocytomas was significantly higher than those with cervical astrocytomas. In both the low- and high-grade groups, the survival rates in groups P/T were significantly higher than those in group B. In the low-grade group, five patients, whose preoperative functional statuses were classified as 'fair' or better, remained 'fair' or better after surgery. In the high-grade group, the postoperative functional statuses were classified as 'no change' or 'aggravated' in all except two patients. No significant difference in the survival rates was detected between patients with and without adjuvant radiotherapy. CONCLUSIONS: The tumor grade and the extent of tumor resection were significant prognostic factors for survival rate. In low-grade malignancy group, good motor function was retained when surgeries were performed before substantial neurological deterioration. The efficacy of postoperative radiotherapy has yet to be determined and needs further study.

Malignant ameloblastic fibro-odontoma in a dog.

An 11-year-old male Collie was presented with a swelling of the face caused by tumor masses arising from the gingiva. Postmortem examination revealed metastases to the lymph nodes, lung, liver, and orbital cavity. Histologically, the tumor represented a combination of fibrosarcomatous proliferation, pulpal mesenchyme, and undifferentiated odontogenic epithelium, with a follicular or plexiform growth pattern. In addition, the follicular areas of the tumor showed a biphasic character, and there were numerous apoptotic cells in plexiform areas. Furthermore, acidophilic material resembling dysplastic dentine or enamel matrix was observed in the metastatic lesion in the lung. Based on the histological characters, the present case was diagnosed as malignant ameloblastic fibro-odontoma. This study is the first known description of a possible malignant ameloblastic fibro-odontoma in a dog with metastasis to distant organs.

Contrasting action of IL-12 and IL-18 in the development of dextran sodium sulphate colitis in mice.

BACKGROUND: Interleukin (IL)-12 and IL-18 are major interferon (IFN)-gamma-inducing factors that collaborate with each other. The present study was conducted to determine the distinct roles of IL-12 and IL-18 in the development of dextran sulphate sodium (DSS) colitis in mice. METHODS: Colitis was induced in IL-12p35(-/-), IL-18(-/-), IL-18 receptor(-/-) and control mice with DSS. Clinical and histopathological analysis was conducted using survival rate, weight loss score, diarrhoea score, bloody stool score and histological score. In addition, cytokine production by lamina propria mononuclear cells (LPMCs) was examined using the specific enzyme-linked immunoassay. RESULTS: IL-12p35(-/-) mice developed only a mild disease associated with no lethality and few histopathological abnormalities. In contrast, IL-18(-/-) and IL-18R(-/-) mice developed more severe colitis associated with high lethality and more histopathological abnormalities compared with control mice. LPMCs from DSS-fed IL-18(-/-) mice produced significantly higher amounts of IFN-gamma, while LPMCs from DSS-fed IL-12(-/-) mice produced lower amounts of IFN-gamma and tumour necrosis factor (TNF)-alpha compared with control mice. CONCLUSION: These results suggest that IL-18 might function with manners different from IL-12 at some pathological conditions in the development of colitis.

Helicobacter pylori infection as a model for gastrointestinal immunity and chronic inflammatory diseases.

Approximately 50% of humanity is infected with Helicobacter pylori. It is a life-long infection that elicits a marked host inflammatory response; however, natural infection fails to yield protective immunity. Rather than providing protection, the chronic inflammatory response associated with natural infection contributes to tissue damage and the pathogenesis of gastroduodenal disease, including atrophic gastritis, peptic ulcer, and gastric cancer. While bacterial factors are important triggers of inflammation, many subjects infected with strains bearing putative virulence factors remain free from disease. Recent genetic studies have implicated the host's immune and inflammatory responses, suggesting that disease results from an interaction between bacterial and environmental factors in genetically susceptible hosts. Other digestive diseases, including celiac disease and inflammatory bowel disease, mimic this paradigm, where it appears that luminal triggers only manifest disease in subjects with the right combination of host and environmental factors. Since infection with H. pylori is relatively common, it is possible to study the impact of a specific etiologic agent on the pathogenesis of disease in humans. This approach has illuminated the complexity of the pathogenic mechanisms, but the advances achieved to date may provide some hints regarding the pathogenesis of chronic inflammatory diseases elsewhere in the digestive tract.

Macrophage- and neutrophil-dominant arthritis in human IL-1 alpha transgenic mice.

To study the effects of IL-1 alpha in arthritis, we generated human IL-1 alpha (hIL-1 alpha). Transgenic mice expressed hIL-1 alpha mRNA in various organs, had high serum levels of hIL-1 alpha, and developed a severe polyarthritic phenotype at 4 weeks of age. Not only bone marrow cells but also synoviocytes from knee joints produced biologically active hIL-1 alpha. Synovitis started 2 weeks after birth, and 8-week-old mice showed hyperplasia of the synovial lining layer, the formation of hyperplastic synovium (pannus) and, ultimately, destruction of cartilage. Hyperplasia of the synovial lining was due to the accumulation of macrophage-like cells expressing F4/80 molecules. hIL-1 alpha was widely distributed in macrophage- and fibroblast-like cells of the synovial lining cells, as well as synovial fluid monocytes. T and B cells were rare in the synovial fluid, and analysis of marker expression suggests that synoviocytes were directly histolytic and did not act as antigen-presenting cells. In the joints of these mice, we found elevated levels of cells of the monocyte/macrophage and granulocyte lineages and of polymorphonuclear neutrophils (PMNs), most of which expressed Gr-1, indicating that they were mature, tissue-degrading PMNS: Cultured synoviocytes and PMNs from these animals overexpress GM-CSF, suggesting that the hematopoietic changes induced by IL-1 and the consequent PMN activation and joint destruction are mediated by this cytokine.

Telomere reduction in human liver tissues with age and chronic inflammation.

Telomere shortening in human liver with aging and chronic inflammation was examined by hybridization protection assay using telomere and Alu probes. The reduction rate of telomere repeats in normal liver (23 samples from patients 17-81 years old) was 120 bp per year, which is in good agreement with the reported reduction rate in fibroblasts of 50-150 bp at each cell division and replacement rate of human liver cells, once a year. Mean telomere repeat length shortened to about 10 kbp in normal livers from 80-year-old individuals. The number of telomere repeats in chronic hepatitis (26 samples) and liver cirrhosis (11 samples) was significantly lower than that in normal liver of the same age (P < 0. 01). Telomere length in all these chronic liver disease samples, other than two exceptions, was not reduced shorter than 5 kbp, which was assumed to give a limit of proliferation (Hayflick's limit) to untransformed cells.

Expression of telomerase component genes in hepatocellular carcinomas.

The aim of the study was to clarify the role of telomerase component genes in hepatocarcinogenesis and to examine both the relationship between the expression of telomerase component genes and histological differentiation in hepatocellular carcinoma (HCC) and the relationship between expression levels of telomerase component genes and telomerase activity in HCCs. Telomerase is a ribonucleoprotein enzyme composed of a template RNA and several proteins. Recently, three such telomerase component genes have been identified: human telomerase reverse transcriptase (hTERT); human telomerase RNA component (hTERC); and telomerase-associated protein 1 (TEP1). The expression of these components was evaluated in 34 HCCs and 24 non-cancerous liver tissues by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of hTERT mRNA was detected in most HCCs, but not in the non-cancerous tissues (P<0.01). Expression of hTERC was detected in both HCCs and non-cancerous tissues, but the expression level in HCCs was higher than that in non-cancerous tissues (P<0.01) and tended to increase as histological differentiation became less marked. The expression level of hTERT mRNA correlated with relative telomerase activity (P<0.01). These results suggest that telomerase reactivation during hepatocarcinogenesis might be regulated by only hTERT and an increase in telomerase activity level in tumour progression might be regulated by both hTERT and hTERC.

Staphylococcal enterotoxin B stimulates expansion of autoreactive T cells that induce apoptosis in intestinal epithelial cells: regulation of autoreactive responses by IL-10.

T cell responses to self Ags and normal microbial flora are carefully regulated to prevent autoreactivity. Because IL-10-deficient mice develop colitis, and this response is triggered by luminal flora, we investigated whether IL-10 regulates the ability of microbial Ags to induce autoreactive T cells that could contribute to intestinal inflammation. T cells from wild-type mice were primed with staphylococcal enterotoxin B (SEB) in vitro, which induced an autoreactive proliferative response to syngeneic feeder cells. The cells were predominately CD3+ and CD4+. T cells from IL-10-deficient mice were constitutively autoreactive, and SEB priming enhanced this further. The autoreactive, proliferative response of T cells from wild-type mice was suppressed by IL-10 in the primary or secondary culture, and this effect was inhibited by neutralizing Abs to the IL-10R. To confirm that an autoreactive repertoire was expanded after SEB priming, we used CBA/J mice (Mls-1a) in which autoreactive T cells recognizing the endogenous viral superantigen are depleted (Vbeta6, 7, 8.1 TCR-bearing cells). However, SEB rescued these autoreactive T cell repertoires. Adding anti-MHC class II Ab blocked the autoreactive response. SEB-primed splenic or colonic T cells also induced apoptosis in syngeneic intestinal epithelial cells that was blocked significantly by IL-10. Thus, microbial Ags have the potential to abrogate self tolerance by stimulating autoreactive T cells that become cytolytic to target cells. IL-10 plays a protective role in maintaining self tolerance after microbial stimulation by preventing the activation of T cells that contribute to epithelial cell damage.

Circulating autoantibodies against purified colonic mucin in ulcerative colitis.

To clarify the role of colonic mucin in the autoimmune process of ulcerative colitis, circulating antibodies against human colonic mucin were investigated. Purified colonic mucin, obtained from human colonic mucosa by gel filtration, using a Bio-Gel A-1.5-m column and CsCl equilibrium density gradient, was divided into soluble mucin (S-mucin) secreted extracellularly and membranous mucin (M-mucin) binding to cell membrane. Sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting analysis showed that antibodies in the serum samples of some patients with ulcerative colitis recognized purified S- and M-mucin of >180-kD. By enzyme-linked immunosorbent assay (ELISA), anti-mucin antibodies were detected in 11 of 60 patients with ulcerative colitis (18%). In contrast, the antibodies were not detected in 22 patients with Crohn's disease. The titers of antimucin antibodies against S-mucin and M-mucin were not different in each patient. By ELISA using mucin in which the sugar chains were destroyed by neuraminidase or NaIO4 treatment, it was demonstrated that anti-mucin antibodies recognized the epitopes of either the sugar chain or the core protein exposed through destruction of the sugar chains. We then investigated the relationship between anti-mucin antibodies and the patients' clinical features. Anti-mucin antibodies were detected in 6 of 15 patients with chronic continuous type ulcerative colitis (40%) and in 5 of 26 patients with relapsing-remitting type (19%), but there was no antimucin antibody-positive serum in patients who had had only one attack without any relapse. These results suggest that anti-mucin antibodies could be a disease marker for ulcerative colitis and that immunological abnormalities in colonic mucin contribute to the persistence of colonic mucosal inflammation.

Precancerous hepatic nodules had significant levels of telomerase activity determined by sensitive quantitation using a hybridization protection assay.

BACKGROUND: Telomeric repeat amplification protocol using internal telomerase assay standard (ITAS) (conventional TRAP) has detected telomerase activity in various malignant tumors. With conventional TRAP, it is difficult to differentiate quantitatively low levels of telomerase activity between well-differentiated hepatocellular carcinomas (HCCs) and dysplastic nodules because of quantitative limitation. To apply a telomerase assay for differential diagnosis, we used a hybridization protection assay combined with TRAP (TRAP/HPA). This combination had better sensitivity and wider linearity than conventional TRAP. METHODS: TRAP/HPA was applied for quantitative measurement of telomerase activity in various hepatic tissues. Telomerase activity was evaluated in 10 precancerous hepatic nodules, 17 well-differentiated HCCs, 19 moderately differentiated HCCs, 5 poorly differentiated HCCs, 22 nontumorous chronic hepatic disease samples, and 2 normal liver tissues. RESULTS: Telomerase activity in HCCs tended to increase according to the malignant transformation. The average relative telomerase activity in 0.6 microg protein, which was expressed as cell equivalent activity of MKN-1, a gastric carcinoma cell line, was 8.5 in precancerous hepatic nodules, 87 in well-differentiated HCCs, 265 in moderately differentiated HCCs, 447 in poorly differentiated HCCs, and 0.4 in nontumorous hepatic tissues, including chronic liver diseases. CONCLUSIONS: TRAP/HPA was sensitive enough to distinguish the telomerase activity in precancerous hepatic nodules from that in other lesions. Telomerase activity in precancerous hepatic nodules was higher than that in nontumorous hepatic tissues. However, the activity in precancerous hepatic nodules was lower than that in well-differentiated HCCs, although statistically not significant. The authors suggest that precancerous hepatic nodules with telomerase activity above the diagnostic cutoff level (twice the highest activity in nontumorous hepatic tissues, or the 2 cell equivalent activity of MKN-1) should be treated as malignancy.

Opposing effects of protein kinase C delta and protein kinase B alpha on H(2)O(2)-induced apoptosis in CHO cells.

H(2)O(2)-induced apoptosis was enhanced in the CHO cell line overproducing protein kinase C delta (PKCdelta) as judged by DNA fragmentation. In response to the H(2)O(2) treatment, PKCdelta was tyrosine phosphorylated and recovered as a constitutively active form, but its proteolytic fragment was not generated. In contrast, H(2)O(2)-induced apoptosis was suppressed in the CHO cell line overexpressing protein kinase B alpha (PKBalpha). Consistently, phosphorylation of BAD, a pro-apoptotic protein negatively regulated by PKBalpha, was sustained in the cells overproducing PKBalpha, but was not changed in the cells overexpressing PKCdelta. In the CHO cell line overproducing both PKCdelta and PKBalpha, H(2)O(2)-induced tyrosine phosphorylation of PKCdelta was suppressed, and DNA fragmentation was diminished concomitantly. These results suggest that PKCdelta contributes to H(2)O(2)-induced apoptosis by a mechanism independent of BAD and that PKCdelta is a target of PKB for the regulation of cell survival.

Regulation of nuclear translocation of forkhead transcription factor AFX by protein kinase B.

The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX was phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB had been replaced by Ala was not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induced transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites had been replaced by Ala was detected exclusively in the cell nucleus. AFX-GFP was in the nucleus when the cells were infected with an adenovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stayed in the cytoplasm when the cells expressed constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation was induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppressed this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhanced DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB.

[Telomere length and telomerase activity in hepatocellular carcinoma].

Telomerase activity and terminal restriction fragment (TRF) length were examined in hepatocellular carcinoma (HCC). Telomerase activity was assayed by telomeric repeat amplification protocol (TRAP) connected with an internal telomerase assay standard (ITAS). The incidence of strong telomerase activity (highly variable level compared with the activity of non-cancerous liver tissue) was 79% in well, 84% in moderately, and 100% in poorly differentiated HCC, while 0% in non-cancerous liver tissues. The incidence of TRF length alteration (reduction or elongation) was 53% in HCC. The incidence of TRF alteration was significantly higher in HCC exceeding 3 cm in diameter, moderately or poorly differentiated in histology. Telomerase activity was not associated with TRF length alteration in HCC. In conclusion, strong telomerase activity and TRF length alteration increased with HCC tumor progressions.