Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement.

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus.

Circulating cell-free DNA as a predictive marker for distant metastasis of hepatitis C virus-related hepatocellular carcinoma.

In a previous study, we showed that levels of cell-free DNA (cfDNA) were significantly higher in sera of patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) than in sera of non-HCC patients with HCV. To confirm this finding, we analysed serum cfDNA levels in a cohort of 96 patients with HCV-related HCC and in 100 HCV carriers without known HCC. Again we found that serum cfDNA levels were significantly higher in HCC patients than in HCV carriers (115.9+/-98.3 vs 34.4+/-40.4 ng ml(-1) (mean+/-s.d.), P<0.0001). Of 87 eligible patients who underwent curative hepatectomy, those with a high cfDNA level had a significantly shorter overall survival (OS) time than those in whom the cfDNA level was not high. Cox proportional hazards model showed the cfDNA level to be an independent prognostic factor for OS and cancer recurrence in distant organs. Our results suggest that the serum cfDNA level reflects the metastatic potential of HCV-related HCC and that it can be a useful predictive biomarker for distant metastasis after curative surgery.

Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology.

Procyclic forms of Trypanosoma brucei brucei remain and propagate in the midgut of tsetse fly where iron is rich. Additional iron is also required for their growth in in vitro culture. However, little is known about the genes involved in iron metabolism and the mechanism of iron utilization in procyclic-form cells. Therefore, we surveyed the genes involved in iron metabolism in the T. b. brucei genome sequence database. We found a potential homologue of vacuole protein sorting 41 (VPS41), a gene that is required for high-affinity iron transport in Saccharomyces cerevisiae and cloned the full-length gene (TbVPS41). Complementation analysis of TbVPS41 in DeltaScvps41 yeast cells showed that TbVPS41 could partially suppress the inability of DeltaScvps41 yeast cells to grow on low-iron medium, but it could not suppress the fragmented vacuole phenotype. Further RNA interference (RNAi)-mediated gene knock-down in procyclic-form cells resulted in a significant reduction of growth in low-iron medium; however, no change in growth was observed in normal culture medium. Transmission electron microscopy showed that RNAi caused T. b. brucei cells to have larger numbers of small intracellular vesicles, similar to the fragmented vacuoles observed in DeltaScvps41 yeast cells. The present study demonstrates that TbVPS41 plays an important role in the intracellular iron utilization system as well as in the maintenance of normal cellular morphology.

Expression level of valosin-containing protein (VCP) as a prognostic marker for gingival squamous cell carcinoma.

BACKGROUND: Valosin-containing protein (VCP) is associated with anti-apoptotic function and metastasis via activation of the nuclear factor-kappaB signaling pathway. In the present study, association of VCP expression with prognosis of gingival squamous cell carcinoma (GSCC) was examined. PATIENTS AND METHODS: VCP expression in 74 patients with GSCC (34 males and 40 females) with ages ranging from 42 to 85 (median 66) years was evaluated by immunohistochemistry, in which staining intensity in tumor cells was categorized as either weaker (level 1) or equal to/stronger (level 2) than that in the endothelial cells. RESULTS: Twenty-four (32.4%) cases showed level 1 and 50 (67.6%) level 2 VCP expression. Patients with level 1 GSCC showed a significantly better 5-year survival rate than those with level 2 GSCC (5-year overall survival: 100% versus 84.9%, P < 0.05). Multivariate analysis revealed VCP expression level, lymph node metastasis and pT(TNM) to be independent factors for overall survival. Patients with GSCC at stages I and II showed favorable prognosis regardless of VCP expression status, whereas at stages III and IV, patients with level 1 VCP expression showed better survival rates than those with level 2 expression. CONCLUSION: Prognostic significance of VCP expression level in GSCC was demonstrated.

Nm23-H1 gene as a molecular switch between the free-floating and adherent states of gastric cancer cells.

The contribution of the nm23-H1 gene to metastasis in malignant tumors, including gastric cancer, is controversial. In this study, we compared nm23-H1 levels in two cell subtypes with different morphologies (floating and adherent states), but that were derived from the same gastric cancer cell line, KATO-III. A real-time quantitative reverse transcription-polymerase chain reaction showed that the number of nm23-H1 mRNA molecules in floating cells was significantly higher than that in adherent cells (P<0.0001). The average of the copies in floating cells was approximately 2.4-fold higher than that in adherent cells. Consistent with mRNA levels, intracellular levels of nm23-H1 protein were higher in floating cells than in adherent cells. There was no difference in cell cycle characteristics between the two subtypes. In conclusion, our present data indicate that expression of nm23-H1 by a tumor could be altered during the different steps in metastases, suggesting that nm23-H1 may act as a molecular switch between the free-floating and adherent states of cancer cells.

Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igkappa leader sequence.

IL-18 is a novel cytokine that induces interferon (IFN)-gamma secretion and plays an important role in antitumor immunity. In the present study, we constructed plasmid vectors encoding the murine mature IL-18 cDNA linked with the Igkappa leader sequence and the pro-IL-18 cDNA to estimate the efficacy of the mature IL- 18 vector and to evaluate IL-18--producing tumor cells as a tumor vaccine. Colon 26 cells were transfected with the abovementioned vectors or with vector alone (mock). Reverse transcription-polymerase chain reaction analysis showed increased expression of murine IL-18 cDNA in both mature IL-18 and pro-IL-18 transfectants in comparison to that in mock transfected cells. The ability of the culture supernatants of mature IL-18 transfectants to induce IFN-gamma secretion was extremely high (40-140 pg/10(6) cells) in comparison to that of pro-IL-18 transfectants (4-18 pg/10(6) cells). When injected into syngeneic BALB/c mice, the growth of mature IL-18 transfectants, but not pro-IL-18 transfectants, was significantly less than that in mock transfected cells ( P< .01, by ANOVA and analysis of covariance). In addition, injection of colon 26 or Meth-A cells into mice immunized with a mature IL-18 transfectant revealed acquired immunity. Depletion of natural killer cells did not affect the growth of transfectants. However, the growth inhibitory effects were partially abrogated following treatment with anti-CD4+ and anti-CD8+ antibodies. These data suggest that the rejection of mature IL-18/colon 26 cells was mediated through T-cell activation. Gene therapy using mature IL-18 transfectants containing a plasmid vector and the Igkappa leader sequence may be a useful tumor vaccine.

Detection of telomerase activity in peritoneal lavage fluid from patients with gastric cancer using immunomagnetic beads.

Cytologic examination of peritoneal lavage fluid is a useful predictor of peritoneal recurrence in gastric cancer. However, this technique is not overly sensitive and requires special abilities in the cytologist. In this study, telomerase activity was used to detect free cancer cells in peritoneal lavage fluid from patients with gastric cancer. In the first part, 12 lavage-fluid samples obtained from 12 patients with gastric cancer were analysed using the conventional telomeric repeat amplification protocol (TRAP) assay. Three of five patients with early gastric cancer had positive telomerase activity. These false-positive results may have been due to lymphocyte contamination. Furthermore, polymerase chain reaction inhibitors were also detected in the lavage-fluid samples. Therefore, we developed a novel method for elimination of haematopoietic cell and Taq polymerase inhibitors to increase the accuracy of the TRAP assay using immunomagnetic beads, which bind to most normal and neoplastic human epithelial cells. Telomerase activity was found in 10 of 20 (50%) lavage-fluid samples from patients with serosal or subserosal invasion. Cytologic examination was positive in nine of 20 (45%) samples. Both the telomerase activity and cytology were negative in all 14 patients without serosal or subserosal invasion. These results suggest that the TRAP assay combined with immunomagnetic beads might be useful for detection of free cancer cells in the peritoneal space in gastric cancer without the aid of an experienced cytologist.

Anticachectic effects of Coptidis rhizoma, an anti-inflammatory herb, on esophageal cancer cells that produce interleukin 6.

Herbs as alternative cancer therapies have attracted a great deal of recent attention due to their low toxicity and costs. In this study, the antitumor activity and anticachectic effect of Coptidis rhizoma, an anti-inflammatory herb, were investigated in nude mice carrying a human esophageal cancer cell line YES-2, which constitutively secretes interleukin-6 (IL-6) and induces cachexia when injected into these mice. In this study, in vivo growth of YES-2 cells was not affected by an oral supplement containing the extract powder of C. rhizoma at a final concentration of 1% (CR supplement). However, in comparison with normal diet, CR supplement significantly attenuated weight loss of tumor-bearing mice without a change in food or water intake. Tumor IL-6 levels were significantly lower in mice treated with CR supplement than in control mice (P<0.001). Serum IL-6 was detectable in four (50%) of eight control mice; IL-6 was not detected in mice treated with CR supplement. We also confirmed that berberine (8-32 microM), a major component of C. rhizoma, dose-dependently inhibited secretion of IL-6 by YES-2 cells in vitro. Moreover, reverse transcription-PCR assay showed that treatment of YES-2 cells with berberine (8-32 microM) for 24 h reduced IL-6 mRNA expression. Our results suggest that C. rhizoma may have an anticachectic effect on esophageal cancer and an effect is associated with the ability of berberine to down-regulate tumor IL-6 production.

HeLa cell transformants overproducing mouse metallothionein show in vivo resistance to cis-platinum in nude mice.

Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.

Inhibitory effect of Coptidis Rhizoma and berberine on the proliferation of human esophageal cancer cell lines.

Our previous study demonstrated that the herbal medicine, Oren-to, had antitumor effects on esophageal cancer cells (ECCs) in vitro. The purpose of this study was to examine which of the seven constituents of Oren-to had antitumor effects on esophageal cancer cells. MTT assay showed that, of the seven constituents, only the aqueous extract of Coptidis Rhizoma had potent inhibitory effect on the proliferation of two types of ECC lines, YES-3 and YES-4. In addition, the proliferation of all six types of ECC lines (YES-1 to YES-6) was inhibited in a dose-dependent manner (P<0.001 for all), when co-cultured at each concentration of Coptidis Rhizoma for 72 h. The ID50 of Coptidis Rhizoma for YES-1 to YES-6 was 2.2 microg/ml, 3.0 microg/ml, 0.25 microg/ml, 2.8 microg/ml, 2.5 microg/ml, and 0.5 microg/ml, respectively, berberine, one of protoberberine components of Coptidis Rhizoma, showed potent antitumor effects on all six types of ECC lines as well as Coptidis Rhizoma. In addition, the ID50 of berberine showed a positive correlation with that of Coptidis Rhizoma in six types of ECC lines examined (r2 = 0.763, P = 0.023). Cell cycle analysis of Coptidis Rhizoma-treated cancer cells showed the accumulation of cells in the G0/G1 phase and relative decrease of the S phase. These results support the possibility that the use of Coptidis Rhizoma containing abundant berberine may be useful as one of alternative therapies for esophageal cancers.

Immunoregulatory effects of the antitumor polysaccharide lentinan on Th1/Th2 balance in patients with digestive cancers.

BACKGROUND: Recent studies demonstrated that patients with advanced cancer may have impaired cell-mediated immunity caused by an imbalance between Th1 and Th2 responses. We evaluated the ability of lentinan (LNT) to modulate Th1 and Th2 responses in patients with digestive cancers. METHODS: Peripheral blood samples were collected preoperatively from 28 patients with digestive cancers before and after intravenous administration of LNT (2 mg x 3 times/week). The proportions of CD4+ T-cells producing intracellular cytokines were determined with flow cytometry. RESULTS: After LNT treatment, CD4+ IFN-gamma+ T-cell percentages increased significantly (p < 0.05), whereas CD4+ IL-4+ T-cell and CD4+ IL-6+ T-cell percentages decreased significantly (p < 0.02). No significant change occurred in proportions of CD4+ IL-10+ T-cells. The after/before LNT treatment percentages ratio of CD4+ IFN-gamma+ T-cells correlated negatively with that of CD4+ IL-4+ T-cells (p < 0.01). The after/before treatment percentage ratio of CD4+ IL-4+ T-cells correlated positively with that of CD4+ IL-6+ T-cells (p < 0.05). CONCLUSION: LNT apparently can cancel Th2-dominant condition in patients with digestive cancers and may improve the balance between Th1 and Th2.

[New hearing test for functional deafness--the procedure to estimate the genuine hearing threshold].

This report describes a new procedure for examining functional deafness with binaural sound stimulation. This new hearing test can estimate the genuine pure tone hearing threshold quantitatively at every frequency by using the ordinary audiometer. In the case of the nonorganic deafness, even if the hearing disorders are bilateral or hemilateral, we believe that measuring auditory threshold separately, causes the deterioration of the threshold. Therefore, this procedure is designed so that the subject may not be aware of testing the auditory acuity of each ear, and utilizes the response of the phantom sound image in the head by simultaneous binaural presentation of sound stimulation. Our strategy is based on the following facts. If the normal subject has the same pure tone threshold level in both ears, the phantom sound image is formed in the median plane of the head by the equal suprathreshold tone level presented simultaneously in each ear. In the case of a unilateral auditory disorder, the sound image is localized to the center of the head only when sound stimulation louder than the threshold level of affected ear is given to both ears at the same time. Simultaneous binaural sound stimulation at a lower level than the threshold of the affected ear forms a lateralized sound image to the unaffected ear in the head. For patients with bilaterally similar hearing loss, the sound image is not formed if the stimulation is less than the threshold level of the pure tone. The band noise in the phase of each frequency with 50 dB HL was given binaurally to 10 normal hearing subjects, and the localization of the sound image formation was examined. This experiment confirmed that around the occipital region of the median plane in all subjects. Furthermore, comparing the formation threshold of the median plane image with the pure tone auditory threshold, proved that there was no significant difference statistically in either value. As a next step, 15 patients with unilateral sensorineural deafness were examined with this technique and we knew that the median sound images would not be formed with stimulation less than the pure tone auditory threshold of the affected ear. For clinical application, patients were classified into two groups with unilateral (6) and bilateral (2) functional deafness, and examined. Midline sound images definitely were formed with the lower magnitude of sound than the pure tone threshold by hemilateral nonorganic deafness. The difference of the forming threshold of the median sound image and the average of pure tone hearing level of the affected ear were maximum 100 dB, minimum 35 dB, and mean 69.4 dB. Furthermore, the difference of the median image forming threshold and the average hearing level of the unaffected ear were maximum 35 dB, minimum 0 dB, and mean 15.4 dB. In bilateral disorders, the midline plane sound image was formed with the corresponding level of the pure tone value in one subject, though the other one was determined by the maneuver method because it did not form a midline sound image. In conclusion, this examination can be readily used to estimate the genuine hearing threshold of the functional deafness.

The nm23-H1 gene as a predictor of sensitivity to chemotherapeutic agents in oesophageal squamous cell carcinoma.

Recently, nm23-H1, an anti-metastasis gene, has been reported to correlate with sensitivity to chemotherapeutic agents including cisplatin in human breast and ovarian carcinoma cells. The aim of this study was to evaluate a role for nm23-H1 in responsiveness to cisplatin-based chemotherapy in patients with oesophageal squamous cell carcinoma (OSCC). The expression of nm23-H1 protein was examined immunohistochemically in 32 eligible patients with OSCC who underwent adjuvant chemotherapy with cisplatin, etoposide, and 5-fluorouracil after tumour resection. Fifteen (46.9%) of 32 patients were positive for nm23-H1 staining and 17 (53.1%) were negative. Both disease-free survival and overall survival rates of nm23-H1-negative patients were significantly shorter than in nm23-H1-positive patients (P < 0.01 for both). There was no significant difference in clinicopathologic characteristics between nm23-H1-positive and nm23-H1-negative groups. Multivariate analysis also showed that nm23-H1 expression was the most significant factor for overall survival of OSCC patients included in this study (P = 0.0007). To further study the role of nm23-H1, a human OSCC cell line (YES-2) was transfected with a plasmid containing a fragment of the nm23-H1 cDNA in an antisense orientation. Reduced expression of nm23-H1 protein in the antisense-transfected (AS) clones was found by Western blot analysis as compared to wild-type YES-2 and YES-2/Neo (clone transfected with the neomycin resistance gene alone). MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay showed that reduced expression of the nm23-H1 protein in AS clones was consistent with the degree of increased resistance to cisplatin but not etoposide or 5-fluorouracil. These data support the conclusion that reduced expression of nm23-H1 may be associated with resistance to cisplatin, suggesting the value of nm23-H1 expression as a prognostic marker for OSCC patients who are to undergo cisplatin-based chemotherapy.

Interleukin-1 receptor antagonist mRNA expression and the progression of gastric carcinoma.

Interleukin-1 receptor antagonist (IL-1ra), an endogeneous inhibitor of IL-1, plays an immunosuppressive role in vivo by blocking the proinflammatory effects of IL-1. In the present study, we examined whether IL-1ra expression in human gastric carcinoma correlates with tumor progression and/or metastatic potential. The reverse transcription-polymerase chain reaction was used to compare the expression of the secreted form of IL-1ra (sIL-1ra) and the intracellular form of IL-1ra (icIL-1ra) mRNA in tumor and corresponding benign tissue obtained from 38 patients with gastric carcinoma. The incidence of sIL-1ra mRNA expression was significantly higher in tumor (52%) than in corresponding benign tissue (18%) (P = 0.002). On the contrary, icIL-1ra mRNA was detected in all tumors and benign tissues. The expression of sIL-1ra mRNA by malignant tissue correlated positively with both lymph node metastasis (P = 0.008) and liver metastasis (P = 0.015). There was no association between tumor sIL-lra mRNA expression and other clinicopathologic factors. The degree of regional lymph node reaction, such as sinus histiocytosis, in tumors expressing sI-1ra mRNA was significantly weaker than that in tumors without sIL-1ra mRNA expression (5/20 vs. 12/18, P = 0.010). These results demonstrate that the altered expression of sIL-1ra by malignant tissue may be related to the progression of gastric carcinoma via modulating host immune response.

Tumour cells engineered to secrete interleukin-15 augment anti-tumour immune responses in vivo.

We examined the effect of interleukin-15 (IL-15) gene transfer into tumour cells on the host's anti-tumour response. In BALB/c mice IL-15 producing Meth-A cells (Meth-A/IL-15) underwent complete rejection, in a response characterized by massive infiltration of CD4+ T-cells and neutrophils. In contrast, Meth-A cells transfected with vector alone (Meth-A/Neo) grew rapidly. Moreover, rechallenged parental cells also were rejected in association with CD8* T-cell infiltration. However, in nude mice there was no drastic difference between Meth-A/IL-15 and Meth-A/Neo cells. These results demonstrate that IL-15-secreting tumour cells can stimulate local and systemic T-cell-dependent immunity and therefore may have a potential role in cancer therapy.

The association between nm23-H1 expression and survival in patients with esophageal squamous cell carcinoma.

The nm23 gene is a potential metastasis suppressor gene originally identified using a murine melanoma cell line. The expression of nm23-H1 protein was examined immunohistochemically in 50 eligible patients with esophageal squamous cell carcinoma (ESCC). The expression was not correlated with other prognostic factors including lymph node metastases; however, overall survival rates of nm23-H1-negative patients were significantly shorter than those of nm23-H1-positive patients (P < 0.05). Furthermore, reduced expression of nm23-H1 was associated with shorter overall survival in patients with involved lymph nodes (P < 0.01), but not in patients without involved lymph nodes. These data support the conclusion that reduced expression of nm23-H1 may be associated with poor prognosis of ESCC patients, suggesting the value of nm23-H1 expression as a prognostic marker for ESCC patients, especially ESCC patients with involved lymph nodes.

Case of gonadoblastoma in a 9-year-old boy without physical abnormalities.

BACKGROUND: A 9-year-old boy was admitted to Jikei University Hospital complaining of gradual enlarging of the left scrotal contents. METHODS/RESULTS: Physical examination was significant for bilateral descended testicles. No abnormalities were detected in the testicles or along the spermatic cords. Scrotal ultrasound showed that hyperechoic shadows were recognized in the central area of the left testicle. Subsequent testicular biopsy and histopathological examination showed intratubular malignant germ cells in the testicular tubules. One week later, left orchiectomy was performed. CONCLUSIONS: Histopathological evaluation revealed gonadoblastoma. Gonadoblastoma, a rare gonadal neoplasm, is composed of germ cells and sex cord derivatives and usually occurs in phenotypically female patients with gonadal dysgenesis. To date, only three cases of gonadoblastoma have been reported in anatomically normal male patients with scrotal testicles. We report on a case of gonadoblastoma unaccompanied by a germ cell tumor in a physically normal male.