Hideyuki Fusamoto

Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy

BACKGROUND Interferon alfa is effective for controlling disease activity in chronic hepatitis C. However, many responders suffer relapse after cessation of therapy. In the present study, the serum concentration of hepatitis C virus RNA was correlated with a sustained response to interferon therapy. METHODS Fifty-three patients with chronic hepatitis C received a 26-week course of interferon alfa. Hepatitis C virus RNA was quantitated in serum at the beginning and end of treatment using a competitive assay that combined reverse transcription and polymerase chain reaction. RESULTS In long-term responders, whose alanine aminotransferase levels remained within the normal range during the 24 weeks after therapy, the titer of viral RNA (logarithmic transformed copy numbers per milliliter of serum) before therapy (7.1 +/- 1.2) was significantly lower (P < 0.001) than that of short-term responders (8.3 +/- 0.5) who had a relapse within the 24 weeks after therapy and nonresponders (8.1 +/- 0.4). Multivariate multiple logistic regression showed that the titer of viral RNA before therapy was the strongest independent predictor of a sustained response to interferon-alfa therapy (P = 0.002). CONCLUSIONS The titer of hepatitis C virus RNA is the most important factor influencing the sustained response to interferon treatment.

Predicting interferon therapy efficacy from hepatitis C virus genotype and RNA titer

We classified 53 Japanese patients with chronic hepatitis C who were treated with natural interferon-α into genotypes and also tested the amounts of hepatitis C virus (HCV) RNA. The rate of the long-term complete response group, whose alanine aminotransferase levels remained within the normal range during the six months after therapy, was significantly higher (P<0.01) in the type-III patients (4/5, 80.0%) than in type-II patients (4/43, 9.3%). For these long-term complete responders, the amounts of HCV RNA was less than 107 copies/ml serum in type-II patients, whereas two type-III patients with relatively high amounts of HCV RNA responded completely. These results confirm that the genotype of HCV is an important factor for predicting the response to interferon therapy. The amounts of HCV RNA can also predict its efficacy in type-II patients.

Circulating matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 as serum markers of fibrosis in patients with chronic hepatitis C : relationship to interferon response

BACKGROUND/AIMS/METHODS The imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to be an important determinant of extracellular matrix deposition and breakdown. We measured serum MMP-1, MMP-2, TIMP-1 and TIMP-2 levels using the respective one-step sandwich enzyme immunoassays in 98 patients with chronic hepatitis C treated with interferon beta to examine their clinical significance for assessment of liver histology and to determine whether they can be useful as predictors of the interferon response. RESULTS Serum TIMP-1 levels showed a positive correlation with the degree of fibrosis (r(s)=0.30, p= 0.004). Serum MMP-2 levels revealed positive relationships with the degree of periportal necrosis (r(s)= 0.32, p=0.002), the degree of fibrosis (r(s)=0.26, p= 0.01) and total score of histological activity index (r(s)=0.24, p=0.02). Serum MMP-2 levels were significantly higher in patients with no response than in those with sustained and transient response (p<0.01 and p<0.05, respectively), while serum MMP-1 levels did not differ among the three groups. Compared with the levels in sustained responders, the total amounts of serum TIMP-1 were significantly lower in transient responders and non-responders (p<0.01 and p<0.001, respectively). As for serum TIMP-2 levels, a significant decrease was found in transient responders and non-responders (p<0.01). The ratios of serum MMP-2 to TIMP-1 levels were significantly higher in transient responders and non-responders than in sustained responders (p<0.001, respectively) even when HCV RNA levels were low in patients with HCV genome subtype 1b or when the HCV genome subtype was 2a or 2b. Sustained response was never found in type 1b patients with ratios of serum MMP-2 to TIMP-1 levels of over 6.0. In logistic multivariate regression analysis, the ratios of serum MMP-2 to TIMP-1 level (p=0.0001), HCV genome subtype (p=0.005) and serum TIMP-2 level (p=0.03) were the independent predictors for sustained response, while serum MMP-2 level (p=0.0006) was the only predictor for no response. CONCLUSIONS Serum MMP-2 and TIMP-1 levels might be useful for estimating the degree of liver fibrosis. The ratio of serum MMP-2 to TIMP-1 levels may serve as a new predictor of interferon response in patients with chronic hepatitis C.

RGM1, A CELL LINE DERIVED FROM NORMAL GASTRIC MUCOSA OF RAT

The epithelium of gastric mucosa undergoes constant renewal. However, the mechanisms of regulation of epithelial cell proliferation and differentiation in the gastric mucosa are poorly understood. Many investigators have used primary cultures of gastric mucosal cells to investigate cell physiology of gastric mucosal epithelium (5,8,14). Because this technique resulted in mixed cell populations of the stomach (5,15), the results may be influenced by complex cell-tocell interactions and are difficult to interpret. For this reason, a cell line made of a single type of cells is more ideal for precise investigation of proliferation or differentiation of gastric mucosal cells. However, cell lines from normal gastric mucosa have not been established. Recently, Matsui and Ohno (in preparation) established a new cell line, RGM1 (rat gastric mucosal cell first), from a rat glandular stomach by a method modified from Matsuoka et ah (7). In brief, the stomach was harvested from an anesthetized Wistar rat aged 4 wk and inverted. After washing the mucosa with phosphate-buffered saline (PBS) at 4 ~ C, the inverted pouch was immersed in a 0.2% pronase E solution at 37 ~ C. The solution was changed every 15 rain and centrifuged to collect the exfoliated gastric cells. Thereafter, the c.ells were washed twice with PBS, and cultivated in a 1:1 mixture of Dulbeccos modified Eagles medium (DMEM) and Hams F-12 medium supplemented with 20% fetal calf serum (FCS). When the cells were passaged to the 10th generation, this cell line was named RGM1. We examined character and usefulness of RGM1 cells using 3 0 4 0 times passaged cells. RGM1 cells are homogeneous epitheliallike cells with large oval nuclei and a polygonal shape (Fig. 1 A). They grow as monolayers of closely opposed cells, and many granules are visible in the cytoplasm. These cells were stained with periodic acid-Schiff (PAS) reagent and were negative for Bowie staining for chief cells (3) and succinic dehydrogenase activity for parietal cells (9). The cells were positive for cytokeratin in cytoplasm by immunohistochemistry. Transmission electron microscopy demonstrated that RGM1 cells had some microvilli on the surface. In the cytoplasm, a few homogeneous, dense cytoplasmic granules, presumably secretory granules, were observed. Junctional complexes were seen at the cell boundaries (Fig. 1 B and C). These data indicate that RGM 1 cells are epithelial in origin and like gastric mucous epithelial cells or mucous neck cells. RGM1 cells grew in an exponential manner with a population doubling time of 15.7 h, with saturation density of 1.97 + 0.38 X

Evidences for involvement of cyclooxygenase-2 in proliferation of two gastrointestinal cancer cell lines.

Cyclooxygenase (COX) consists of two isozymes, COX-1 and COX-2. The roles of these isozymes in the gastrointestinal tract are unknown. We investigated messenger RNA expression of the COX-1 and COX-2 genes in the gastrointestinal cancer cell lines MKN28, MKN45, KATO III CACO-2, DLD-1 and LoVo. These cell lines expressed comparable levels of COX-1 mRNA, although their expression of COX-2 varied. Therefore, we studied the effects of NS-398 and indomethacin, specific and non-specific inhibitors for COX-2, on proliferation of the cell lines. Both of the inhibitors suppressed proliferation of the two cell lines that highly expressed COX-2 (MKN45 and CACO-2). However, these inhibitors exerted minimal effects on proliferation of the other cell lines, which expressed significantly lower levels of COX-2. Therefore, it was proposed that COX-2 participates in proliferation of cancer cells because of over expression of the COX-2 gene.

Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy

BACKGROUND/AIMS Hepatitis C virus (HCV) genome heterogeneity by sequence analysis in association with interferon (IFN) inefficacy has been reported. This study was performed to establish a convenient method for detecting the HCV quasispecies complexity and to determine the correlation between the complexity and the responsiveness to IFN therapy in patients with chronic hepatitis C. METHODS The quasispecies complexity of HCV hypervariable region 1 in patients treated with IFN-alpha was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism (SSCP). RESULTS Seven of 25 patients (28%) with low complexity (SSCP band number of < or = 2) were HCV RNA negative after treatment, whereas in 24 patients with high complexity (SSCP band number of > or = 3), the response to IFN was almost insignificant because only 1 patient (4.5%) remained HCV RNA negative after treatment (P < 0.05). Among type 1b patients, IFN therapy was only effective for patients with low amounts of HCV RNA (< or = 10(7.5) copies/mL serum) and low complexity. In contrast, most type 2a patients tended to respond to the therapy with exceptions being those with high amounts of HCV RNA and high complexity. CONCLUSIONS The complexity of the hypervariable region 1 quasispecies may be a factor for predicting IFN inefficacy in patients with chronic hepatitis C.

Gastric Mucosal Hemodynamics after Thermal or Head Injury: A Clinical Application of Reflectance Spectrophotometry

In order to elucidate the role of mucosal blood flow in stress ulceration in critically ill patients with thermal or head injury, we applied reflectance spectrophotometry to the human gastric mucosa. During gastrofiberscopy, the spectra of the corpus and antral gastric mucosa were taken using a flexible coaxial optic bundle and a computer-equipped spectrophotometer. The subjects were 27 patients with thermal or head injury, and they were compared with 8 young healthy volunteers and 9 age-matched patients who had GI symptoms but showed no gastric lesion endoscopically. In 8 patients whose gastric mucosal blood volume at the time of admission decreased to about 27% of the level of young healthy volunteers or 36% of that of age-matched controls, the acute gastric mucosal lesions appeared in the corpus mucosa mostly within a few days after the measurement of mucosal blood volume. In contrast, in cases with thermal or head injury and without acute gastric mucosal lesions, the mucosal blood volume did not decrease significantly as compared with that of the young healthy volunteers and age-matched controls. In conclusion, analysis of mucosal has revealed that ischemia in the gastric mucosa is the main cause of the acute gastric mucosal lesions in patients with thermal or head injury. Further, this study represented noninvasive methodology for the measurement of the gastric mucosal blood volume in patients with gastric diseases.

Hepatitis C virus genotype and RNA titer in the progression of type C chronic liver disease

Hepatitis C virus genotype and the amounts of circulating HCV RNA are the most important factors in determining the efficacy of interferon therapy for chronic hepatitis C. To clarify the correlation of these two factors to the progression of liver disease, we classified 148 Japanese patients with type C chronic liver disease into genotypes and also measured their HCV RNA titers (logarithmic transformed copy number/ml serum) by competitive reverse transcription-polymerase chain reaction. We found type II in 23 (76.7%) of 30 patients with chronic persistent hepatitis, 34 (79.1%) of 43 with chronic active hepatitis, 29 (72.5%) of 40 with cirrhosis and 30 (85.7%) of 35 with hepatocellular carcinoma. Thus, there was no significant difference in the prevalence of type II among the various stages of chronic liver disease. We also found the RNA titer to be significantly higher in patients with chronic active hepatitis (8.0 +/- 0.8) than in those with chronic persistent hepatitis (7.0 +/- 1.0, p < 0.001), and also those with cirrhosis (7.6 +/- 0.8, p < 0.05) or hepatocellular carcinoma (7.7 +/- 0.8, p < 0.05). When the titers were compared among genotypes, there was no significant difference between type II and III at any stage (type II vs. type III: chronic persistent hepatitis, 7.2 +/- 1.0 vs. 6.7 +/- 0.8; chronic active hepatitis, 8.1 +/- 0.7 vs. 7.8 +/- 1.0; cirrhosis, 7.7 +/- 0.8 vs. 7.8 +/- 0.7; hepatocellular carcinoma, 7.7 +/- 0.8 vs. 7.8 +/- 0.5). In conclusion, although genotype affects interferon therapy efficacy, it seems to have little influence on serum RNA levels and the progression of type C chronic liver disease.

Endogenous Nitric Oxide Modulates Ethanol-Induced Gastric Mucosal Injury in Rats

BACKGROUND/AIMS Endothelium-derived relaxing factor regulates vascular tone via vasodilation. The relative contribution of endogenous nitric oxide to the pathophysiology of ethanol-induced gastric mucosal microcirculatory disturbances was investigated in anesthetized rats. METHODS Macroscopic and microscopic gastric mucosal damage and gastric mucosal hemodynamics including blood flow and hemoglobin oxygen saturation (ISO2) were assessed by pretreatment with a specific NO synthase inhibitor, N omega-nitro-L-arginine (L-NNA), before and after intragastric administration of ethanol. RESULTS Pretreatment with L-NNA significantly increased macroscopic (7.7-fold) and microscopic damage caused by 30% ethanol. Concurrent administration of L-arginine, but not D-arginine, significantly reduced the increase in mucosal damage. Similar results were obtained with 60% ethanol. Pretreatment with L-NNA decreased both mucosal blood flow and ISO2 in the basal period and enhanced decreases in both mucosal blood flow (2.7-fold) and ISO2 (4.3-fold) induced by 30% ethanol compared with controls. Concurrent administration of L-arginine, but not D-arginine, significantly inhibited the effect of L-NNA on blood flow and ISO2 in the basal period as well as after intragastric administration of 30% ethanol. CONCLUSIONS Endogenous NO modulates ethanol-induced gastric mucosal injury through the regulation of gastric mucosal microcirculation.

Hepatitis C virus antibody and hepatitis C virus replication in chronic hepatitis B patients

We assessed hepatitis C virus infection in 156 chronic hepatitis B patients using second-generation hepatitis C virus antibody (anti-HCV). Active virus replication was further investigated in anti-HCV-positive cases by means of polymerase chain reaction assay for the detection of serum hepatitis C virus RNA. Anti-HCV prevalence was higher in patients negative for hepatitis B e antigen (HBeAg) (10/48, 21%) than in HBeAg-positive patients (10/108, 9%) (p < 0.05), and the reactivity (cut-off index) in anti-HCV enzyme-linked immunosorbent assay of the positive cases was significantly higher in HBeAg-negative patients (4.1 +/- 0.1) than in -positive ones (3.6 +/- 0.6) (p < 0.05). The prevalence of hepatitis C virus RNA in anti-HCV-positive cases was also higher in the HBeAg-negative group (9/10, 90%) than in the -positive group (3/10, 30%) (p < 0.01). Viremia was found in association with high reactivity in anti-HCV ELISA (cut-off index > 3.5) in both groups. Nine (90%) of 10 such cases were viremic in the HBeAg-negative group compared with three (43%) of seven in the HBeAg-positive group (p < 0.05). These results suggest that hepatitis C virus replication may be influenced by hepatitis B virus replicative states, indicating possible interference between hepatitis B and C viruses.