Promoting notification and linkage of HBs antigen and anti-HCV antibody-positive patients through hospital alert system.

BACKGROUND: In Japan, approximately 0.9% and 1% of the whole population are infected with HBV and HCV, respectively. Doctors from departments other than gastroenterology often order viral hepatitis tests before an invasive examination or an operation. However, the notification of positive results to the patients and linkage to care is not appropriately performed. The in-hospital alert system was constructed to promote the notification and referral to gastroenterologists for patients with positive viral hepatitis tests, and its efficacy was evaluated. METHODS: The patients who tested HBsAg and anti-HCV antibody by chemiluminescent enzyme immunoassays and chemiluminescent immunoassays were investigated for whether they were notified of the positive results and if they were referred to gastroenterologists at our hospital. The notification and referral rate was compared before (from January to December 2014) and after the introduction of the alert system (from February to September 2016). RESULTS: HBsAg-positive rate was 1.1% (69/6543) before the introduction of the alert system and 0.8% (41/5403) after it. The notification rate has significantly improved from 46% to 73% (p = 0.0061) and the referral rate has improved from 16% to 27%, while not significant. Positive rate of anti-HCV antibody was 2.1% (139/6481) before the introduction of the alert system and 2.4% (128/5322) after it. The rate of notification and referral has significantly improved from 35% to 62% (p < 0.0001) and from 6% to 23% (p < 0.0001), respectively. CONCLUSIONS: The in-hospital alert system increased the rates of notification and referral of the patients with positive viral hepatitis tests. Enlightenment of doctors other than gastroenterologists on viral hepatitis and cooperation of medical staffs would be helpful to improve the notification and referral rates.

Virological characteristics of hepatitis B genotype G/A2 recombination virus in Japan.

AIM: We identified four cases of infection with hepatitis B virus genotype G and A2 recombinant (HBV/G/A2) strains, which were initially overlooked by enzyme immunoassay-based genotyping. The patients were all men who have sex with men (MSM) and inhabited several metropolitan areas of Japan, suggesting that the recombinant strains may be circulating among high-risk groups such as MSM. Here, we investigated the genomic structure and virological properties of the HBV/G/A2 strains. METHODS: Complete genome sequences of the isolates were determined and phylogenetically analyzed. Replication efficiency of HBV/G/A2 was investigated by transfecting plasmids containing 1.24-fold viral genome. The in vivo viral kinetics of HBV/G/A2 were investigated using chimeric mice with humanized livers. RESULTS: Phylogenetic analysis revealed that the four strains were almost identical (>99.7% homologous). The preS2/S region of these strains was highly homologous to that of genotype A2 and the remaining region was almost identical to that of genotype G, reflecting inter-genotypic recombination. Interestingly, in all four cases, genotype A was co-infected as a minor population. In vitro analysis revealed that HBV/G/A2 had a low replication rate. Although detectable viremia was not measurable following the inoculation of HBV/G/A2 into chimeric mice, subsequent superinfection of HBV genotype A greatly enhanced HBV/G/A2 replication and viral spread. CONCLUSION: We found that four cases of HBV/G/A2 recombinant among MSM patients in the metropolitan areas of Japan, and HBV/A co-infections are required for its efficient replication. High-risk groups such as MSM should be carefully tested for infection of genotype G-derived variants.

Real-time air monitoring of mustard gas and Lewisite 1 by detecting their in-line reaction products by atmospheric pressure chemical ionization ion trap tandem mass spectrometry with counterflow ion introduction.

A new method enabling sensitive real-time air monitoring of highly reactive chemical warfare agents, namely, mustard gas (HD) and Lewisite 1 (L1), by detecting ions of their in-line reaction products instead of intact agents, is proposed. The method is based on corona discharge-initiated atmospheric pressure chemical ionization coupled with ion trap tandem mass spectrometry (MS(n)) via counterflow ion introduction. Therefore, it allows for highly sensitive and specific real-time detection of a broad range of airborne compounds. In-line chemical reactions, ionization reactions, and ion fragmentations of these agents were investigated. Mustard gas is oxygenated in small quantity by reactive oxygen species generated in the corona discharge. With increasing air humidity, the MS(2) signal intensity of protonated molecules of mono-oxygenated HD decreases but exceeds that of dominantly existing intact HD. This result can be explained in view of proton affinity. Lewisite 1 is hydrolyzed and oxidized. As the humidity increases from zero, the signal of the final product, namely, didechlorinated, dihydroxylated, and mono-oxygenated L1, quickly increases and reaches a plateau, giving the highest MS(2) and MS(3) signals among those of L1 and its reaction products. The addition of minimal moisture gives the highest signal intensity, even under low humidity. The method was demonstrated to provide sufficient analytical performance to meet the requirements concerning hygienic management and counter-terrorism. It will be the first practical method, in view of sensitivity and specificity, for real-time air monitoring of HD and L1 without sample pretreatment.

Sensitive and comprehensive detection of chemical warfare agents in air by atmospheric pressure chemical ionization ion trap tandem mass spectrometry with counterflow introduction.

A highly sensitive and specific real-time field-deployable detection technology, based on counterflow air introduction atmospheric pressure chemical ionization, has been developed for a wide range of chemical warfare agents (CWAs) comprising gaseous (two blood agents, three choking agents), volatile (six nerve gases and one precursor agent, five blister agents), and nonvolatile (three lachrymators, three vomiting agents) agents in air. The approach can afford effective chemical ionization, in both positive and negative ion modes, for ion trap multiple-stage mass spectrometry (MS(n)). The volatile and nonvolatile CWAs tested provided characteristic ions, which were fragmented into MS(3) product ions in positive and negative ion modes. Portions of the fragment ions were assigned by laboratory hybrid mass spectrometry (MS) composed of linear ion trap and high-resolution mass spectrometers. Gaseous agents were detected by MS or MS(2) in negative ion mode. The limits of detection for a 1 s measurement were typically at or below the microgram per cubic meter level except for chloropicrin (submilligram per cubic meter). Matrix effects by gasoline vapor resulted in minimal false-positive signals for all the CWAs and some signal suppression in the case of mustard gas. The moisture level did influence the measurement of the CWAs.

Sensitive monitoring of volatile chemical warfare agents in air by atmospheric pressure chemical ionization mass spectrometry with counter-flow introduction.

A new method for sensitively and selectively detecting chemical warfare agents (CWAs) in air was developed using counter-flow introduction atmospheric pressure chemical ionization mass spectrometry (MS). Four volatile and highly toxic CWAs were examined, including the nerve gases sarin and tabun, and the blister agents mustard gas (HD) and Lewisite 1 (L1). Soft ionization was performed using corona discharge to form reactant ions, and the ions were sent in the direction opposite to the airflow by an electric field to eliminate the interfering neutral molecules such as ozone and nitrogen oxide. This resulted in efficient ionization of the target CWAs, especially in the negative ionization mode. Quadrupole MS (QMS) and ion trap tandem MS (ITMS) instruments were developed and investigated, which were movable on the building floor. For sarin, tabun, and HD, the protonated molecular ions and their fragment ions were observed in the positive ion mode. For L1, the chloride adduct ions of L1 hydrolysis products were observed in negative ion mode. The limit of detection (LOD) values in real-time or for a 1 s measurement monitoring the characteristic ions were between 1 and 8 µg/m(3) in QMS instrument. Collision-induced fragmentation patterns for the CWAs were observed in an ITMS instrument, and optimized combinations of the parent and daughter ion pairs were selected to achieve real-time detection with LOD values of around 1 µg/m(3). This is a first demonstration of sensitive and specific real-time detection of both positively and negatively ionizable CWAs by MS instruments used for field monitoring.

[Different sensitivity to chemotherapy between adenocarcinoma and adenosquamous carcinoma in a case of synchronous multicentric gastric cancer].

A 60-year-old man presented with upper gastrointestinal bleeding. We diagnosed double gastric cancer (adenocarcinoma and adenosquamous carcinoma) based on an endoscopic examination. Due to uncontrollable bleeding, total gastrectomy was performed after 4 courses of chemotherapy with S-1+cisplatin. Histological investigation revealed that no obvious anti-cancer effect was observed in adenosquamous carcinoma (Grade 1), while tumor cells were eliminated in the area of adenocarcinoma (Grade 3). This case clearly demonstrated that sensitivity to chemotherapy was different between adenocarcinoma and adenosquamous carcinoma of the stomach.

Inflammatory cytokines modulate chemokine production patterns of HepG2 cells toward initially inclined direction.

AIM: Human hepatocytes are known to express an array of inflammatory cytokines and chemokines. In this study, we examined the potential roles of hepatocytes in regulating immune responses in the liver, by assessing the induction of Th1- or Th2-specific chemokines in HepG2 cells after various inflammatory stimulations. METHODS: HepG2 cells were stimulated with IL-1alpha, IFN-gamma, IL-4, IL-10, and/or CCL2, harvested at several time points, and served for the analyses of cytokine/chemokine mRNA expressions by semi-quantitative RT-PCR. RESULTS: (i) IL-1alpha up-regulated mRNA levels of CXCL8, CXCL10, and CCL2. IFN-gamma increased those of CXCL9, CXCL10, and CCL5, while IL-4 or IL-10 had no effect. (ii) Addition of IL-4 to the culture of IFN-gamma-stimulated cells, down-regulated CXCL9 and CXCL10 mRNA levels. (iii) Addition of IFN-gamma to the culture of IL-1alpha-stimulated cells, further up-regulated CXCL9 and CXCL10 mRNA levels. Addition of IL-4 decreased CXCL8 and CXCL10 levels, and increased CCL2 level in IL-1alpha-stimulated cells. (iv) CCL2 induced IL-4 mRNA expression. CONCLUSIONS: IFN-gamma augmented mRNA expression of Th1-specific chemokines (CXCL9 and CXCL10) in HepG2 cells. IL-4 had no effect on those of Th2-spesific chemokines (CCL17 and CCL22); however, it was supposed to augment Th2 response indirectly through the induction of CCL2 under the inflammatory condition. The findings suggest that hepatocytes have ability to promote immune responses in the liver toward the direction, initially determined by the cytokine balances in the local inflammatory region.

Usefulness of radiofrequency ablation with micro-convex probe for hepatocellular carcinoma.

BACKGROUND/AIMS: It was aimed to assess whether a micro-convex probe is superior to the present conventional probe for ultrasonography from the points of safety and efficacy during percutaneous radiofrequency ablation therapy for hepatocellular carcinoma. METHODOLOGY: Twenty-one patients with 23 hepatocellular carcinoma lesions who had one or 2 lesions, each 4 cm or less in diameter, and liver function of Child-Pugh class A or B were enrolled. All the patients except for 2 patients were seropositive for hepatitis C virus. Radiofrequency ablation was carried out under a real-time US guidance. The cooled-tip electrodes used were single and clustered. RESULTS: It was possible to perform safe and accurate percutaneous radiofrequency ablation procedure using micro-convex probes for the treatment of all hepatocellular carcinoma nodules. It was also possible to treat hepatocellular carcinoma located in the right subphrenic region without artificial pleural effusion under intercostal ultrasonography guide. Improved clustered needles were successfully applied to treat the nodules more than 3 cm in diameter with less resistance for penetration compared with the conventional needle. The findings of advanced dynamic flow image on ultrasonography to assess the therapeutic efficacy indicated the consistency with those of dynamic CT which was done 3 to 5 days later radiofrequency ablation. Major complication of radiofrequency ablation procedure was noted in none. CONCLUSIONS: These results suggest that micro-convex probe with clustered tips is superior to conventional probe for ultrasonography from the points of safety and efficacy during radiofrequency ablation for hepatocellular carcinoma nodule located in the right subphrenic region and for larger sized nodule more than 3 cm.

[Acute pancreatitis associated with the administration of ceftriaxone in an adult patient].

We report a case of a 35-year-old patient with acute pancreatitis after administration of ceftriaxone. She was given ceftriaxone (2g/day) for 9 days because of diverticulitis of the colon. She was admitted to our hospital again because of epigastralgia 12 days after the first administration of ceftriaxone. Laboratory examination showed markedly elevated serum amylase, and CT scan demonstrated findings consistent with acute pancreatitis, in addition to sludge in the common bile duct and gall bladder, which was not identified before the administration of ceftriaxone. We should be aware of the fact that administration of ceftriaxone sometimes results in the formation of biliary sludge and can cause severe adverse events such as cholecystitis and pancreatitis, not only in children, but also in adult patients.

Alanine Aminotransferase as the First Test Parameter for Wilson's Disease.

Background and Aims: The liver is the first organ affected by toxic copper in the classical and severe hepatic forms of Wilson's disease (WD). Because their associated chronic liver damage is mostly asymptomatic, an intervention using a special test including serum alanine aminotransferase (ALT) activity is needed for detecting WD. Methods: Using the modified international criteria for the diagnosis of WD, 45 patients were selected from the collective databases of our institutions, and 7 infants were reviewed from the literature. Two patients had the severe hepatic form, with normoceruloplasminemia and no mutations in ATP7B. The rapid ALT change during hemolytic anemia was adjusted for a baseline. The diagnostic potential of the ALT test was assessed from the age-dependent natural course of the liver damage of WD. Results: The natural course had three stages. ALTs were still low in some infants younger than 4 years-old. They were high in all children between the ages of 4 and 8 years-old; then, they reduced to low levels in some patients over 9 years of age. The high ALT stage represents chronic active hepatitis, and the subsequent low ALT stage is due to silent cirrhosis. The hepatic copper content is a reliable but invasive test, while urinary copper secretion is an alternative, non-invasive test for copper toxicosis of WD. The serum ceruloplasmin and ATP7B analyses are subtype tests of WD. The response to anti-copper regimens is the final test result. Conclusions: ALT could be the first parameter to test to detect WD in children between the ages of 4 and 8 years.

Use of the Model for End-Stage Liver Disease (MELD) score to predict 1-year survival of Japanese patients with cirrhosis and to determine who will benefit from living donor liver transplantation.

BACKGROUND: Consideration of the prognosis of patients with liver cirrhosis is important when determining the appropriate timing of liver transplantation. Especially in Japan, where 99% of liver transplants are from living donors, timing is very important not only for the patient but also for the family, who need time to consider the various factors involved in living donations. METHODS: To clarify the applicability of the Model for End-Stage Liver Disease (MELD) score in Japanese patients with cirrhosis, changes in the MELD score over 24 months were reviewed in 79 patients with cirrhosis who subsequently died of liver failure (n=33) or who survived 24 months (n=46). All patients had Child class B or C cirrhosis at the start of follow-up. We also compared their survival with that of 30 patients treated by living donor liver transplantation (LDLT) in our institute to determine the proper timing of transplantation in patients with cirrhosis. RESULTS: Significant stratification of survival curves was observed for MELD scores of <12, 12-15, 15-18, and >18 (P=0.0018). A significant survival benefit of LDLT was observed in patients with MELD score >or=15 (P=0.0181), and significantly more risk with transplantation was observed in those with MELD score <15 compared with that of patients in whom the disease followed its natural course (P=0.0168). CONCLUSIONS: MELD score is useful for predicting 1-year survival in Japanese patients with cirrhosis. MELD scores of 15 had discriminatory value for indicating a survival benefit to be gained by liver transplantation and thus can be used to help patients and their families by identifying patients who would benefit from LDLT.

Deficiency of forkhead box P3 and cytotoxic T-lymphocyte-associated antigen-4 gene expressions and impaired suppressor function of CD4(+)CD25(+) T cells in patients with autoimmune hepatitis.

AIM: Recently, forkhead box P3 (Foxp3), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR), and CD28 were identified as the key molecules that control the development and activation of CD4+CD25+ regulatory T cells (T-reg). We investigated the expression pattern of these molecules on T-reg, and investigated the ability of T-reg to produce cytokines in patients with autoimmune hepatitis (AIH). METHODS: Fifteen patients with AIH and nine healthy patients were included. To determine the frequency of T-reg, a two-color flow cytometry analysis was performed. T-reg were isolated using immunomagnetic beads, and the mRNA levels of Foxp3, CTLA-4, GITR, and CD28 were quantified by real-time polymerase chain reaction (PCR). The ability of T-reg to produce interferon-gamma, interleukin (IL)-10, transforming growth factor-beta, and tumor necrosis factor-alpha after stimulation by OKT3 was evaluated by measuring the levels of mRNA in T-reg by real-time PCR. RESULTS: The frequency of T-reg was increased in AIH. The mRNA levels of Foxp3 and CTLA-4 were significantly lower in AIH. The ability of T-reg to produce IL-10 was impaired in AIH. CONCLUSION: We speculate that the inferiority of the Foxp3 and CTLA-4 gene expressions on T-reg results in the impaired suppressor function of T-reg, and eventually in the breakdown of self-tolerance.

Site specific mineral composition and microstructure of human supra-gingival dental calculus.

Dental calculus has been implicated in the aetiology of several periodontal conditions. Its prevention and removal are therefore desirable clinical goals. While it is known that calculus is very variable in chemical composition, crystallinity and crystallite size little is known about site specific variability within a dentition and between individuals. With this in mind, a study was undertaken to investigate the comparative site specific nature and composition of human dental supra-gingival dental calculus obtained from 66 male patients visiting for their dental check-up using fluorescent X-ray spectroscopy, X-ray diffractometry and Fourier transform infrared spectroscopy. The supra-gingival dental calculus formed on the lingual surfaces of lower anterior teeth and the buccal surfaces of upper molar teeth were classified into four types based on calcium phosphate phases present. There was significant difference in composition of the crystal phase types between lower and upper teeth (p<0.01). There was no significant difference in crystal size between dental calculus on anterior or molar teeth of all samples. The degree of crystallinity of dental calculus formed on the upper molar teeth was higher than that formed on the lower anterior teeth (p<0.01). The CO(3)(2-) contents in dental calculus formed on the lower anterior teeth were higher than on upper molar teeth (p<0.05) which might explain the difference in crystallinity. Magnesium and Si contents and Ca:P ratio on the other hand showed no significant difference between lower and upper teeth. It was concluded that the crystal phases, crystallinity and CO(3)(2-) contents of human dental supra-gingival dental calculus is related to its location in the mouth.

Evaluation of anti-hepatitis E virus (HEV) immunoglobulin A in a serological screening for HEV infection.

BACKGROUND: Several formulations of serological diagnostic kits were developed recently in Japan for detecting hepatitis E virus (HEV) infection. The present study was conducted to evaluate a novel anti-HEV serological kit based on detection of class A immunoglobulin antibody (anti-HEV IgA). METHODS: Serum samples from 81 acute hepatitis (AH) and 112 chronic hepatitis (CH) patients were tested for anti-HEV IgG, anti-HEV IgM, and anti-HEV IgA by enzyme immunoassay, and HEV RNA was detected by reverse transcription-polymerase chain reaction. RESULTS: Eight of 81 (9.9%) AH patients were positive for anti-HEV IgG; 6/81 (7.4%) were positive for anti-HEV IgM; and 3/81 (3.7%) were positive for anti-HEV IgA. HEV RNA was detected only in two patients, and both were positive for anti-HEV IgA and negative for hepatitis A, B, and C virus markers. Of 112 CH patients, reactivity to anti-HEV IgM and anti-HEV IgG was found in two and four patients, respectively. None of these six patients was positive for anti-HEV IgA or HEV RNA. For these six CH patients, serial serum samples stored during the clinical follow-up (1994-2003) were further subjected to anti-HEV IgG, IgM, IgA, and HEV RNA examinations. None of the examined stored samples was reactive for anti-HEV IgA or HEV RNA despite reactivity to anti-HEV IgM and IgG. CONCLUSIONS: Serological examination for anti-HEV IgA together with IgM and IgG allows sensitive and specific determination of acute or past infection with HEV. Although its prevalence is low, HEV infection must be investigated in acute hepatitis patients even in nonendemic HEV countries.

Usefulness of live 3D echocardiography during radiofrequency ablation in a case of hepatocellular carcinoma.

We report a case of hepatocellular carcinoma (HCC) with chronic hepatitis C virus (HCV) infection, which was successfully treated with percutaneous radiofrequency ablation (RFA) under live three-dimensional (3D) echocardiography guidance. Recently, it was reported that live 3D echocardiography was able to enhance the efficacy of catheter-based endomyocardial injection since the 3D images made it possible to observe the target from multiple directions so that it guided more accurately. A 63-year-old Japanese man had an HCC nodule of 3.0 cm in diameter at the S8 region of the liver. Here we applied live 3D echocardiography during RFA therapy with a LeVeen needle electrode. The echocardiography guidance allowed for easier and accurate approach for needle puncture. The guidance was also effective to confirm whether an enough safety margin for the nodule was obtained. Thus, live 3D echocardiography appears to safely guide RFA needles by accurate targeting for HCC nodule, providing real-time visualization when combined with echocardiography contrast.

Noninvasive assessment of liver disease - measurement of hepatic fibrosis using tissue strain imaging.

The development of hepatic fibrosis in patients with liver disease is associated with an increased risk of liver cancer. Assessing the degree of hepatic fibrosis is therefore one of the most important factors in treatment planning. Liver biopsy is commonly performed to assess hepatic fibrosis, but this method is associated with complications such as hemorrhage. Recently, a number of studies on the noninvasive assessment of hepatic fibrosis have appeared in the literature. The present study was conducted to determine whether an easily performed myocardial examination technique can also be applied to the assessment of hepatic fibrosis. The statistical software Minitab, which performs hypothesis testing based on the P value, was used for statistical analysis. The mean strain values were 0.26 in the normal adult group, 0.155 in the chronic hepatitis group, and 0.058 in the cirrhosis group. Statistically significant differences were observed between the groups. The results of the present study suggest that noninvasive tissue strain imaging may become the method of choice for assessing hepatic fibrosis in routine clinical practice.

Plasma amino acid profiles applied for diagnosis of advanced liver fibrosis in patients with chronic hepatitis C infection.

UNLABELLED: Although biopsy has been considered mandatory in diagnosing liver fibrosis, there is a high demand for alternative effective and noninvasive methods. In this study we aimed to develop a noninvasive and effective method using the plasma amino acid profiles for the diagnosis of liver fibrosis. MATERIALS AND METHODS: Fifty-three patients with chronic hepatitis C infection were included in the study. Plasma amino acid concentration was analyzed and severity of fibrosis was staged based on biopsy. We employed a previously published amino acid-based approach to develop a discriminator (designated as an "amino-index") for the diagnosis of patients with advanced liver fibrosis. The area under the receiver operating characteristic curve (AUC) was used for evaluation of the diagnosis. RESULTS: (Phe)/(Val)+(Thr+Met+Orn)/(Pro+Gly) was derived as the optimal amino index. The AUCs were 0.92+/-0.04 (SE) and 0.99+/-0.01 for discriminating advanced fibrosis (fibrosis stages F3 and F4) and for discriminating cirrhosis, respectively. By use of the optimal cut-off values, both the sensitivity and specificity achieved a score over 0.88. CONCLUSION: Fibrosis index based on amino acid concentration could be applied to diagnose liver fibrosis as a convenient noninvasive approach.

Alteration of serum cytokine balances among different phases of chronic hepatitis B virus infection.

To understand the pathogenesis of chronic hepatitis B virus (HBV) infection, we examined the serum levels of IL-10, TNF-alpha IL-12 p70, and IL-12 p40 in 77 patients chronically infected with HBV and 19 controls. The patients were classified into four groups: asymptomatic carriers (ASC), patients with chronic hepatitis (CH), patients with liver cirrhosis (LC), and patients with hepatocellular carcinomas (HCC). The cytokine values among these groups were compared and their relations to clinical parameters were investigated. All these cytokine values were higher in the patient groups than in controls. IL-10 and TNF-alpha became higher in accordance with the progress of the disease phases, from ASC to LC, and lowest when the patients had HCC. IL-12 p40 was also lowest in HCC, however, the group with highest levels was CH. IL-12 p70 was unchanged among ASC, CH, and LC, but were raised in HCC. Serial analyses for the cytokine values in the same patients showed the similar tendencies. Regression analysis showed the significant correlations between ALT and IL-10. Serum cytokine values well reflected the pathological differences of the individual disease phases, and may become useful indices to understand the pathogenesis of chronic HBV infection.

The SHAP-hyaluronan complex in serum from patients with chronic liver diseases caused by hepatitis virus infection.

Our previous study suggested that the serum-derived hyaluronan associated protein (SHAP)-hyaluronan (HA) complex in the sera of patients with rheumatoid arthritis is useful as a marker that directly correlates with the degree of inflammation. Here, we have investigated the serum levels of the SHAP-HA complex in patients at various clinical stages of chronic hepatitis (CH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) caused by infection with the hepatitis C or hepatitis B virus. Both serum levels of the SHAP-HA complex and HA in those patients were significantly higher than those of the controls and increased in the order of CH