The role of high-risk HPV testing in cervical cancer surveillance.

OBJECTIVES: While high-risk HPV (hrHPV) testing is not formally recommended as a surveillance modality in patients with a history of cervical cancer, it is often performed in routine practice. It is unclear whether the presence of hrHPV infection after cervical cancer treatment is associated with recurrent disease. METHODS: Patients with a cervical cancer diagnosis who were seen in a single institution between May 2012 and December 2019 were retrospectively identified. Squamous cell, adenocarcinoma, adenosquamous, and neuroendocrine histologies were included. Those with cancer progression within 3 months of treatment or < 1 year of documented surveillance were excluded. Patients who had hrHPV testing performed were included in the primary outcome analysis. RESULTS: Of the 262 patients meeting inclusion criteria, 58 (22%) recurrences were diagnosed, and recurrence was most commonly detected by a surveillance imaging study (71%). Among the 169 patients that were tested for hrHPV during the surveillance period, 41 (24%) had at least one positive hrHPV test. Recurrent disease was diagnosed in 24 (14%). Of the 24 patients with recurrent disease, 5 (21%) had at least one positive hrHPV test during surveillance, versus 36 (24%) of 145 patients without recurrent disease (p = 0.67). No recurrences were detected by hrHPV testing. CONCLUSIONS: Positive hrHPV testing in the surveillance setting was not associated with cervical cancer recurrence but did lead to additional studies and procedures. Our findings do not support the routine use of hrHPV testing for the evaluation of cervical cancer recurrence.

Magnetic anisotropy manipulation of the femtosecond laser-excited spin wave modes in full-Heusler Co2Fe1-x Mn x Al films.

Spin-wave dynamics in full-Heusler Co2Fe1-x Mn x Al films have been investigated using all-optical pump-probe magneto-optical polar Kerr spectroscopy. We find magnetic damping and anisotropy can be modulated by composition x. Damon-Eshbach (DE) spin wave occurs only in the samples which present intrinsic magnetic damping and have huge uniaxial magnetic anisotropy, implying that intrinsic magnetic damping and huge uniaxial magnetic anisotropy is the necessary conditions to excite coherent DE spin wave. Kittel spin wave appears in low uniaxial magnetic anisotropic samples and presents extrinsic magnetic damping. Therefore, laser-excited spin-wave modes can be manipulated by magnetic anisotropy, whose physical picture is discussed phenomenologically.

Mixed pH-sensitive polymeric micelles for combination drug delivery.

PURPOSE: To prepare mixed polymeric micelles that can carry two different drugs, doxorubicin (DOX) and 17-hydroxyethylamino-17-demethoxygeldanamycin (GDM-OH), for combination cancer chemotherapy. METHODS: The pH-sensitive micelles were prepared from poly(ethylene glycol)-poly(aspartate hydrazide) block copolymers to which either DOX or GDM-OH is conjugated through acid-labile hydrazone bond (individual micelles). Mixed micelles were formed not only by simply mixing two different individual micelles in aqueous solutions (aqueous mixed micelles) but also by evaporating organic solvents from the organic/aqueous mixed solvents in which two block copolymers possessing different drugs were dissolved homogeneously (organic mixed micelles). Particle size measurements, pH-dependent drug release tests, cytotoxicity assays and western blot analysis were subsequently conducted. RESULTS: Individual and aqueous/organic mixed micelles showed clinically relevant particle size (<100 nm) and pH-dependent drug release patterns. Mixed polymer micelles suppress cancer cell growth effectively in a drug concentration, mixing method and schedule-dependent way. CONCLUSION: Combination chemotherapy using polymeric micelles seems to minimize a schedule-dependent change in combination drug efficacy in comparison to drug combination using DMSO formulations.

Effect of tissue transglutaminase on the solubility of proteins containing expanded polyglutamine repeats.

The expansion of a polyglutamine (polyQ) domain in neuronal proteins is the molecular genetic cause of at least eight neurodegenerative diseases. Proteins with a polyQ domain that is greater than 40 Q (Q40) residues form insoluble intranuclear and cytoplasmic inclusions. Expanded polyQ proteins self-associate by non-covalent interactions and become insoluble. They can also be covalently cross-linked by tissue transglutaminase (TTG), a calcium-dependent enzyme present in cells throughout the nervous system. However, it remains unclear whether TTG cross-linking directly contributes to the insolubility of the expanded polyQ proteins. Using an in vitro solubility assay, we found TTG cross-linked Q62 monomers into high molecular weight soluble complexes in a calcium-dependent reaction. Inhibition of TTG cross-linking by primary amine substrates including putrescine and biotinylated pentylamine antagonized TTG's ability to form soluble complexes. In contrast, primary amines (histamine and lysine) that were less effective inhibitors of TTG cross-linking did not inhibit Q62 from becoming insoluble. In summary, TTG can increase the solubility of expanded polyQ proteins by catalyzing intermolecular cross-links. This demonstrates directly that TTG will reduce the ability of expanded polyQ proteins from becoming insoluble. Furthermore, the effectiveness of a primary amine substrate at inhibiting formation of insoluble inclusions may be related to their ability to inhibit intermolecular cross-linking by TTG.

Biliary tract disease and acute non-A-E hepatitis in Hong Kong: prospective study.

OBJECTIVE: To investigate the role of biliary tract disease in patients with acute non-A-E hepatitis. DESIGN: Prospective study. SETTING: Infectious diseases unit, government hospital, Hong Kong. PATIENTS: Sixty-one consecutive patients, admitted with the diagnosis of acute hepatitis and negative hepatitis serology for hepatitis A, B, C, D, and E virus. MAIN OUTCOME MEASURES: Abdominal ultrasound and endoscopic retrograde cholangiopancreatography findings; clinical outcome. RESULTS: Ultrasonographic abnormalities indicating biliary tract disease were found in 30% (18/61) of patients. Endoscopic retrograde cholangiopancreatography performed in 78% (14/18) of patients with abnormal ultrasound finding(s) confirmed the presence of biliary tract disease. Age, sex, serum alanine aminotransferase level, and serum albumin level were independent predictors of biliary tract disease in the patients studied. CONCLUSION: Biliary tract diseases were found in 20% of patients with acute non-A-E hepatitis. Serum amylase and abdominal ultrasonography should be performed for all patients presenting with acute non-A-E hepatitis. Endoscopic retrograde cholangiopancreatography is indicated for those with apparent gallstones or abnormal biliary tract findings.

Asymmetric inter- and intramolecular cyclopropanation of alkenes catalyzed by chiral ruthenium porphyrins. Synthesis and crystal structure of a chiral metalloporphyrin carbene complex.

Extensive investigations of asymmetric intermolecular cyclopropanation of terminal alkenes with diazoacetates catalyzed by ruthenium porphyrin [Ru(P*)(CO)(EtOH)] (1, H2P = 5,10,15,20-tetrakis[(1S,4R,5R,8S)-1,2,3,4,5,6,7,8-octahydro-1,4:5,8-dimethanoanthracene-9-yl]porphyrin) and the application of catalyst 1 to asymmetric intramolecular cyclopropanation of allylic or homoallylic diazoacetates are described. The intermolecular cyclopropanation of styrene and its derivatives with ethyl diazoacetate afforded the corresponding cyclopropyl esters in up to 98% ee with high trans/cis ratios of up to 36 and extremely high catalyst turnovers of up to 1.1 x 10(4). Examination of the effects of temperature, diazoacetate, solvent, and substituent in the intermolecular cyclopropanation reveals that (i) both enantioselectivity and trans selectivity increase with decreasing temperature, (ii) sterically encumbered diazoacetates N2CHCO2R, such as R = Bu(t), and donor solvents, such as diethyl ether and tetrahydrofuran, are beneficial to the trans selectivity, and (iii) electron-donating para substituents on styrene accelerate the cyclopropanations, with the log(k(X)/k(H)) vs sigma(+) plot for para-substituted styrenes p-X-C6H4CH=CH2 (X = MeO, Me, Cl, CF3) exhibiting good linearity with a small negative rho(+) value of -0.44 +/- 0.09. In the case of intramolecular cyclopropanation, complex 1 promoted the decomposition of a series of allylic diazoacetates to form the cyclopropyl lactones in up to 85% ee, contributing the first efficient metalloporphyrin catalyst for an asymmetric intramolecular cyclopropanation. Both the inter- and intramolecular cyclopropanations were proposed to proceed via a reactive chiral ruthenium carbene intermediate. The enantioselectivities in these processes were rationalized on the basis of the X-ray crystal structures of closely related stable chiral carbene complexes [Ru(P*)(CPh2)] (2) and [Ru(P*)(C(Ph)CO2CH2CH=CH2)] (3) obtained from reactions of complex 1 with N2CPh2 and N2C(Ph)CO2CH2CH=CH2, respectively.

Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase.

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

Tissue transglutaminase is expressed, active, and directly involved in rat dermal wound healing and angiogenesis.

Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.

Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa.

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis. However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.

Tissue transglutaminase is expressed as a host response to tumor invasion and inhibits tumor growth.

A stable extracellular matrix (ECM) constitutes an important part of host response mechanism against tumor growth and invasion. Tissue transglutaminase (TG), a calcium-dependent enzyme, can cross-link all major ECM proteins to form a stable ECM, because these cross-links are resistant to proteolytic and mechanical damage. TG can also enhance stability and strength of the ECM by its ability to facilitate the activation of transforming growth factor-beta. We hypothesized that TG ECM-promoting abilities form an important part of the host response mechanism against tumor growth. Increased expression of TG was observed in the ECM of the host tumor interface of subcutaneously implanted rat mammary adenocarcinoma R3230 Ac. TG expression was also detected in the endothelial cells and macrophages. We also detected the cross-link product at the host tumor interface and within the tumor tissue, showing that TG was active. Western blots showed TG was degraded into three fragments of 55-, 50-, and 20-kDa forms. When recombinant wild-type TG was applied to R3230 Ac implanted in rat dorsal skin flap window chamber, it caused significant growth delay at day 7 compared with recombinant inactive TG controls. Collagen was detected in increased amounts in TG treated tumors, suggesting augmentation of production and stability of the ECM. We conclude that TG forms a distinct part of host response system against and acts to inhibit tumor growth.

Regulation of human tissue transglutaminase function by magnesium-nucleotide complexes. Identification of distinct binding sites for Mg-GTP and Mg-ATP.

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.

Sphingosylphosphocholine reduces the calcium ion requirement for activating tissue transglutaminase.

Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase reaction resulting in the formation of gamma-glutamyl-epsilon-lysine bonds and is activated during apoptosis to catalyze the formation of apoptotic body. We investigate whether lipids that are membrane components and involved in cell signaling could modify the Ca2+-dependent activation of tTG. We found that sphingosylphosphocholine (lyso-SM) was the only lipid to activate transglutaminase at low Ca2+ concentrations. In the presence of lyso-SM (125 microM), transglutaminase was detectable at 10 microM Ca2+, whereas in the absence of lyso-SM, similar activity was obtained at 160 microM Ca2+. Furthermore, in the presence of lipid vesicles lyso-SM retained the ability to enhance the Ca2+-dependent activation of tTG. Lyso-SM did not significantly change the Km for the glutamyl and primary amine substrates. However, the Kact for Ca2+ was reduced from 300 microM to 90 microM. Structure-function studies of lyso-SM analogs indicate that phosphocholine group on C1, the free amino group at C2 and a C4-C5 double bond are critical for the activation of transglutaminase activity. This is the first demonstration that a specific sphingolipid could enhance the activity of tTG and could play a role in vivo in activation of the tTG at physiologic Ca2+ levels.

C-terminal deletion of human tissue transglutaminase enhances magnesium-dependent GTP/ATPase activity.

Tissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (DeltaS538, DeltaE447, DeltaP345, DeltaC290, DeltaV228, and DeltaF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent Km value for either GTP or ATP. In contrast, the Kcat value for GTP was increased by 4.6- and 3-fold for the DeltaS538 and DeltaE447 mutants, respectively. The DeltaP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1. 9-fold for the DeltaC290, DeltaV228, and DeltaF185 mutants, respectively. The Kcat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the DeltaP345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the DeltaS538 and DeltaE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.

Tissue transglutaminase expression in human breast cancer.

Tissue transglutaminase (tTG) is postulated to play a role in apoptosis, cell adhesion, metastasis, and extracellular matrix (ECM) assembly. In this study, the distribution and expression of tissue transglutaminase was investigated in normal human mammary tissue and in intraductal and invasive human breast cancer by immunohistochemistry and in situ hybridization. Frozen and formalin-fixed paraffin-embedded sections of normal, intraductal, and invasive human breast carcinoma were examined with an avidin-biotin complex immunoperoxidase method for tTG antigen and by in situ hybridization to determine the cell types expressing tTG mRNA. The expression of tTG in normal and malignant mammary epithelium in culture was evaluated by quantitative immunoblot analysis. Low-level expression of tTG was found in normal tissues with the antigen located in the ECM surrounding the ducts and in the endothelium. In intraductal cancer, there was a marked increased expression of the tTG antigen, and the increased staining was found in the ECM and was also localized in a distinct pattern at the boundary between the in situ tumor cells and the normal tissue. Further immunohistochemical analysis revealed that the cells in this boundary also stained for the endothelial cell markers CD31, CD34, and von Willebrand factor. In invasive tumors, the tTG antigen was no longer localized to the normal tissue/tumor boundary but dispersed around the tumor cells. In situ hybridization studies revealed three distinct compartments of tTG synthesis: (a) tumor cells, (b) endothelial cells, and (c) stromal cells. In addition, normal and malignant epithelial cells in culture expressed variable amounts of tTG, and the expression of tTG in these epithelial cells was at least 17-fold less than endothelial cells. The up-regulation of tTG in intraductal and invasive human breast cancer and its localization to the ECM and neovasculature suggest that tTG may regulate tumor growth and metastasis.

Resonators for self-mode-locking Ti:sapphire lasers without apertures.

It is advantageous to achieve stable self-mode locking without hard apertures by designing resonators to minimize cavity-dispersion noncoaxiality in Brewster-cut gain media. The cavity-loss modulation introduced by Kerr effects is then optimized.

Carboxyl-terminal truncation of recombinant factor XIII A-chains. Characterization of minimum structural requirement for transglutaminase activity.

A series of truncation mutants lacking 218, 229, 250, and 269 amino acid residues from the carboxyl terminus of blood coagulation factor XIII A-chains (FXIII A), designated as delta K513, delta A502, delta Y481, and delta K462, respectively, were expressed in Escherichia coli to define the minimum structure required for transglutaminase activity. delta K513 and delta A502 displayed a 3.8-4.7-fold reduction in the Kcat with no change in the Km for the glutamine substrate and a 2-fold increase in the Km of the primary amine substrate. There was no detectable transglutaminase activity for either thrombin-activated delta Y481 or delta K462. The rate of ammonia release of thrombin-activated delta K513 and delta A502 was reduced 6- and 4-fold, respectively, whereas ammonia release was not detected for the delta Y481 and delta 462 mutants. The Kact for calcium ions of the delta K513 mutant was similar to recombinant FXIIIa, whereas, it was increased by approximately 3-fold for the delta A502 mutant. The rate of fibrin gamma-chain dimer formation for the delta K513 and delta A502 mutants was reduced by approximately 19-fold. delta K462 did not bind to fibrin, while all of the other thrombin-cleaved mutants were bound. In conclusion, these results documented that the carboxyl-terminal calcium binding domain (Asp468-Glu495) was important for FXIIIa to adopt the correct conformation to ensure that efficient catalysis occurred.

Purification and characterization of recombinant human coagulant factor XIII A-chains expressed in E. coli.

The purpose of this study was to develop an Escherichia coli expression system to facilitate study of the structure and function of blood coagulation factor XIII (FXIII) A-chains. We engineered an NcoI site into the full-length FXIII A-chain cDNA and subcloned it into pKK233-2 expression vector. A low level of full-length FXIII A-chain and a 30-kDa FXIII A-chain-related antigen were expressed in the JM 105 strain of E. coli. Protein sequencing of the 30-kDa protein demonstrated that it was synthesized by internal translation starting at either Met474 or Met475. We mutated the internal ribosome-binding sequences from AGGA to TGGT (pKF13A2 construct) and found that it yielded a 30-fold increase in the production of full-length FXIII A-chains. JM105 harboring pKF13A2 produced 20 mg of soluble FXIII A-chains antigen from 1 liter culture in TB medium. The recombinant FXIII A-chain was readily purified to homogeneity through PEG fractionation, Q-Sepharose, and mono-P column chromatography with a 2100-fold increase in specific activity and a yield of 150 to 200 micrograms of FXIII A-chains per liter of culture. The purified FXIII A-chains behaved as a dimer on gel filtration analysis, were thrombin- and calcium-activated, cross-linked fibrin, and bound to fibrin to the same extent as purified plasma FXIII A-chains and recombinant FXIII A-chains purified from yeast. These results document that FXIII A-chains can be readily expressed and purified from E. coli culture and that they retained properties similar to those of purified human factor XIII A-chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Seroepidemiological survey of hepatitis E in Hong Kong by recombinant-based enzyme immunoassays.

The agent that causes the enterally transmitted form of non-A, non-B hepatitis has been cloned and called hepatitis E virus (HEV). We have carried out a seroepidemiological survey on the prevalence of hepatitis E in Hong Kong. In a retrospective study, serum from 394 patients with acute viral hepatitis and 355 healthy subjects was tested for antibodies to HEV (anti-HEV) with a recombinant-based enzyme immunoassay. 65 (16.5%) patients with hepatitis were positive for IgM anti-HEV and 23 (5.8%) were also positive for IgM anti-HEV. Of 18 patients diagnosed as having acute non-A, non-B, non-C hepatitis, 6 were IgM anti-HEV positive. 17 (6%) patients in whom acute hepatitis A was diagnosed were also infected with HEV. None of 70 patients with acute hepatitis B or C or exacerbation of chronic hepatitis B was IgM anti-HEV positive. 57 (16.1%) of the healthy subjects were positive for IgG anti-HEV. The prevalence of IgG anti-HEV was higher in subjects over 20 years old than in younger subjects (24% vs 4%, p < 0.0001). IgG anti-HEV was detected in 26% of subjects who were positive for IgG antibody to HAV and in 7% of those negative for that antibody (p < 0.0001). We demonstrated the validity of the recombinant-based enzyme immunoassays for the diagnosis of hepatitis E. Our results suggest that hepatitis E accounts for a third of non-A, non-B, non-C hepatitis in Hong Kong and that coinfection of hepatitis A and E can occur.