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The AKR1A1 protein is a member of the aldo-keto reductase superfamily that catalyzes the transformation of D-glucuronate to L-gulonate in the synthesis of L-ascorbic acid (vitamin C, Vit C). We previously demonstrated that AKR1A1 knockout mice (AKR1A1

Fractures are the result of fragile bone structures after trauma caused by direct or indirect external impact or strong muscular contraction. Most fracture patients undergo surgical fixation to accelerate the healing process and restore the function of mutilated bone. Promoting the healing process remains an important issue for the treatment of bone fractures. Our previous studies demonstrated the remarkable bone-protective effects of kefir peptides (KPs) in ovariectomized rats and mice. In this study, we further evaluate the efficacy of KPs on fracture healing using a rat model of femoral fracture.Fifteen 8-week-old male Sprague Dawley (SD) rats were divided into the sham, mock, and KPs groups, in which the mock and the KPs groups underwent femur-fracture surgery with nail fixation, while the sham group underwent a sham operation. The next day, rats were orally administered with daily 400 mg/kg of KPs (KPs group) or distilled water (sham and mock groups) for four weeks. X-ray imaging, histochemical staining and serum osteogenic markers were applied for fracture healing evaluation. In vitro, mouse bone marrow mesenchymal stem cells (BMMSCs) and MC3T3-E1 line were subjected to osteoblast differentiation in the presence of KPs and compared with no KPs treatment.The results demonstrated that KPs treatment improved the progression of the fracture healing process (p 0.05) and significantly increased the expressions of Col1a1, Alp, Spp1, Vegfa and Cox2 mRNA in the femurs of the KPs-treated fractured rats compared to those of the mock-treated fracture rats. In vitro, KPs treatment promoted bone regeneration factor (Col1a1, Alp, M-csf and Phospho1) expression in MC3T3-E1-derived osteoblast cultures (on Day 3) and enhanced osteogenic differentiation and mineralization in BMMSC-derived osteoblast cultures (on Day 17 and Day 21).This is the first study to show that KPs can help with fracture healing by promoting osteogenic differentiation, and it also suggests that KPs can be used as a nutritional supplement to accelerate fracture healing.

Ethnopharmacological relevance The medicinal mushroom Antrodia cinnamomea has been used to treat cancer but its anti-angiogenic effects have not been studied in detail. Aim of the study The main objective of this study was to determine the molecular mechanism of activity underlying the anti-angiogenic effects of A. cinnamomea. Materials and methods The effects of an A. cinnamomea ethanol extract (ACEE) on cell migration and microvessel formation were investigated in endothelial cells in vitro and Matrigel plugs implanted into mice in vivo. Activation of intracellular signaling pathways was examined using Western blotting. Protein expression was assessed using immunohistochemistry in a mouse model of lung metastasis. Results We show that treatment with ACEE inhibits cell migration and tube formation in human umbilical vein endothelial cells (HUVECs). ACEE suppresses phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and expression of pro-angiogenic kinases in vascular endothelial growth factor (VEGF)-treated HUVECs, in addition to reducing expression of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3). ACEE treatment inhibits VEGF-induced microvessel formation in Matrigel plugs in vivo. In addition, ACEE significantly reduces VEGFR2 expression in Lewis lung carcinoma cells and downregulates the expression of cluster of differentiation 31 (CD31) and VEGFR2 in murine lung metastases. Conclusion These results indicate that A. cinnamomea produces anti-angiogenic effects by inhibiting the VEGFR2 signaling pathway.

Ethnopharmacological evidence Antrodia camphorata, a highly valued polypore mushroom native only to Taiwan, has been traditionally used as a medicine for anti-inflammation. Aim of the study In this study, anti-inflammatory effects of Antrodia camphorata (AC) and its active compound, ergostatrien-3β-ol (ST1), were investigated in a mouse skin ischemia model induced by skin flap surgery on the dorsal skin. Materials and methods A U-shaped flap was elevated on the dorsal skin of the nine-week-old male mice. Mice were randomly assigned to six groups for treatment (n=6) including normal skin/propylene glycol (PG), surgical skin flap/PG, solid-state-cultured AC (S/AC), wood-cultured AC (W/AC), high-dose ST1 (H-ST1), low-dose ST1 (L-ST1). Antrodia camphorata was dissolved in 25 μL PG and smeared on the skin flap every six hours for 24 h. At the end of the experiment, each mouse was anesthetized, and skin tissues were collected from their back for histopathological analysis, extracting RNA and protein according to our previous reports. Results Skin-flap-induced ischemia damage significantly increased the expression of the iNOS, COX2, and IL-6 proteins and decreased the expression of IκB protein. In addition, focal, moderate coagulative necrosis with inflammatory cell infiltration was found in the epidermis, and moderate inflammatory cells and necrosis with slight edema was noted in the sub-dermis at 24 h after skin flap surgery. However, treatment with solid-state-cultured or wood-cultured AC, or with its derived ST1 active compound, significantly reduced the necrosis and inflammatory cell infiltration in both the epidermis and sub-dermis of the skin flap. The treatments also reduced the inflammatory response by decreasing the expression of inflammation-related genes including iNOS, IL-6, TNF-α, and NF-κB, as shown by changes in RNA and protein expression, when compared with the surgical skin flap procedure alone. Conclusions These results demonstrated that methanolic extracts of wood-cultured fruiting bodies and solid-state-cultured mycelia from Antrodia camphorata have excellent anti-inflammatory activities and thus have great potential as an addition for hydrocolloid dressings.

Summary: Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs) have been efficiently achieved by somatic cell nuclear transfer (SCNT). We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/−) mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells. : Sung et al. demonstrate in a mouse model that telomeres of telomerase haplo-insufficient cells can be elongated by somatic cell nuclear transfer. Moreover, ntESCs derived from Terc+/− cells exhibit pluripotency evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency.

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder characterized by dysregulation of various genes. Recently, microRNAs (miRNAs) have been reported to be involved in this dysregulation, suggesting that manipulation of appropriate miRNA regulation may have a therapeutic benefit. Here, we report the beneficial effects of miR-196a (miR196a) on HD in cell, transgenic mouse models, and human induced pluripotent stem cells derived from one individual with HD (HD-iPSCs). In the in vitro results, a reduction of mutant HTT and pathological aggregates, accompanying the overexpression of miR-196a, was observed in HD models of human embryonic kidney cells and mouse neuroblastoma cells. In the in vivo model, HD transgenic mice overexpressing miR-196a revealed the suppression of mutant HTT in the brain and also showed improvements in neuropathological progression, such as decreases of nuclear, intranuclear, and neuropil aggregates and late-stage behavioral phenotypes. Most importantly, miR-196a also decreased HTT expression and pathological aggregates when HD-iPSCs were differentiated into the neuronal stage. Mechanisms of miR-196a in HD might be through the alteration of ubiquitin-proteasome systems, gliosis, cAMP response element-binding protein pathway, and several neuronal regulatory pathways in vivo. Taken together, these results show that manipulating miR-196a provides beneficial effects in HD, suggesting the potential therapeutical role of miR-196a in HD.

Spinal epidural abscess is rare in patients following dental extraction. Only seven cases have been described in the literature. We report the first case of an epidural abscess in the lumbar spine following dental extraction, and present a review of the relevant literature. A 53-year-old man presented with low back pain 1 week following dental extraction, and imaging revealed the presence of a lumbar epidural abscess. He underwent surgical drainage by decompressive laminectomy with evacuation of pus and debridement of the infected bone, and he was treated with a prolonged course of intravenous antibiotics. The patient demonstrated no neurologic sequelae at the 6-month follow-up examination. A search of the relevant literature showed that, of the seven epidural abscesses that occurred following dental extractions, five were cervical and two were intracranial. An epidural abscess in the lumbar spine following dental extraction had not been reported. Thus, this is the first report of an epidural abscess in the lumbar spine following dental extraction. It is also the first case of epidural abscess following dental extraction that was determined to be caused by Streptococcus suis . Our findings indicate that epidural abscess must be considered as a diagnosis for all patients presenting with intractable low back pain, with or without fever and neurologic impairment, after a recent dental extraction. We conclude that appropriate imaging must be conducted for early diagnosis.

Ethnopharmacological evidence Gexia-Zhuyu Tang (GZT), also called Gexiazhuyu decoction (GXZYD), is a traditional Chinese herbal medicine for chronic liver diseases such as cirrhosis and liver fibrosis. Aim of the study In this study, we have investigated the affects of GZT on a rat model of dimethylnitrosamine (DMN)-induced liver fibrosis. Materials and methods In this study, the protective effects of GZT on DMN-induced liver fibrosis were measured using a rat model. Following 5 weeks of DMN-treatment (8 mg/kg, i.p., given 3 consecutive days each week), oral administration of GZT at 1.8 g/kg daily via oral gavage for 2 weeks beginning at week 13. Results Both body and liver weights were significantly decreased. The reductions in body and liver weights corresponded with increasing liver damage severity. Furthermore, GZT-treatment remarkably decreased the levels of serum GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase), and the mRNA expression levels of collagen alpha- 1( I ) and alpha-smooth muscle actin ( alpha-SMA ) in DMN-induced hepatic fibrosis. In addition, hepatic stellate cells (HSCs) play a major role in various types of liver fibrosis through initial myofibroblast transformation. The proliferation of HSCs was inhibited by GZT. Treatment with GZT also induced HSC apoptosis in a dose- and time-dependent manner. GZT treatment induced HSC apoptosis by facilitating Ca 2+ release from the mitochondria within 6 h. Subsequently, caspases 3 and 12 were elevated by 72 h after treatment. Conclusions Our studies indicate that GZT exhibited both hepatoprotective and antifibrogenic effects in DMN-induced hepatic injury. These findings suggest that GZT may be useful in preventing the development of hepatic fibrosis.

Rats are used extensively in abdominal disease research. To monitor disease progress in vivo, high-frequency ultrasound (HFU) can be a powerful tool for obtaining high-resolution images of biological tissues. However, there is a paucity of data regarding the correlation between rat anatomy and corresponding HFU images. Twenty-four adult male Sprague–Dawley (SD) rats underwent abdominal scans using HFU (40 MHz) surgical procedures to identify abdominal organs and major vessels as well as in situ scanning to confirm the imaging results. The results were compared with those of human abdominal organs in ultrasonographic scans. The rat liver, paired kidneys, stomach, intestines, and major blood vessels were identified by HFU and the ultrasonic morphologies of the liver and kidneys showed clear differences between rats and humans. Clinically relevant anatomical structures were identified using HFU imaging of the rat abdomen, and these structures were compared with the corresponding structures in humans. Increased knowledge with regard to identifying the anatomy of rat abdominal organs by ultrasound will allow scientists to conduct more detailed intra-abdominal research in rodents.

Aims of the study Yi Guan Jian (YGJ) has long been employed clinically to treat liver fibrosis in traditional Chinese Medicine but the mechanism underlying the regulation has not been clarified in detail. The present investigation was designed to assess the involvement of the fibrosis pathway in dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Materials and methods Liver fibrosis was induced by DMN injection (10 mg/kg, i.p., given three consecutive days each week) following 4 weeks. YGJ was oral administered (1.8 g/kg daily via gastrogavage for two weeks). Liver sample were subjected to histological and western blot studies. For evaluation of hepatic fibrosis-related factors, collagen α1-I , tissue inhibitor of metalloproteinase-1 ( TIMP-1 ), and α-smooth muscle actin ( α-SMA ) mRNA and protein levels were analyzed. Results YGJ remarkably prevented body weight loss and DMN damage in the liver, and it inhibited the elevation of serum glutamate oxaloacetate transaminase (GOT), and glutamic pyruvic transaminase (GPT). Oral administration of YGJ extract significantly reduced the accumulation of collagen α1-I , TIMP-1 , and α-SMA in liver tissues. Conclusions Taken together, these findings indicate that the YGJ Chinese herb showed hepatoprotective and anti-fibrogenic effects against DMN-induced hepatic injury. Our data suggest that the YGJ may be useful in reversing the development of hepatic fibrosis.

Many factors have been implicated in the onset of type 2 diabetes mellitus (T2DM). Recently, immune response and inflammation were suggested to play certain roles in the development and complications of T2DM. The aim of this study is to investigate the putative correlation between the promoter polymorphisms of interleukin-4 (IL-4), one of the immune-regulatory type 2 helper T-cell cytokines, and T2DM. Genomic DNA from 425 Taiwanese T2DM patients and 148 nondiabetic control study subjects were extracted, and their IL-4 promoter polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Both of the distribution of IL-4 C-589T (P = .013) and C-34T (P = .05) genotypes were significantly different between T2DM patients and control subjects. Significant association between IL-4 C-589T alleles (P = .002) and T2DM, as well as C-34T alleles and T2DM (P =.024), was also identified. In addition, a statistically significant association between homologous IL-4 -589 C/C genotype and lower circulatory high-density lipoprotein cholesterol levels was observed. Our results suggested that IL-4 promoter polymorphisms are associated with T2DM. A significant association between IL-4 -589 C/C genotype and lower circulatory high-density lipoprotein cholesterol level was observed as well. The above results suggested that IL-4 may participate in lipid metabolism and diabetic susceptibility.

The purpose of this study was to investigate the effects of dietary supplementation with recombinant porcine lactoferrin (rPLF) produced by yeast culture on peripheral lymphocyte proliferation and serum antibody titers in chickens vaccinated against the infectious bursal disease (IBD) virus. Treatment groups were fed with rPLF powder in their diet (2.0%, w/w), and the IBD vaccine was administrated at 1 and 3 weeks of age. At 8, 12, and 16 weeks after vaccination, serum IBD antibody titers were measured via the micro-method and T cell proliferation rates were evaluated. In gene expression analyses, rPLF-treated chicken peripheral T lymphocytes were stimulated with concanavalin A (ConA) for 24h. The mRNA expression levels of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and interleukin-12 (IL-12) were determined using a semi-quantitative RT-PCR assay. The results revealed that the rPLF additive led to significant increases in serum IgG and IBD-specific antibody titers (P

The human Enterovirus genus of the piconavirus family causes most of the febrile illnesses that affect children during the summer season in Taiwan. Enterovirus type 71 (EV71) plays a key role in patients with hand-foot-and-mouth disease (HFMD) combined with severe paralysis or encephalitis. It is important to find a method for preventing infection with EV71 since there is no antiviral agent or vaccine for humans. In this study, we developed a transgenic mouse model for demonstrating the protective effects of recombinant lactoferrin (LF) against EV71 infection. Transgenic mice carrying alpha-lactalbumin-porcine lactoferrin (alphaLA-pLF) and BALB/c wild-type mice were subjected to EV71 inoculation. First, we analyzed the expression efficiencies of recombinant pLF (rpLF) in hemizygous and homozygous transgenic mice. Following EV71 inoculation on the 4th day of life, pups ingesting transgenic milk showed the significantly higher survival rate and heavier body weight compared to wild-type mice. RT-PCR analysis for EV71 viral RNA showed that the recombinant pLF had a blocking effect on EV71 infection. Our data suggest that oral intake of pLF-enriched milk exhibited the ability to prevent infection with EV71. The study also provides an animal model for validating the protective effects of pLF.

From January 1993 to September 2002, 931 patients suffered from intertrochanteric fracture and subrochanteric fracture received open reduction and internal fixation with APGN in our institute. Among these patients, 16 patients (1.7%) developed a femoral shaft fracture after the initial fixation with APGN. Removal of the APGN, closed reduction and fixation with long Gamma nail (LGN) was performed in all the 16 patients. The patients were followed for 12-60 months (average, 39.8 months). The union time of fracture was 12-24 weeks (average, 18.5 weeks) for femoral shaft fractures and 12-20 weeks (average, 16 weeks) for peritrochanteric fractures. Two early complications were noted, including one superficial (6%) infection and one deep (6%) infection. Two malunions (12.5%) developed with no definite functional impairment. The functional results using the Harris hip score were good to excellent. In conclusion, closed reduction and internal fixation with a LGN is very effective in the management of a femoral shaft fracture, a complication of a previous APGN that had been initially used for stabilisation of a pertrochanteric fracture.

Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.

Differential methylation hybridization (DMH) is a high-throughput microarray technique designed to identify changes in DNA methylation patterns commonly observed in cancer and other disease states. The DMH methodology comprises three fundamental components: the arraying of CpG island clones on glass slides, the preparation of the sample amplicons under investigation, and the hybridization of amplicon targets onto the CpG island microarray. Herein, we outline the DMH protocol and illustrate its utility and the validation approaches used in analyzing the hypermethylation profile of breast tumor and normal samples.

In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine. A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant. Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran. Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.

Seventy-two displaced acetabular fractures managed surgically were evaluated retrospectively. The follow-up period was 10 (6–14) yr. The commonest fractures were posterior wall (28) and both columns (10). The surgical approaches were Kocher–Langenbeck (47), ilioinguinal (19) and extended iliofemoral (6). No neural monitoring was used in operations and no preventive agents for heterotopic ossification or thromboembolism were used perioperatively. Reduction was rated congruent in 59 (81.9%) and noncongruent in 13 (18.1%). The early postoperative complications were 1 vascular injury, 1 iatrogenic sciatic nerve injury, 1 deep vein thrombosis and 2 wound infections. The late complications were heterotopic ossification in 20 patients, avascular necrosis of the femoral head in 4 and symptomatic arthritis in 10. Functional outcomes were rated as excellent in 31, good in 23, fair in 7 and poor in 11. Our results show that traditional management is effective enough for displaced acetabular fractures.

Between 1994 and 1997, 180 cases of primary total hip arthroplasty (THA) were performed with the posterior (Moore) approach for a variety of indications and studies prospectively. The cases were separated randomly into 2 groups to evaluate the effect of posterior capsulorrhaphy in the prevention of postoperative dislocation. In group 1 (96 cases), closure of the arthroplasty was performed with a posterior capsulorrhaphy; in group 2 (84 cases), closure was performed without capsulorrhaphy. The follow-up period was 38 months (range, 12-60 months). No dislocations occurred in group 1, whereas 2 dislocations (2.3%) occurred in group 2. Although the factors affecting dislocation in primary THA are many, a posterior capsulorrhaphy may be helpful in the prevention of posterior dislocation of primary THA performed with a posterior approach.

We describe here structural and expression analysis of the jun-B oncogene in two cervical cancer cell lines. In the CC7T-a cell line, results from both Southern analysis and cDNA cloning studies revealed the existence of two structurally altered jun-B alleles besides the normal gene. One of the altered alleles was due to a type 16 human papillomavirus (HPV-16) integration event, whereas the other allele was a consequence of a chromosomal translocation involving chromosome 19 (jun-B) and an EST182 locus residing in chromosome 15. In the HeLa cell line, which contains integrated HPV-18, an apparent structural aberration, a 3-fold amplification and a 3-fold overexpression of the jun-B gene were observed. Our observations suggest that deregulation of the jun-B gene expression may have contributed to the transformation process in these two cervical cancer cell lines.

The effect of semirigid Ender nails (EN) in the treatment of closed humeral shaft fractures was reviewed and analyzed. Clinical study was set retrospectively with detailed parameters. One hundred and eighteen closed humeral shaft fractures, treated with closed reduction and internal fixation with ENs, were collected. The follow-up period was 78 (24–175) months. The average operation blood loss was 105 cc, operation time was 57 min, hospital stay was 6.5 days, and union time was 10.5 weeks. The postoperative complications included three superficial infections, one iatrogenic radial nerve palsy, eight nail backouts, and eight nonunions. In our experience, for closed humeral shaft fractures fixed surgically, EN is a good choice for its simplicity and efficacy, but the fracture gap should be minimized after fixation and postoperative care should be closely observed.

Three hundred and five undisplaced subcapital femoral neck fractures managed by pinning in situ with Knowles' pins were evaluated to elucidate the role and effect of such treatment. The protocol of management and follow up, and evaluation, both radiographically and functionally, were set up prospectively. The duration from injury to management was 3.5 (1-14) days, the operation time was 22 (9-48) min and most of the patients were discharged without hospitalization. The follow-up period was 75 (28-136) months. The final results showed 282 (92.5 per cent) fractures united without complications (mean union time: 20 weeks), 14 (4.6 per cent) limbs with non-union, and 9 (2.9 per cent) had implant problems. Twenty-two (7.2 per cent) developed avascular necrosis after union. Percutaneous pinning of undisplaced subcapital femoral neck fractures as day cases is a simple, safe, effective and economic method.

The outcome of hepatitis D virus (HDV) superinfection varies among patients and in different geographical areas. To find out whether HDV genotype affects outcome, we used a simple genotyping method based on restriction-fragment length polymorphism with enzymes XhoI and SacII for cleavage of PCR products of the HDV genome. Of samples from 88 patients studied, the genotypes of 61 were confirmed by two methods--analysis with both enzymes or by combined restriction-enzyme and direct sequencing analyses--with consistent results. No genotype III HDV was detected among these patients. The majority of patients with acute HDV infection (35/41 [85%]) had genotype II HDV. Among the 41 patients with acute infection, four of six with genotype I had fulminant disease compared with two of 35 with genotype II. Among patients in chronic stage, cirrhosis or hepatocellular carcinoma were found in 12 of 18 with genotype I HDV and eight of 29 with genotype II. Thus genotype II was the predominant HDV genotype in this study in Taiwan. Genotype II HDV was less frequently associated with fulminant hepatitis at the acute stage or with an unfavourable long-term clinical outcome at the chronic stage than was genotype I.