Clinical course and predictive factors of virological response in long-term lamivudine plus adefovir dipivoxil combination therapy for lamivudine-resistant chronic hepatitis B patients.

The aims of this study were to assess the long-term efficacy of lamivudine (LAM) plus adefovir dipivoxil (ADV) combination therapy in patients with chronic hepatitis B resistant to LAM, to identify predictive factors of complete viral response (HBV-DNA <2.6 log copies/ml at 12 months of combination therapy), and to analyze amino acid substitutions associated with treatment resistance in the hepatitis B virus (HBV) genome. Seventy-two patients who received ADV in addition to LAM for breakthrough hepatitis were enrolled. Undetectable HBV-DNA was observed in 61%, 74%, 81%, 84%, and 85% at 12, 24, 36, 48, and 60 months of combination therapy, respectively. On multivariate analysis, undetectable HBV-DNA during the preceding LAM monotherapy (P < 0.0001), alanine aminotransferase value ≥ the upper limit of normal × 6 (P = 0.006) and HBV-DNA level < 6.0 log copies/ml at the initiation of combination therapy (P = 0.007) were independent significant predictors of complete viral response. The cumulative rate of undetectable HBV-DNA was significantly higher in patients with response to the preceding LAM monotherapy than in those with poor response to it. Breakthrough hepatitis occurred in three patients without complete viral response and with poor response to the preceding LAM monotherapy, and rtA181A/V substitution was detected in one of the three patients. In conclusion, undetectable HBV-DNA during the LAM monotherapy was the strongest independent predictor of complete viral response to the following combination therapy. The efficacy of LAM plus ADV combination therapy may be determined by viral response to the preceding LAM monotherapy.

A mutation of the start codon in the X region of hepatitis B virus DNA in a patient with non-B, non-C chronic hepatitis.

There are cases of hepatitis involving occult hepatitis B virus (HBV) infection in which, even though the HB surface antigen (HBsAg) is negative, HBV-DNA is detected by a polymerase chain reaction (PCR). We conducted a sequence analysis of the entire HBV region in a case of non-B non-C chronic hepatitis in a 46-year-old female. A diagnosis of non-B non-C chronic hepatitis was made. Although HBV markers, such as HBs antibody (anti-HBs), anti-HBc, HBeAg and anti-HBe, were negative, HBV-DNA was positive. Nested PCR was performed to amplify the precore region of HBV-DNA and all remaining regions by long nested PCR. Sequence analysis of the two obtained bands was conducted by direct sequencing. Compared with the control strains, the ATG (Methionine) start codon in the X region had mutated to GTG (Valine). It is assumed that a mutation at the start codon in the X region may be the reason why HBV markers are negative in some cases of hepatitis that involve occult HBV infection.

IQGAP1 and vimentin are key regulator genes in naturally occurring hepatotumorigenesis induced by oxidative stress.

To identify key genes involved in the complex multistep process of hepatotumorigenesis, we reduced multivariate clinicopathological variables by using the Long-Evans Cinnamon rat, a model with naturally occurring and oxidative stress-induced hepatotumorigenesis. Gene expression patterns were analyzed serially by profiling liver tissues from rats of a naive status (4 weeks old), through to those with chronic hepatitis (26 and 39 weeks old) to tumor development (67 weeks old). Of 31 099 probe sets used for microarray analysis, 87 were identified as being upregulated in a stepwise manner during disease progression and tumor development. Quantitative real-time reverse transcription-polymerase chain reaction and statistical analyses verified that IQGAP1 and vimentin mRNA expression levels increased significantly throughout hepatotumorigenesis. A hierarchical clustering algorithm showed both genes clustered together and in the same cluster group. Immunohistochemical and western blot analyses showed similar increases in protein levels of IAGAP1 and vimentin. Finally, pathway analyses using text-mining technology with more comprehensive and recent gene-gene interaction data identified IQGAP1 and vimentin as important nodes in underlying gene regulatory networks. These findings enhance our understanding of the multistep hepatotumorigenesis and identification of target molecules for novel treatments.

A novel magnetic crystal-lipid nanostructure for magnetically guided in vivo gene delivery.

Cancer gene therapy requires a safe and effective gene delivery system. Polymer- and lipid-coated magnetic nanocrystals have been used to deliver silencing RNA, but synthesizing these magnetic vectors is difficult. Here, we show that a new nanoparticle formulation can be magnetically guided to deliver and silence genes in cells and tumours in mice. This formulation, termed LipoMag, consists of an oleic acid-coated magnetic nanocrystal core and a cationic lipid shell. When compared with the commercially available PolyMag formulation, LipoMag displayed more efficient gene silencing in 9 of 13 cell lines, and better anti-tumour effects when systemically administered to mice bearing gastric tumours. By delivering an optimized sequence of a silencing RNA that targets the epidermal growth factor receptor of tumour vessels, the intended therapeutic benefit was achieved with no evident adverse immune reaction or untoward side effects.

Bovine lactoferrin potently inhibits liver mitochondrial 8-OHdG levels and retrieves hepatic OGG1 activities in Long-Evans Cinnamon rats.

BACKGROUND/AIMS: To assess the effect of lactoferrin on oxidative liver damage and its mechanism, we used Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant-like hepatitis and lethal hepatic failure. METHODS: Four-week-old female LEC rats were divided into the untreated and treated groups. The latter was fed bovine lactoferrin at 2% mixed with conventional diet. RESULTS: The cumulative survival rates were 75.0% vs. 100% at 14 weeks, 37.5% vs. 91.7% at 15 weeks, and 12.5% vs. 91.7% at 16 weeks, respectively, for untreated and treated rats (P=0.0008). The 8-OHdG levels in liver mitochondrial DNA and malondialdehyde in plasma and liver tissues were significantly lower in treated than untreated rats (P<0.001, =0.017 and 0.034, respectively). Mitochondrial DNA mutations were more common in untreated rats. OGG1 mRNA and protein expression levels were significantly lower in untreated than treated rats (P=0.003 and 0.007, respectively). Hypermethylation of the second CpG island located upstream of OGG1 gene was observed in untreated rats. CONCLUSIONS: Our findings indicated that lactoferrin inhibits oxidative liver damage in LEC rats. Lactoferrin could be potentially useful for the treatment of oxidative stress-induced liver diseases.

Rapid identification and specific quantification of Staphylococcus epidermidis by 5' nuclease real-time polymerase chain reaction with a minor groove binder probe.

A species-specific quantitative detection method involving 5' nuclease real-time polymerase chain reaction using a minor groove binder probe that was designed from the sodA gene was developed for Staphylococcus epidermidis. This method distinguished S. epidermidis from other staphylococci and specifically quantified the bacterium. This study shows that the method is useful for the identification and quantitative detection of S. epidermidis.

[A case of leptospirosis caused by Leptospira borgpetersenii serovar sejroe infected in Bali Island, Indonesia].

We report a patient with leptospirosis caused by Leptospira borgpetersenii serovar Sejroe infection on Bali Island, Indonesia. This 33-year-old Japanese man had stayed at a resort hotel on the island from July 8 to July 13 2004. At the hotel, he swam in the pool, walked barefoot, and lied down in the grass. He developed a high fever and headache 7 days after completing his trip, and was admitted to our hospital on July 23. On admission he showed conjunctival suffusion and complained of myalgias. Laboratory findings included granulocytosis and elevated CRP. Plasmodium spp. were not found in blood smears, and no pathogenic bacteria were isolated from blood or fecal cultures. We diagnosed the patient as leptospirosis upon detection of slender coiled organisms with characteristic morphology by darkfield examination of blood sample. Minocycline 100 mg i.v.b.i.d. showed excellent efficacy. A microscopic agglutination test (MAT) during the convalescent stage demonstrated a significant increase in antibodies against L. borgpetersenii serovar Sejroe, confirming the diagnosis of leptospirosis. Despite occurrence of a pandemic of leptospirosis in certain Southeast Asian countries including Indonesia, information concerning pandemic disease is limited. In addition serovars of "imported" cases representing infection in pandemic areas differ widely from those in domestic cases. Adequate laboratory support therefore is crucial for accurate diagnosis of leptospirosis.

[Evaluation of the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA by the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN)].

The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We developed the simultaneous detection system for Chlamydia trachomatis/Neisseria gonorrhoeae DNA, combined with luminescence detection by a probe hybridization. In the performance tests, this system was able to detect 10 to 100 copies of C. trachomatis/N. gonorrhoeae DNA for only 3.5 hours, and was highly specific to C. trachomatis/N. gonorrhoeae without any cross-reaction to C. pneumoniae, N. lactamica, N. sicca or N. meningitidis. When we tested 60 clinical samples of urine and cervical swabs, the interpretive results were completely consistent with those obtained by Roche PCR system. Of 13 positive samples by the ICAN and PCR systems for C. trachomatis, four were negative by EIA method(IDEIA Chlamydia). These results indicate that the ICAN system is an efficient and sensitive system to simultaneously detect C. trachomatis/N. gonorrhoeae DNA.

[Fatal septic shock and rhabdomyolysis following transfusion of platelet concentrates contaminated with Streptococcus pneumoniae].

We report on a 58-year-old male diagnosed as having primary myelofibrosis with thrombocytopenia, who died of fatal septic shock and rhabdomyolysis after platelet concentrates (PCs) transfusion. The initial diagnosis of primary myelofibrosis was established by splenomegaly, leukoerythroblastosis and bone marrow fibrosis. PCs were transfused because of thrombocytopenia with marked bleeding tendency. Soon after the PCs transfusion in March 2000, he had attacks of chest pain, back pain and myalgia, then went into shock and died of unknown causes. PCs were suspected as being the cause of death, because Streptococcus pneumoniae was found in the culture of PCs in the WBC-reduction in-line filter and fresh frozen plasma from the same donor preserved in the Japan Red Cross Center. Rhabdomyolysis, neutrophil infiltration and phagocytosed bacteria were found from the autopsy materials, which were identified by DNA analysis as the same species found in the PCs. PCs are kept at room temperature because platelet function is lost in the cold. When PCs are contaminated with bacteria, marked multiplication induces fatal bacteremia. This is a rare report in Japan of fatal septic shock caused by PCs with bacterial contamination. We must pay strict attention to bacterial contamination in blood components.