Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics.

There is an urgent need for novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology or serve as therapeutic targets for neurodegenerative diseases. Cerebrospinal fluid (CSF) is in direct contact with the CNS and reflects the biochemical state of the CNS under different physiological and pathological settings. Because of this, CSF is regarded as an excellent source for identifying biomarkers for neurological diseases and other diseases affecting the CNS. Quantitative proteomics and sophisticated computational software applied to analyze the protein content of CSF has been fronted as an attractive approach to find novel biomarkers for neurological diseases. This review will focus on some of the potential pitfalls in biomarker studies using CSF, summarize the status of the field of CSF proteomics in general, and discuss some of the most promising proteomics biomarker study approaches. A brief status of the biomarker discovery efforts in multiple sclerosis, Alzheimer's disease, and Parkinson's disease is also given.

Completely or nearly identical hepatitis B virus strains replicate between patients with acute or fulminant hepatitis B and their respective infectious sources.

Five patients with acute hepatitis B and four with fulminant hepatitis B were selected for sequencing of the precore/core gene of the virus strains. Furthermore, identical sequencing was done with the HBV of the infectious sources, i.e., the sexual partner in eight cases and a natural child (chronic carrier) infecting the mother in one case. Of the subjects responsible for the infection, four were healthy HBV carriers, three suffered from chronic hepatitis B, and one from acute and one from fulminant hepatitis B. The nucleotide sequences of HBV from both the patients and the implicated sources of infection exhibited perfect identity of the precore region and perfect or high identity of the core region. The completely or nearly identical strain of virus seemed to proliferate successively in the patients following the transmission from the infecting individuals regardless of sequence variations and infectious status. In two cases a peculiar pattern of infection and disease was found: In one married couple the husband, during the incubation period of acute hepatitis B, infected his wife, who developed fulminant hepatitis. In another married couple, both partners ultimately developed fulminant hepatitis (the wife being the source of the infection).

Sequence and gene structure of the hepatitis E virus isolated from Myanmar.

Hepatitis E virus (HEV) is a causative agent of enterically transmitted non-A, non-B hepatitis. Hepatitis E occurs not only in sporadic forms but also in epidemic outbreaks in the developing world. We have revealed the nucleotide and predicted amino acid sequences of full cDNA of HEV isolated from sporadic hepatitis E of Myanmar. The genome is 7194 nucleotides long, followed by a poly(A) tail, and has three open reading frames. The nonstructural gene is located in the 5' terminus, while the structural gene is situated in the 3' terminus. Our HEV strain has 98.5% nucleic acid identity with the HEV strain cloned by workers at Genelabs Incorporated from Myanmar. The difference is point nucleotide substitutions. There is a high degree of nucleotide relatedness among HEVs isolated from the same geographical location.

An epidemic outbreak of hepatitis E in Yangon of Myanmar: antibody assay and animal transmission of the virus.

An epidemic outbreak of hepatitis E occurred in an army recruit camp of Yangon, Myanmar, in October 1989. One hundred and eleven patients among 600 residents were hospitalized. As high as 83.7% of these patients were positive for the acute phase antibody against hepatitis E virus by an enzyme-linked immunosorbent assay developed in our laboratory. Also, 30.6% of 49 symptom-free residents examined were positive for the antibody. We prepared a stool extract from six patients and inoculated it into 10 rhesus monkeys for a series of three sub-passages. All of them developed acute biochemical hepatitis along with an elevation of antibody levels. A rechallenge with viruses of the present outbreak failed to provoke hepatitis in two monkeys that had previously recovered from acute hepatitis caused by an isolate of sporadic hepatitis E of the same area. Similarly, the rechallenge of the sporadic strain did not induce hepatitis in two monkeys that had been previously infected with the epidemic virus. These data suggested that the subjects would obtain neutralizing antibodies against the hepatitis E virus once infected, and many adult inhabitants of the endemic area had no protective antibodies and were still susceptible to hepatitis E infection.

Evolution of the hepatitis B virus gene during chronic infection in seven patients.

We analyzed the evolution of the precore/core gene of hepatitis B virus (HBV) over a period of 6 to 11 years in seven patients with chronic HBV infection, who exhibited a variety of clinical features. Sequence analysis revealed the following results: (1) HBeAg to anti-HBe seroconversion was correlated roughly with the occurrence of precore-defective mutants, and several years appeared to be required for complete replacement of wild types by mutants; (2) core gene mutations preceded precore-defective mutations and tended to increase with time, although not cumulatively. They occurred not only during serum alanine aminotransferase (ALT) elevations but also after ALT returned to normal; (3) ALT fluctuations appeared to subside with complete replacement of the wild type by the mutant type and/or substantial accumulation of core gene mutations; (4) unexpectedly, the anti-HBe-positive "healthy" carrier was found to harbor the wild type precore gene, as did the HBeAg-positive "healthy" carrier; however, the core gene of the former evolved at a rapid rate; and (5) a partial deletion was recognized in the core gene at the onset of fatal hepatic failure in one patient. Thus, the precore/core mutation was closely related with the clinical features in the patients.

Sequence comparison of the capsid region of hepatitis E viruses isolated from Myanmar and China.

Hepatitis E viruses (HEVs) were isolated during epidemics, one from Myanmar (formerly called Burma) and one from China and were partially sequenced. Another HEV Myanmar strain from sporadic hepatitis was previously sequenced by us. A cDNA sequence comparison was performed among them in the 3'-terminal region, approximately 750-base long. This region contained at least two immunological epitopes and was considered to correspond to the structural protein. The nucleotide sequence identity was 97.2% between the two Myanmar strains and 93.3 and 92.5% between the two Myanmar and the China strain. The deduced amino acid sequence identity ranged from 98.4 to 100.0% among the three strains. Thus this segment was well conserved on the amino acid level among the different strains isolated from these two Asian countries, although the China strain diverged more from the Myanmar strains on the nucleotide sequence level. This data may provide important information for the development of a vaccine and for identification of the virological link between different geographical locations.

Hepatitis B virus polymerase mutations during antiviral therapy in a patient following liver transplantation.

BACKGROUND/AIMS: The purpose of this study was to investigate possible resistance mutations which arose in the polymerase gene of hepatitis B virus (HBV) in a patient with severe recurrent HBV infection following liver transplantation. The patient's management included antiviral chemotherapy for almost 4 years comprising ganciclovir, foscarnet and famciclovir. In the last 2.5 years of famciclovir treatment, an increase in serum HBV DNA levels and a reduced sensitivity of the virion-associated DNA polymerase to penciclovir triphosphate were observed. METHODS: The viral polymerase gene and X gene were sequenced from serum samples collected at representative time intervals covering the entire treatment period. RESULTS: No mutations were detected in the X gene. Three nucleotide mutations, each of which resulted in an altered amino acid sequence, were detected in the polymerase gene after 816 days of total antiviral therapy (370 days of famciclovir). Two of these mutations were detected by direct sequencing and the third was detected after cloning and was present in 10% of the clones. All three mutations occurred in "region B" of RNA-dependent DNA polymerases. The HBV polymerase has similarities to both RNA and DNA polymerases. These mutations in the HBV polymerase gene were located in a similar area to the penciclovir-induced mutations observed in the herpes simplex virus DNA polymerase gene. CONCLUSIONS: Three mutations within the HBV polymerase gene were detected which were associated with a reduced penciclovir sensitivity.

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B.

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5' terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.

Vitamin B12 R-binder localization in the human uterus: an immunohistochemical study.

The localization of vitamin B12 R-binder in the uterus was studied by use of an immunoperoxidase technique. Positive staining by anti-R-binder antiserum was observed in the columnar epithelium of the endocervix (18/18 cases) and in the surface epithelium of the endometrium (8/21 cases). Staining was usually seen in the apical portion of the epithelium; cytoplasmic staining in the endocervical columnar epithelium was intense. The secretory products in the endocervical glands showed positive staining. The endometrial glandular epithelium did not stain (0/24 cases). Metaplastic squamous epithelium of the endocervix showed positive staining (3/18 cases). The native squamous epithelium as well as the stromal components of the cervix, endometrium, and myometrium were negative for R-binder. This study shows that R-binder is localized in the uterus, especially in the endocervical glands. The R-binder in the endocervix may have antimicrobial activity in the uterus as in other organs, such as the intestines and mammary glands.

Detection of precore/core-mutant hepatitis B virus genome in patients with acute or fulminant hepatitis without serological markers for recent HBV infection.

To confirm the possibility that some hepatitis B virus (HBV) variants do not induce HB s antigen (HBsAg), anti-HB core antibody (anti-HBc) and anti-HBc IgM in a transient infection, polymerase chain reaction (PCR) was performed in 20 patients with acute hepatitis and 7 patients with fulminant hepatitis. Patients were diagnosed with non-A, non-B hepatitis by serological markers at admission. PCR successfully amplified the precore/core gene in 5 (25%) of the patients with acute hepatitis and 2 (29%) of the patients with fulminant hepatitis. Subsequent sequencing revealed frequent mutations including precore-defects in the precore/core gene.