Stochastic dynamic modeling of short gene expression time-series data.

In this paper, the expectation maximization (EM) algorithm is applied for modeling the gene regulatory network from gene time-series data. The gene regulatory network is viewed as a stochastic dynamic model, which consists of the noisy gene measurement from microarray and the gene regulation first-order autoregressive (AR) stochastic dynamic process. By using the EM algorithm, both the model parameters and the actual values of the gene expression levels can be identified simultaneously. Moreover, the algorithm can deal with the sparse parameter identification and the noisy data in an efficient way. It is also shown that the EM algorithm can handle the microarrary gene expression data with large number of variables but a small number of observations. The gene expression stochastic dynamic models for four real-world gene expression data sets are constructed to demonstrate the advantages of the introduced algorithm. Several indices are proposed to evaluate the models of inferred gene regulatory networks, and the relevant biological properties are discussed.

Phenotypic and population differences in the association between CILP and lumbar disc disease.

BACKGROUND: Lumbar disc disease (LDD) is one of the leading causes of disability in the working-age population. A functional single-nucleotide polymorphism (SNP), +1184T-->C, in exon 8 of the cartilage intermediate layer protein gene (CILP) was recently identified as a risk factor for LDD in the Japanese population (odds ratio (OR) 1.61, 95% CI 1.31 to 1.98), with implications for impaired transforming growth factorbeta1 signalling. AIM: To validate this finding in two different ethnic cohorts with LDD. METHODS: This SNP and flanking SNPs were analysed in 243 Finnish patients with symptoms of LDD and 259 controls, and in 348 Chinese subjects with MRI-defined LDD and 343 controls. RESULTS AND CONCLUSION: The results showed no evidence of association in the Finnish (OR = 1.35, 95% CI 0.97 to 1.87; p = 0.14) or the Chinese (OR = 1.05, 95% CI 0.77 to 1.43; p = 0.71) samples, suggesting that cartilage intermediate layer protein gene is not a major risk factor for symptoms of LDD in Caucasians or in the general population that included individuals with or without symptoms.

Selective plasma filtration for treatment of fulminant hepatic failure induced by D-galactosamine in a pig model.

BACKGROUND: Plasma exchange may be useful for treating patients with fulminant hepatic failure but during the procedure growth factors that are important for hepatic regeneration are discarded. Addition of a selective plasma filter to the plasmapheresis circuit could eliminate protein bound toxic substances and retain growth factors for hepatic regeneration. This process is called selective plasma filtration. AIMS: To determine if selective plasma filtration could be a useful treatment modality for fulminant hepatic failure. METHODS: The system was tested in five groups of pigs with fulminant hepatic failure induced by galactosamine: group I, diseased control group (n=5); group II, sham control, (n=6); group III, plasma exchange (n=6); group IV, treatment with AC-1770 selective plasma filter (n=7); and group V, treatment with AC-1730 selective plasma filter which had a smaller pore size than AC-1770 (n=7). Fresh pig plasma was given to replace filtered plasma in pigs of groups III, IV, and V. Treatment was initiated 48 hours after administration of 0.75 g/kg galactosamine. The efficacy of selective plasma filtration was assessed by survival rate and improvement in haematological, biochemical, and immunohistological parameters. RESULTS: Pigs treated with AC-1770 or AC-1730 selective plasma filters survived longer than the other groups (group I: 55 (10) hours; group II: 68 (7) hours; group III: 91 (10) hours; group IV: 269 (156) hours; group V: 950 (555) hours). One pig in group IV survived for 50 days; one pig in group V survived for 77 days and another pig in group V is still alive (>150 days). After treatment, plasma levels of aspartate aminotransferase, bilirubin, bile acid, ammonia, lactate dehydrogenase, and alpha-glutathione-S-transferase decreased. Substantial amounts of tumour necrosis factor alpha (TNF-alpha) and endotoxin were found in the filtrate. The selective plasma filtration groups retained significantly higher amounts of hepatocyte growth factor than plasma exchange alone. Similar TNF-alpha clearance was observed in the selective plasma filtration groups and the plasma exchange group. On day 4, significant improvement in liver function, as measured by the indocyanine green clearance test, was observed in groups IV and V but not in the other groups. A higher regeneration index of hepatocytes was also observed in the groups treated with AC-1770 and AC-1730 selective plasma filters. CONCLUSION: Selective plasma filtration improved survival time and expedited liver regeneration in pigs with fulminant hepatic failure.

The immune response of mice and cynomolgus monkeys to macaque mucin 1-mannan.

Mice immunised with human epithelial mucin MUC1 coupled to oxidised mannan produce MUC1 specific MHC Class 1 restricted CD8(+) cytotoxic T cells and are completely protected from the development of MUC1(+) tumours; such therapy may be applicable to humans. In this light we describe pre-clinical studies in cynomolgus monkeys (Macaca fascicularis), to test the efficacy of mannan-MUC1 in higher primates. Monkey MUC1 genomic clones were isolated from a macaque library, peptides and fusion protein synthesised and mice and monkeys immunised with macaque MUC1-mannan. In mice CTL responses were induced (as has been found with human MUC1 mannan conjugates), but in contrast monkeys produced a humoral response, with no T cell proliferative, cytotoxic responses or CTLp found. In spite of the presence of anti-MUC1 auto-antibodies, there was no toxicity or induction of autoimmunity.

Comparison of a commercial enzyme-linked immunosorbent assay with immunofluorescence and complement fixation tests for detection of Coxiella burnetii (Q fever) immunoglobulin M.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Q fever (PanBio Coxiella burnetii immunoglobulin M [IgM] ELISA, QFM-200) was compared to the indirect fluorescent antibody test (IFAT) for C. burnetii IgM and the complement fixation test (CFT). The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (n = 6), chlamydia (n = 5), or legionella (n = 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PanBio C. burnetii (Q fever) IgM ELISA (F = 187) is superior to that of CFT (F = 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever.

Induction of humoral and cellular responses in cynomolgus monkeys immunised with mannan-human MUC1 conjugates.

Mice immunised with oxidised mannan conjugated to the human mucin 1 (MUC1), produce MHC Class 1 restricted CD8+ cytotoxic T-cells which eradicate MUC1 + tumours, indicating potential for the immunotherapy of MUC1 + cancers in humans. We now describe preclinical studies performed in cynomolgus monkeys immunised with human or murine MUC1 conjugated to oxidised mannan, where immune responses and toxicity were examined. High titred antibodies specific for MUC1 were produced, MUC1 specific CD4+ and CD8+ T-cell proliferative responses and specific cytotoxic precursor cells (CTLp) were found, but not MUC1 specific cytotoxic T-cells (CTL). There was no toxicity and monkeys can be immunised against human MUC1 with mannan-MUC1 conjugates, but a humoral response (Th2 type) predominates. The results contrast with those obtained in mice when a CTL response (Th1 type) predominates.

Herpes simplex virus type 2 infection of heterosexual men attending a sexual health centre.

OBJECTIVE: To identify risk factors, particularly circumcision status, associated with serological evidence of herpes simplex virus type 2 (HSV-2) infection of heterosexual men. DESIGN: A cross-sectional case-control study employing an anonymous delinked interviewer-administered questionnaire, clinical examination, and a type-specific serological test for HSV-2. PARTICIPANTS AND SETTING: Three hundred consecutive heterosexual male patients at a public sexually transmissible diseases (STD) clinic in Sydney, Australia. MAIN OUTCOME MEASURES: Associations between serological evidence of HSV-2 infection and history of genital herpes or contact with genital herpes, history of other common STDs, and demographic and behavioural factors such as age, education level, number of sexual partners and lack of circumcision. RESULTS: One hundred and ninety-four patients (64.7%) had antibodies to HSV-2 but only 24% of these gave a history of genital herpes. A history of genital herpes or sexual contact with genital herpes, reported total lifetime number of sexual partners, failure to complete high school and a history of non-gonococcal urethritis or genital warts were associated with serological evidence of HSV-2 infection at the univariate level. Neither increasing age nor lack of circumcision was associated with HSV-2 infection. Following multivariate analysis only the lifetime number of partners and failure to finish high school were significantly strong predictors of HSV-2 infection. CONCLUSION: This is the highest prevalence of HSV-2 infection ever detected in an Australian population and one of the highest recorded globally. As younger men were as commonly infected as older men, and an earlier (1985) study involving the same clinic yielded a lower prevalence, it appears that a high level of ongoing HSV-2 transmission is occurring among Sydney heterosexuals. Increased awareness of this fact could enhance safer sex campaigns.

Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections.

Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections.

Mucin 1 antigens in the serum and bronchial lavage fluid of patients with lung cancer.

BACKGROUND: Lung cancer (LC) is the most common fatal malignancy, but there are no useful tumor markers for diagnosis or monitoring. Mucin 1 has an established role as a marker in other malignancies, but has undergone limited assessment in LC. METHODS: Serum from 86 patients with LC and 24 with benign pulmonary disease (BPD), and bronchial lavage fluid from 55 LC patients and 21 BPD patients were tested using the Mucin 1 assays mammary serum antigen (MSA) and cancer-associated serum antigen (CASA). RESULTS: For LC, serum CASA achieved sensitivity of 57%, specificity of 93% relative to normals, and 63% specificity relative to BPD. For MSA the same parameters were 19%, 95%, and 92%. Serum CASA levels were significantly higher in LC patients compared with BPD (P = 0.024) but there was no difference for MSA (P = 0.635). CASA showed excellent correlation with tumor stage and in patients with changing status of disease, while MSA did not. By contrast there was no difference in bronchial lavage fluid tumor marker levels from LC and BPD patients (CASA, P = 0.87; MSA, P = 0.89). CONCLUSIONS: In a small series serum CASA appears to be a useful agent in detecting LC because it is elevated in all types and stages of LC, and its level correlates with stage and progress of disease. Some patients with BPD have elevated levels suggesting a greater value for monitoring rather than diagnosis. Both serum MSA testing and measurements of either marker in bronchial lavage fluid are of no value.

The reliability of serological tests for the diagnosis of genital herpes: a critique.

The reliability and limitations of the currently used routine tests for herpes simplex virus type 2 (HSV-2) antibody in Australia are reviewed. Six case reports illustrate the clinical dangers of overinterpretation of the currently available kits and the need for a readily available specific HSV-2 antibody test. In Sydney, HSV-2 causes approximately 85% of primary genital herpes and > 95% of recurrent genital herpes. Due to the extensive serological cross-reactivity between HSV-1 and HSV-2, currently available "type specific" commercial assays cannot reliably distinguish between the 2. Isolation of herpes simplex virus (HSV) or detection of HSV antigen in vesicle fluid is the preferred diagnostic test but may be overlooked or patients may have no visible lesions. The only accurate techniques for detecting HSV-2 specific antibody are the Western blot assay and an enzymatic immunoassay using glycoprotein G (gG-2), a component of the HSV-2 envelope. These tests, which still are restricted to research laboratories can be used to accurately identify people with previous exposure to HSV-2 (IgG) or to diagnose primary infection where virus isolation has not been performed or is impossible. Current commercially available antibody tests may have extensive cross-reactivity.

Herpes simplex virus type 2 antibody in patients attending antenatal or STD clinics.

OBJECTIVE: To determine the prevalence of antibody to herpes simplex virus type 2 (HSV-2) in patients attending a general public antenatal clinic and three public sexually transmitted disease (STD) clinics in Sydney. BACKGROUND: Highly specific tests for herpes simplex type 2 antibody, using the glycoprotein G2, have been recently introduced, allowing determination of past asymptomatic infection. Overseas studies have confirmed the long held suspicion that asymptomatic infection is more common than clinical genital herpes. The seroprevalence of HSV-2 in antenatal and STD clinic patients varies markedly in different countries. These are the first data available for Australia by means of this highly specific test. DESIGN: Cross-sectional study of seroprevalence in these two patient groups. Sera used in the antenatal study were those submitted for routine antenatal screening for viral markers. PARTICIPANTS: Two hundred and twenty-nine consecutive patients attending the Westmead Hospital antenatal clinics, and 107 consecutive patients attending three public STD clinics. HYPOTHESES: That Australian populations show a relatively high prevalence of past asymptomatic infection with HSV-2; and that higher rates of infection will be found in patients attending STD clinics and with past or current histories of STDs. MAIN OUTCOME MEASURES: Comparison of HSV-2 seroprevalence between antenatal clinic patients and STD clinic patients; and associations of HSV-2 antibody with age, sex, occupation, country of birth, a history of current or past STDs and antibody to HSV-1. RESULTS: Antibody to HSV-2 was found in 14.5% of antenatal clinic patients and 40% of STD clinic patients. None of the antenatal patients and less than half of the seropositive STD clinic patients reported clinical genital herpes. Associations with age, socioeconomic status and previous HSV-1 infection were less marked than in studies from the United States. Female STD clinic patients had a significantly higher seroprevalence than males and three times the seroprevalence of age-matched antenatal clinic patients. The correlation between HSV-2 antibody and current gonorrhoea was more marked than that between HSV-2 and other STDs. CONCLUSION: Asymptomatic infection with HSV-2 is quite common in Australian antenatal patients and more common in patients with STDs, who have higher rates of sexual exposure.

The diagnosis of recent herpes simplex virus type 2 genital infections by the simplex-2 test.

The prevalence of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) was determined in patients with culture confirmed HSV-2 genital herpes and control groups using a commercial HSV-2 early antigen (Simplex-2; Gene Link Australia Ltd). Eighty seven per cent of 39 sera collected between 14 and 28 days after confirmed primary and recurrent HSV-2 infection were positive. In acute sera collected between 2-10 days after onset the Simplex-2 test was negative in all 90 patients with presumed primary infection but positive in 53% of 230 sera from recurrent infection. A specificity of 90-94.5% was obtained by testing 36 patients with recent proven HSV-1 infection and 331 control group patients. The Simplex-2 test may be useful in some cases of culture-negative, clinically suspected genital HSV-2 lesions only when sera are collected between 14-28 days after primary and recurrent infection. Its lack of specificity makes it unsuitable for the routine diagnosis of recent HSV-2 infection in the general population.

Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.

Direct determination of lead in urine by flameless atomic absorption spectrophotometry with platform atomisation.

A direct method of urinary lead assay by flameless atomic absorption spectrophotometry is described for routine use in the clinical laboratory. It employs a simple sample pre-treatment procedure preceding automated analysis. Reproducibility, linearity and recovery were satisfactory without the need of standard addition. Mean and reference intervals of normal urine were similar to values reported in the literature.

Rapid diagnosis of acute Epstein-Barr virus infection by an indirect enzyme-linked immunosorbent assay for specific immunoglobulin M (IgM) antibody without rheumatoid factor and specific IgG interference.

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of Epstein-Barr virus-specific immunoglobulin M (IgM) antibody was developed with commercial reagents. Sera containing rheumatoid factor (RF) (as little as 0.5 IU/ml) coupled with specific IgG resulted in false-positives in the ELISA. This interference was eliminated by the use of anti-human IgG antibodies to remove RF and IgG. Thus, pathogen-specific IgG complexes to which IgM-RF could be bound during the subsequent test were inhibited, and competition between specific IgG and IgM was also prevented. Of the 1,672 serum specimens tested, 353 were found to be Epstein-Barr virus IgM antibody positive by indirect immunofluorescence (IF). Compared with the IF test, the ELISA showed 96.6% sensitivity, 99.7% specificity, and 99% accuracy. Further evidence indicated that most of the 12 ELISA false-negatives were IF false-positives. There was a linear correlation between mean ELISA values and increasing IF titers (r = 0.96). However, the IF test has the disadvantages that it lacks automated reading and requires considerable technical expertise, both of which restrict the range of laboratories performing the test. The indirect ELISA has the advantages that it is simple and rapid and can be automated. All the reagents used in this assay are commercially available, have been prestandardized, and are stable.

Evaluation of rubella immune status by three commercial enzyme-linked immunosorbent assays.

Three commercial indirect enzyme-linked immunosorbent assays (ELISAs) (Enzygnost-Rubella, RUBELISA, and ORTHO Rubella) were evaluated for the determination of immune status by testing 1,090 serum specimens, 410 of which were from nonimmune patients. In comparison with the standard reference technique, the hemagglutination inhibition (HAI) test, the sensitivities of ORTHO Rubella (100%) and Enzygnost-Rubella (99.26%) were excellent, whereas the sensitivity of RUBELISA (95.60%) was marginally lower because of the inability of this assay to detect antibody in 22% of the serum specimens with HAI titers of 10 and 11% of sera with HAI titers of 20. The specificity of all three systems was greater than 97%. There was a linear correlation between mean ELISA values and increasing HAI titers (r greater than or equal to 0.94). Both ORTHO Rubella and Enzygnost-Rubella were shown to be suitable replacements for the HAI test, provided that an equivocal zone is incorporated in the ORTHO system and only unheated sera are used in the Enzygnost system.

Immunoglobulin responses to echovirus type 11 by enzyme linked immunosorbent assay: single-serum diagnosis of acute infection by specific IgM antibody.

An indirect solid-phase enzyme-linked immunosorbent assay (ELISA) for the determination of specific IgM and IgG antibodies to echovirus type 11 in a single dilution of serum was developed using partially purified echovirus type 11 bound to microplates. Whole serum was used for IgG antibody but prior to assaying for IgM antibody interfering IgG was removed by ion exchange chromatography. The ELISA for echovirus type 11 IgG antibody was a more sensitive, rapid, technically easier and less costly alternative to the neutralisation test. With the IgG ELISA 12 of 132 sera (10.6%) known to contain enterovirus antibodies other than echovirus type 11 were positive but it could not be determined to what extent this was due to the greater sensitivity of the ELISA or cross-reactions. The IgM ELISA was even more sensitive than the IgG ELISA with acute sera, and showed a reactivity in 4 of 36 sera (11.1%) with no detectable echovirus type 11 neutralising antibodies. Echovirus type 11 IgM antibody was detected in all sera collected after the first week of infection and up to 30 days after infection. However, it was only detected in 58% of sera collected during the first week after onset thus limiting its use for rapid diagnosis. The echovirus type 11 IgM ELISA appears to have considerable laboratory diagnostic potential when a rising antibody level cannot be demonstrated in paired sera or when virus is not cultured.