Urinary endothelin-like immunoreactivity in patients with cirrhosis

BACKGROUND/AIMS: To investigate a possible relationship between the renal production of endothelin and the presence of renal dysfunction and activation of vasoactive systems in cirrhosis, the urinary excretion and the circulating plasma levels of immunoreactive endothelin (irET) and the plasma levels of vasoactive hormones were measured in 19 healthy subjects, 12 cirrhotic patients without ascites and 39 patients with ascites and different degrees of renal dysfunction. METHODS: The urinary excretion and the circulating levels of irET were assessed after 5 days on a 40 mEq sodium diet and off diuretics. Renal function parameters and the plasma levels of vasoactive hormones were also measured. RESULTS: Patients with and without ascites had similar values of urinary irET as compared with healthy subjects (30+/-3, 31+/-3 and 29+/-2 ng/day, respectively, p>0.10). By contrast, patients with ascites had higher circulating levels of irET (15+/-1.2 pg/ml) than patients without ascites and healthy subjects (11+/-1.6 and 5+/-0.4 pg/ml, p<0.01). In patients with cirrhosis, no correlation was found between urinary irET and circulating irET. Moreover, urinary irET did not correlate with liver tests, serum and urine sodium, glomerular filtration rate or vasoactive substances. Patients with hepatorenal syndrome had similar urinary irET to patients with ascites without hepatorenal syndrome. CONCLUSIONS: Urinary excretion of irET is not increased in cirrhotic patients with ascites and does not correlate with abnormalities in renal function.     

Chemiluminescence: sensitive detection technology for reporter gene assays

A series of enzyme-activated chemiluminescence-based assays of reporter gene expression, useful in many biomedical applications, has been developed. The chemiluminescence detection systems for beta-galactosidase, beta-glucuronidase (GUS), and secreted placental alkaline phosphatase (SEAP) reporter enzymes are all based on use of 1,2-dioxetane substrates. This detection technology also permits the combined luminescence detection of two different reporter enzymes in the same tube, e.g., a dual assay for beta-galactosidase and luciferase. The sensitivity of these chemiluminescence assays is several orders of magnitude greater than that of conventional colorimetric or fluorometric detection methods; e.g., the detection limit for beta-galactosidase by the chemiluminescence assay is 8 fg and by a fluorometric assay is 2 pg. Furthermore, chemiluminescence enables detection of beta-galactosidase, GUS, and SEAP enzyme concentrations over a dynamic range of more than five to six orders in magnitude. These assays offer highly sensitive, quantitative, rapid, nonisotopic detection of reporter enzymes that are widely used in both mammalian cells and plant cells.     

Comparisons of ELISA and Western blot assays for detection of Cryptosporidium antibody

A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurred in Talent, Oregon 6 months earlier. The ELISA, which tested for combined IgG, IgA and IgM, and the WB, which tested separately for IgG and IgA, detected an almost twofold increase in serological response for persons who consumed Talent drinking water during the previous 11 months. The increases, however, were statistically significant (P < 0.05) only for the WB. The identification of serological evidence of infection, using sera collected 6 months after the end of the outbreak in a population not selected because of cryptosporidiosis-like illness, suggests that assays of Cryptosporidium-specific IgG and IgA may assist in estimating the magnitude of asymptomatic infections in the population.     

Comparison of ELISA and HPLC for the determination of desmosine or isodesmosine in aortic tissue elastin

We developed a rapid and simple method for estimating tissue elastin content by measuring desmosine (D) in tissue hydrolysates by competitive ELISA. We compared the ELISA previously reported HPLC methods. When D or isodesmosine (ID) in hydrolysate of the same elastin preparation were measured by the two different methods, a good linear relationship was obtained (r = 0.854 for human aorta or r = 0.938 for rabbit aorta, respectively). The ELISA method can detect as little as 6 pmol/ml and it may be useful in monitoring elastin metabolism in patients with various connective tissue diseases.     

Comparison of three molecular assays for rapid detection of rifampin resistance in Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is an emerging problem of great importance to public health, with higher mortality rates than drug-sensitive TB, particularly in immunocompromised patients. MDR-TB patients require treatment with more-toxic second-line drugs and remain infectious for longer than patients infected with drug-sensitive strains, incurring higher costs due to prolonged hospitalization. It is estimated that 90% of United Kingdom rifampin-resistant isolates are also resistant to isoniazid, making rifampin resistance a useful surrogate marker for multidrug resistance and indicating that second- and third-line drugs to which these isolates are susceptible are urgently required. Resistance in approximately 95% of rifampin-resistant isolates is due to mutations in a 69-bp region of the rpoB gene, making this a good target for molecular genotypic diagnostic methods. Two molecular assays, INNO-LiPA Rif.TB (Innogenetics, Zwijndrecht, Belgium) and MisMatch Detect II (Ambion, Austin, Tex.), were performed on primary specimens and cultures to predict rifampin resistance, and these methods were compared with the resistance ratio method. A third method, the phenotypic PhaB assay, was also evaluated in comparison to cultures in parallel with the genotypic assays. In an initial evaluation 16 of 16, 15 of 16, and 16 of 16 rifampin-resistant cultures (100, 93.8, and 100%, respectively), were correctly identified by line probe assay (LiPA), mismatch assay, and PhaB assay, respectively. Subsequently 38 sputa and bronchealveolar lavage specimens and 21 isolates were received from clinicians for molecular analysis. For the 38 primary specimens the LiPA and mismatch assay correlated with culture and subsequent identification and susceptibility tests in 36 and 38 specimens (94.7 and 100%), respectively. For the 21 isolates submitted by clinicians, both assays correlated 100% with routine testing.     

Automated high-performance liquid chromatographic determination of hydroxylysylpyridinoline and lysylpyridinoline in urine using a column-switching method

17 right-handed males volunteered for an experiment that compared task-related patterns of electromyographic (EMG) activation with data from muscle biopsy on proportion of slow-twitch ((ST) aerobic) to fast-twitch ((FT) anaerobic) muscle fibers. The biopsy was taken from the right-leg gastrocnemius muscle after EMG measurement from that area of the leg muscle. EMG was also recorded from the left forearm flexor carpi radialis area. Recordings were obtained from pre- and post-task resting periods and during 150 s of video-task performance when the right hand operated a joy-stick. The results showed a highly significant tonic EMG activation in the leg muscle of subjects with predominance of ST fibers, and this relationship generalized to the EMG from the 'passive' forearm. The proportion of ST to FT fibers is genetically defined and not altered by exercise. Therefore, our results lend support to a genetic differentiation between individuals with high vs. low probability of unintended build-up of muscle tension during perceptual-motor task performance.     

The contribution of assays for lymphocyte capping and creatine kinase to detection of the Becker-type dystrophy trait

Members of three unrelated families with the mild Becker type of muscular dystrophy were subjected to lymphocyte capping tests and measurements of serum creatine kinase activity. Both tests correctly identified all nine affected males, but only the capping test was abnormal in seven of eight obligate carriers. The number of capped cells in carriers and affected persons with the Becker-type dystrophy was generally intermediate between those observed for individuals with the Duchenne trait and normal controls, thus potentially aiding in the differential diagnosis between the two myopathies. The lack of sensitivity of measurements of serum creatine kinase activity in identifying carriers is further complicated by the difficulty of establishing reliable reference intervals for this enzyme in 204 healthy controls. Detailed directions for the performance of the capping test are presented.     

Molecular heterogeneity for bovine alpha-mannosidosis: PCR based assays for detection of breed-specific mutations

Given the elective nature of photorefractive keratectomy (PRK), the high expectations of patients, and misconceptions of the general public about refractive surgery, the use of the excimer laser for PRK opens the door to new liability risks for ophthalmologists and, in the comanagement environment, referring optometrists. The authors discuss informed consent, marketing, comanagement, and off-label use guidelines and protocols to help protect ophthalmologists against claims and better defend those that might arise.     

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the method of choice in the analysis of clinical specimens of BRSV.     

Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products

Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.     

Highly sensitive and specific polymerase chain reaction assays for detection of baboon and pig cells following xenotransplantation in humans

Pig and baboon xenotransplants in humans require assays that discriminate source from human cells to investigate engraftment and identify true infection of recipients with xenogeneic endogenous retroviruses. We developed two polymerase chain reaction assays that target a porcine-specific sequence in the beta-globin gene and a baboon-specific sequence in the mitochondrial cytochrome oxidase subunit II gene. The sensitivity and specificity of both assays were evaluated on DNA lysates from baboon, pig, and human peripheral blood lymphocytes. Both assays detected single cells in backgrounds of human DNA. Additionally, both assays were highly specific and yielded negative results in reactions containing only human DNA. The two assays reliably detected peripheral blood lymphocyte samples from 14 baboons and 16 pigs. The baboon- and pig-specific target sequences are gender independent and nonpolymorphic and allow universal applicability. The high sensitivity and specificity is of particular importance in assessing low-level engraftment in human xenotransplant chimeras.     

Comparative evaluation of chlamydiazyme, PACE 2, and AMP-CT assays for detection of Chlamydia trachomatis in endocervical specimens

We conducted a comparative evaluation of the Chlamydiazyme (Abbott Laboratories), PACE 2 (Gen-Probe), and AMP-CT (Gen-Probe) assays for the detection of Chlamydia trachomatis in endocervical samples. Specimens from 787 females were included in the study. The sensitivities of the PACE 2 and Chlamydiazyme assays in comparison to the results of the AMP-CT assay were 79.3 and 63.4%, respectively. The specificities of the Chlamydiazyme and PACE 2 assays were 100%. All of the positive specimens detected in this study were positive by the AMP-CT assay. On the basis of the final results of the comparison, the prevalence of C. trachomatis in the population was 10.4%. Retesting of specimens whose results were in the intermediate zone by the PACE 2 assay by a probe competition assay identified some additional true-positive specimens. Amplification assay testing of such specimens did not significantly increase the yield. The majority of specimens which tested positive by the AMP-CT assay only were not in the intermediate zone by the PACE 2 assay. We were unable to identify demographic or clinical factors which could predict those individuals who tested positive by amplified tests but not by nonamplified tests. The Gen-Probe PACE 2 assay proved to be superior to the Chlamydiazyme assay for the screening and diagnosis of C. trachomatis infections in female endocervical specimens.     

CT and MRI in detection of intrahepatic portosystemic shunts in patients with liver cirrhosis

OBJECTIVE: Our goal was to determine the prevalence and anatomic location of intrahepatic portosystemic shunts (IPSs) in patients with hepatic cirrhosis as shown by CT and MRI. MATERIALS AND METHODS: We retrospectively reviewed CT and MR scans of 33 cirrhotic patients who had IPSs. In addition, two series of 100 consecutive CT or MR were reviewed to determine the prevalence of IPSs and the percentage of intrahepatic and extrahepatic paraumbilical veins. RESULTS: Intrahepatic portosystemic shunts were divided into three groups according to the intrahepatic course: paraumbilical shunt between the left portal vein and the paraumbilical vein anterior to the liver (n = 29); inferior vena caval shunt between the posterior branch of the right portal vein and the inferior vena cava (n = 2); and miscellaneous (n = 2). Shunts of the paraumbilical type ran through the medial (n = 23), lateral (n = 3), or both medial and lateral (n = 3) segments of the left lobe of the liver. Twenty-five patients had one shunt, and four had more than one. Six cases were also associated with extrahepatic paraumbilical veins. CONCLUSION: Intrahepatic portosystemic shunts, especially the paraumbilical type, were not infrequently visualized in patients with hepatic cirrhosis.     

Reproducibility of hepatitis C virus antibody detection with various confirmatory assays

Angiostrongylus costaricensis (classified under the more specific genus Parastrongylus by different authors) is a filiform intestinal nematode endemic to Central and South America, which afflicts primarily the pediatric population 1 to 13 years of age. Only three cases of adult infections have been previously documented outside the endemic region. To our knowledge, we report the first such case in the western United States, as well as the oldest patient yet recorded. Our patient is a 73-year-old woman living in Los Angeles who presented with an acute abdomen 4 weeks following a 5-month visit to El Salvador. Examination of the gross ileum removed at exploratory laparotomy revealed a perforation associated with a segmentally thickened intestinal wall. Microscopic examination showed eosinophilic ileitis, arterial lumens containing nematodes morphologically consistent with A costaricensis, eggs in the submucosa, and arteriolitis with thrombosis.     

Thyroid stimulating hormone assays based on the detection of gold conjugates by scanning force microscopy

Carcinoma of the gallbladder a gastrointestinal malignancy with an extraordinarily poor prognosis. However, aggressive surgery, with special reference to hepatic resection, may improve survival. To prove this, we performed a retrospective analysis over an 18-year period to investigate the experience of a center that began employing liver resection in patients with gallbladder cancer in 1978. The analysis was based on patients' documentation and regular follow-up to January 1996. The standard procedures were extended cholecystectomy (cholecystectomy with lymphadenectomy and wedge hepatic resection), anatomic segmentectomy of segments IVa and V, and extended hepatectomy. Significance was assessed by the log-rank test. Thirty-nine patients were resected, curatively in 41% (n = 22; group I) and palliatively in 31% (n = 17; group 2). In 28% (n = 15; group 3) a palliative or no operation was performed. Only curatively resected patients were analyzed and followed up to January 1996. No patients in group 1 died postoperatively. The actuarial 5-year survival rate of the patients with curative resection was 55%. Four patients had stage I, two had stage II, four had stage III, and two had stage IV disease according to TNM-classification. Six of the 16 patients without lymph node metastasis survived more than 5 years. A significant difference in long-term survival was recognised between stage II and stage IV patients and between stage (pT1a)- and (look table 1b) (pT1b)-patients (P < 0.01). Diagnostic efforts should focus on detecting early stages I and II gallbladder cancer. In advanced cases, aggressive surgery, particularly with hepatic resection, is the method of choice and is successful even in patients 70 years and older.     

Serial changes in serum alpha-fetoprotein prior to detection of hepatocellular carcinoma in liver cirrhosis

In 49 liver cirrhosis patients with hepatocellular carcinoma in which the hepatocellular carcinoma nodule was smaller than 30 mm in size, serial changes in serum alpha-fetoprotein levels prior to the detection of the hepatocellular carcinoma were studied retrospectively, and compared with those of 70 cirrhotic patients with no hepatocellular carcinoma. The changes in alpha-fetoprotein levels were classified into the 4 types normal, low plateau, high plateau and spiky type. The spiky type (spiky elevation during a short period) was more frequently noted in the patients with hepatocellular carcinoma than in those with liver cirrhosis. In a prospective analysis of 39 patients with liver cirrhosis, hepatocellular carcinoma was detected in 6, and was also more frequently noted in the spiky type than the other types. Moreover, in the hepatocellular carcinoma patients with the spiky type of alpha-fetoprotein, hepatocellular carcinoma was suspected, on the basis of the tumor doubling time, to exist at the time of the first elevation of alpha-fetoprotein. These results suggest that the liver cirrhotics with the spiky type of alpha-fetoprotein may be considered to be a high-risk group of hepatocellular carcinoma.