Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Lung cancer mortality in workers exposed to sulfuric acid mist and other acid mists.

Mortality patterns were studied in 1,165 workers exposed to sulfuric acid mist and other acid mists (primarily hydrochloric acid mist) in steel-pickling operations. Standardized mortality ratio (SMR) analysis of the full "any acid exposure" cohort (n = 1,165), with the use of U.S. death rates as a standard, showed that lung cancer was significantly elevated, with a mortality ratio of 1.64 [95% confidence interval (CI) = 1.14-2.28, based on 35 observed deaths]. The lung cancer mortality ratio for workers exposed only to sulfuric acid (n = 722) was lower (SMR = 1.39), but further restriction to the time 20 years and more from first employment in a job with probable daily sulfuric acid exposure (approximately equal to 0.2 mg/m3) yielded a mortality ratio of 1.93 (95% CI = 1.10-3.13). An excess lung cancer risk was also seen in workers exposed to acids other than sulfuric acid (SMR = 2.24; 95% CI = 1.02-2.46). When comparison was made to other steel workers (rather than to the U.S. general population) to control for socioeconomic and life-style factors such as smoking, the largest lung cancer excess was again seen in workers exposed to acids other than sulfuric acid (SMR = 2.00; 95% CI = 1.06-3.78). Adjustment for potential differences in smoking habits showed that increased smoking was unlikely to have entirely explained the increased risk. Mortality from causes of death other than lung cancer was unremarkable, with the exception of significantly low rates for deaths due to digestive system diseases.

Target-directed discovery and production of pharmaceuticals in transgenic mutant plant cells.

Plants are a source of complex bioactive compounds, with value as pharmaceuticals, or leads for synthetic modification. Many of these secondary metabolites have evolved as defenses against competing organisms and their pharmaceutical value is "accidental", resulting from homology between target proteins in these competitors, and human molecular therapeutic targets. Here we show that it is possible to use mutation and selection of plant cells to re-direct their "evolution" toward metabolites that interact with the therapeutic target proteins themselves. This is achieved by expressing the human target protein in plant cells, and selecting mutants for survival based on the interaction of their metabolome with this target. This report describes the successful evolution of hairy root cultures of a Lobelia species toward increased biosynthesis of metabolites that inhibit the human dopamine transporter protein. Many of the resulting selected mutants are overproducing the active metabolite found in the wild-type plant, but others overproduce active metabolites that are not readily detectable in non-mutants. This technology can access the whole genomic capability of a plant species to biosynthesize metabolites with a specific target. It has potential value as a novel platform for plant drug discovery and production, or as a means of optimizing the therapeutic value of medicinal plant extracts.

Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes.

The macromolecular synthesis (MMS) operon consists of three genes: rpsU, which encodes the S21 ribosomal protein in Bacillus subtilis (Bs), rpsU is replaced by orfP23 which encodes a protein of unknown function), dnaG, encoding the DNA primase involved in the initiation of chromosome replication, and rpoD, which encodes the principal sigma subunit of RNA polymerase. The operon was cloned in three segments from Listeria monocytogenes (Lm), initially using a probe designed from a highly conserved region of RpoD. Analysis of the nucleotide sequence revealed three genes: orfP17 (whose product, P17, is homologous to Bs P23), dnaG and rpoD. The Lm DnaG resembles the primase from Escherichia coli through the first two-thirds of the sequence. C-terminal similarity was observed between DnaG from Lm and Bs. Lm RpoD is similar to Bs SigA, shares identical DNA-binding domains with SigA, and is a member of the sigma 43 subgroup of the sigma 70 family.

Environmental epidemiologic investigations in the styrene-butadiene rubber production industry.

A review of the literature and an update that is in progress of a previous retrospective cohort mortality study of the styrene-1,3-butadiene industry are discussed. The follow-up has now been extended from April 1, 1976, through December 31, 1981, for plant B and December 31, 1982, for plant A. The person-years at risk of death have gone from 34,187 to 43,341 in plant A and from 19,742 to 26,314 in plant B. Among the death certificates received to date, observed deaths have increased in both plants, with increases in cancers of the trachea, bronchus and lung and in lymphosarcomas, reticulosarcomas, and cancers of the overall lymphatic and hematopoietic system.

New antibiotics from genetically engineered actinomycetes. I. 2-Norerythromycins, isolation and structural determinations.

Novel macrolide antibiotics have been isolated from a genetically manipulated actinomycete. The major components produced have been isolated and identified as 2-norerythromycins A, B, C and D by mass spectrometry and extensive 1D and 2D NMR experiments.

Mortality of workers employed at organochlorine pesticide manufacturing plants--an update.

A previous mortality study among four organochlorine pesticide manufacturers was updated through 1987. The organochlorine pesticides included chlordane; heptachlor and endrin; aldrin, dieldrin and endrin; and dichlorodiphenyltrichloroethane. The mortality for all causes and all malignant neoplasms at each of the plants was lower than expected. There was a statistically significant increase in liver and biliary tract cancer among workers at plant 3 (5 observed, standardized mortality ratio 393, 95% confidence interval 1.27-9.20). These results are somewhat consistent with experimental animal findings showing benign and malignant tumors of the liver after exposure to aldrin and dieldrin. However, the deaths were due to a mixture of intra- and extra-hepatic tumors, and the dose-response analysis was limited because of the small number of deaths and lack of exposure data. Additional study of this group should include continued follow-up of the total cohort and a case-referent analysis of the deaths from liver and biliary tract cancer.

Mortality study of workers employed at organochlorine pesticide manufacturing plants.

A retrospective cohort study was conducted to examine the mortality of workers employed in the manufacture of the chlorinated hydrocarbon pesticides, chlordane, heptachlor, dichloro-diphenyl-trichloro-ethane (DDT) and aldrin/dieldrin/endrin. Four manufacturing plants were selected for study, and each cohort included all workers employed for at least six months prior to January 1964. The entire study group totaled approximately 2,100 individuals. Vital status ascertainment for these cohorts ranged from 90 to 97% complete; the cut-off date for follow-up was 31 December 1976. In general there were too few deaths in this study on which to draw any meaningful conclusions. The standardized mortality ratio (SMR) for all causes in each cohort was below the expected level (100) and ranged from 66 to 82, probably a reflection of the "healthy worker effect." For "all malignant neoplasms" the SMRs ranged from 68 to 91 and for respiratory cancer from 55 to 132. In the aldrin/dieldrin/endrin cohort observed deaths due to pneumonia and "other respiratory diseases" were significantly above the expected number of deaths. For several other specific cancer sites (stomach in plant 1, esophagus, rectum, liver and lymphatic/hematopoietic system in plant 3), the observed deaths were more than the expected number and should be examined in more detail. It is recommended that these cohorts be followed for several more years and the mortality patterns be reexamined.

Auditory P300 abnormalities and leukocyte activation in HIV infection.

To evaluate whether P300 testing might serve as a screening modality for the early detection of HIV-related neuropathology, we tested 26 HIV-infected men (23 without neurologic symptoms, 2 with peripheral neuropathy, 1 with AIDS-associated dementia) and 15 controls. Although they had no overt neurologic symptoms, the P300 latency was delayed or undetectable in 30% of patients without clinically evident neurologic disease. P300 latencies did not correlate with peripheral blood CD4 T-cell count, serum quinolinic acid or p24 antigen levels, or the numbers of activated peripheral blood monocytes. Three individuals with abnormal P300 latencies had been HIV-seropositive for < or = 1 year, suggesting that delayed evoked responses detect early neurologic dysfunction. P300 responses do not predict imminent dementia. In only one previously asymptomatic individual with abnormal P300 waveforms have overt neurologic symptoms developed during a 2-year followup. Extended longitudinal studies will be necessary to define the predictive value of P300 latencies in the development of AIDS-related dementia. However, the sensitivity, quantitative nature, and speed of administration of this test suggest that it may be useful for identification of early neurologic involvement in HIV infection.

Pediatric applications of serial auditory brainstem and middle-latency evoked response recordings.

Serial auditory brainstem (ABR) and middle-latency (AMR) response recordings were made for 12 children (8 male, 4 female) ranging in age from 2 weeks to 10 years. A total of 40 ABR and 32 AMR assessments were carried out at bedside in varied hospital environments, including a pediatric intensive care unit (ICU), a neonatal ICU and an operating room. Clinical entities were distributed as follows: acute, severe head injury (5), hydrocephalus (2), meningomyelocele (2), hyperbilirubinemia (1), ototoxic drug overdose (1), severe developmental delay (1). Auditory evoked responses were applied in monitoring peripheral and central auditory system status, and contributed to medical, surgical and audiologic management. Abnormalities of the ABR were reversed in some children, such as those with hydrocephalus, with medical or surgical therapy. In other cases, such as a hyperbilirubinemic child, a marked ABR abnormality apparently reversed spontaneously. We present five cases to illustrate diverse applications of serial auditory evoked response measures in children.

Speech recognition in recurrent otitis media: results in a set of identical twins.

Performance-intensity functions for Pediatric Speech Intelligibility Test (PSI) word and sentence materials presented in competition were obtained for a set of identical female twins, aged 4 years, 2 months. Twin B had a medical history significant for recurrent otitis media, while Twin A had only one reported occurrence of the disease. Twin A yielded normal functions for both word and sentence materials. Twin B, however, yielded an essentially normal function for sentences, but the function for monosyllabic words was grossly depressed. These findings support the hypothesis that transient conductive hearing loss and auditory deprivation resulting from recurrent otitis media can affect the processing of phonetic information essential to the development of word recognition skills.

Audiologic findings in a set of fraternal twins with CHARGE association.

CHARGE association refers to a constellation of congenital malformations of unknown etiology that includes ocular Coloboma, Heart defects, choanal Atresia, Retarded growth/development, Genital hypoplasia, and Ear anomalies and/or hearing loss. This paper discusses audiologic results in a set of fraternal twins with a diagnosis of CHARGE. Both infants exhibit external ear defects and audiologic patterns characteristic of CHARGE and demonstrate the variable and often asymmetric findings associated with this disorder. Early identification and evaluation are imperative for appropriate audiologic management of individuals with a diagnosis of CHARGE and for persons with CHARGE-like anomalies.

Mortality of workers exposed to polychlorinated biphenyls--an update.

A retrospective cohort mortality study of workers exposed to polychlorinated biphenyls (PCBs) in two plants manufacturing electrical capacitors was reported in 1981. The study was conducted primarily to examine the risk of cancer mortality associated with exposure to PCBs. Based on animal data, liver cancer was the disease of most interest. Due to the small number of deaths and a relatively short observation period, the study was considered inconclusive. This study has been updated by adding 7 yr of observation. The number of deaths in the study cohort has increased from 163 to 295. Mortality from all causes was found to be lower than expected (295 observed vs. 318 expected deaths) as well as mortality from all cancers (62 observed vs. 80 expected deaths). A statistically significant excess in deaths was observed in the disease category that includes cancer of the liver (primary and unspecified), gall bladder, and biliary tract (5 observed vs. 1.9 expected; p less than .05). Most of this excess was observed in women employed in one plant. Due to the small number of deaths and the variability of specific cause of death within this category, it remains difficult to interpret these findings in regard to PCB exposure.

Mortality and industrial hygiene study of workers exposed to polychlorinated biphenyls.

Because of the demonstrated toxic effects on animals resulting from exposure to polychlorinated biphenyls (PCBs), the National Institute for Occupational Safety and Health conducted a retrospective cohort mortality study of 2,567 workers in two plants where PCBs were used in the manufacture of electrical capacitors. All workers included in the study were employed for at least 3 months in areas of the plants where PCBs were used. The vital status of 98% of the two cohorts was determined, and 39,018 person-years were accumulated. All-cause mortality was lower than expected (163 obs. vs 182.4 exp.) as well as all cancer mortality (39 obs. vs 43.8 exp.). Excess mortality was noted for rectal cancer (4 obs. vs 1.19 exp.) and liver cancer (3 obs. vs 1.07 exp.), although neither excess was statistically significant. In one of the plants the observed mortality due to cirrhosis of the liver was also elevated. The results of detailed industrial hygiene surveys conducted in each plant are also presented.

Religiosity and unmarried black adolescent fatherhood.

This paper examines the relationship between religiosity and unmarried adolescent fatherhood in a select population of unwed black adolescent fathers and their counterparts. The results indicated that the fathers did not differ so much from the nonfathers in the degree that they are religiously oriented but in the manner that they give expression to their religious involvement. The fathers were more likely to be responsive to nongroup modes of institutionalized religion, i.e., media forms; whereas the nonfathers religious involvement was likely to be within institutionalized groups.

Lung cancer mortality among asbestos textile workers: a review and update.

In an update of the mortality of the cohort of 1200 South Carolina textile workers, of whom almost half died, there were 185 excess deaths (SMR = 1.44), which included 71 cardiovascular diseases (SMR = 1.37), 43 non-malignant respiratory diseases (SMR = 2.25) and 41 lung cancers (SMR = 2.25). Only two definite mesotheliomas were observed. Other possible cases may have occurred but no confirmatory pathology was available. Strong exposure-response relationships have been found for lung cancer and for non-malignant respiratory diseases. The data suggest a doubling of the lung cancer risk at an exposure of approximately 30 fibre years. Mortality from pneumoconiosis and other respiratory diseases was elevated at even the lowest cumulative exposure category (< 2 f ml-1 years). A nested case-control analysis failed to demonstrate a significant role for mineral oil exposure in the etiology of lung cancer. Differences in airborne fibre sizes may be important in explaining different lung cancer and pneumoconiosis risks in various industries. In particular, the data on airborne fibres in textile manufacturing industries suggested 11-27% were longer than 5 microns compared to 2-5% for mining and milling.