Purine receptors modulate chondrocyte extracellular inorganic pyrophosphate production.

OBJECTIVE: Extracellular inorganic pyrophosphate (ePPi) plays a key role in the regulation of normal and pathologic mineralization. The purpose of this work was to evaluate the role of P1 and P2 purine receptors in modulating ePPi production by articular chondrocytes. METHODS: Porcine cartilage explants and chondrocyte monolayers were cultured in the presence of P1 agonists, or a P2 agonist or antagonist and inhibitors of P2 signaling. Ambient media ePPi concentrations were measured after 48-96h. RESULTS: The P1 agonists NECA and CGS 21680 significantly decreased ePPi concentrations surrounding chondrocytes and cartilage explants. The P2 agonist, ADP, increased ePPi levels, and the P2 antagonist, suramin, decreased ePPi concentrations. Thapsigargin and 1,2 bis-(2-aminophenoxy)ethane-N,N,N'N'-tetra acetic acid (BAPTA), which dampen Ca(2+)-related P2 signaling, suppressed the response to ADP. CONCLUSIONS: Purine receptors are important regulators of ePPi production by chondrocytes. P1 receptor stimulation diminishes and P2 receptor stimulation enhances ePPi production. Alterations in receptor signaling or aberrations of extracellular purine nucleotide metabolism resulting in abnormal quantities or proportions of P1 and P2 receptor ligands could foster changes in ePPi production that in turn affect mineralization. We propose a homeostatic role for extracellular purine nucleotides and purine receptors in stabilizing ePPi concentrations.

Modeling particle exposure in U.S. trucking terminals.

Multi-tiered sampling approaches are common in environmental and occupational exposure assessment, where exposures for a given individual are often modeled based on simultaneous measurements taken at multiple indoor and outdoor sites. The monitoring data from such studies is hierarchical by design, imposing a complex covariance structure that must be accounted for in order to obtain unbiased estimates of exposure. Statistical methods such as structural equation modeling (SEM) represent a useful alternative to simple linear regression in these cases, providing simultaneous and unbiased predictions of each level of exposure based on a set of covariates specific to the exposure setting. We test the SEM approach using data from a large exposure assessment of diesel and combustion particles in the U.S.trucking industry. The exposure assessment includes data from 36 different trucking terminals across the United States sampled between 2001 and 2005, measuring PM2.5 and its elemental carbon (EC), organic carbon (OC) components, by personal monitoring, and sampling at two indoor work locations and an outdoor "background" location. Using the SEM method, we predict the following: (1) personal exposures as a function of work-related exposure and smoking status; (2) work-related exposure as a function of terminal characteristics, indoor ventilation, job location, and background exposure conditions; and (3) background exposure conditions as a function of weather, nearby source pollution, and other regional differences across terminal sites. The primary advantage of SEMs in this setting is the ability to simultaneously predict exposures at each of the sampling locations, while accounting for the complex covariance structure among the measurements and descriptive variables. The statistically significant results and high R2 values observed from the trucking industry application supports the broader use of this approach in exposure assessment modeling.

Polymorphisms in GSTT1 and p53 and urinary transitional cell carcinoma in south-western Taiwan: a preliminary study.

Little is known about the relevance of genetic polymorphisms to arsenic-related bladder cancer. A preliminary case-control study was conducted to explore the association between genetic polymorphisms of GSTT1, p53 codon 72 and bladder cancer in southern Taiwan, a former high arsenic exposure area. Fifty-nine urinary transitional cell carcinoma (TCC) patients from a referral centre in south-western Taiwan and 81 community controls matched on residence were recruited from 1996 to 1999. A questionnaire was administered to obtain arsenic exposure and general health information. Genotypes of p53 codon 72 and GSTT1 were analysed by polymerase chain reaction-restriction fragment length polymerase. The combined variant genotypes (heterozygous or homozygous variant) of p53 codon 72 and GSTT1 null were observed in 29% of cases and in 44% of controls, respectively. In this preliminary study, bladder cancer risk was slightly elevated for subjects carrying the variant genotype of p53 codon 72 or in subjects carrying the GSTT1 null genotype. Variants in p53 codon 72 increased the risk of bladder cancer among smokers. However, the results were not statistically significant and larger confirmatory studies are needed to clarify the role of candidate gene polymorphisms and bladder cancer risk in arsenic exposed populations.

Synthesis of evidence from epidemiological studies with interval-censored exposure due to grouping.

We describe a method for assessing dose-response effects from a series of case-control and cohort studies in which the exposure information is interval censored. The interval censoring of the exposure variable is dealt with through the use of retrospective models in which the exposure is treated as a multinomial response and disease status as a binary covariate. Polychotomous logistic regression models are adopted in which the dose-response relationship between exposure and disease may be modeled in a discrete or continuous fashion. Partial conditioning is possible to eliminate some of the nuisance parameters. The methods are applied to the motivating study of the relationship between chorionic villus sampling and the occurrence of terminal transverse limb reduction.

Fluorouracil modulation in colorectal cancer: lack of improvement with N -phosphonoacetyl- l -aspartic acid or oral leucovorin or interferon, but enhanced therapeutic index with weekly 24-hour infusion schedule--an Eastern Cooperative Oncology Group/Cancer and Leukemia Group B Study.

PURPOSE: To investigate mechanism-directed regimens in maximizing the efficacy of fluorouracil (5-FU) in advanced colorected cancer. PATIENTS AND METHODS: Based on promising phase II data, a randomized comparison of various methods for the biochemical modulation of 5-FU was undertaken in patients with advanced colorectal cancer. The control group received single-agent 5-FU as a 24-hour infusion weekly. Patients (N = 1,120) with no prior chemotherapy for metastatic disease were randomized to one of the following arms: arm A, 5-FU 2,600 mg/m2 by 24-hour infusion, weekly; arm B, N-phosphonoacetyl-l-aspartic acid 250 mg/m2 day l, 5-FU 2,600 mg/m2 by 24-hour infusion day 2, weekly; arm C, 5-FU 600 mg/m2 with oral leucovorin (LV) 125 mg/m2 hourly for the preceding 4 hours, weekly; arm D, 5-FU 600 mg/m2 with intravenous (IV) LV 600 mg/m2, weekly; arm E, 5-FU 750 mg/m2/d IV by continuous infusion for 5 days, then 750 mg/m2 weekly, and recombinant interferon alfa-2a 9 million units subcutaneously three times weekly. Median follow-up was 4.8 years. RESULTS: Of the 1,098 assessable patients, 57% had measurable disease. The toxicity of all the regimens was tolerable. Grade 4 or worse toxicity occurred in 11%, 11%, 30%, 24%, and 22% on each arm, respectively; diarrhea was the most common adverse effect. These toxicity patterns favored significantly (P <.001) the 24-hour infusion arms. Median survival (months) by arm was A, 14.8; B, 11.9; C, 13.5; D, 13.6; and E, 15.2. These survival durations did not differ significantly. CONCLUSION: We conclude that a weekly infusion regimen of 5-FU is significantly less toxic than and as effective as 5-FU bolus regimens modulated by either LV or interferon in patients with metastatic colorectal cancer.

Occupational pesticide exposure and semen quality among Chinese workers.

This study investigated the association between occupational pesticide exposure and semen quality among Chinese workers. Male workers, 32 who were exposed to organophosphate pesticides and 43 who were not exposed were recruited from two nearby factories and interviewed. Following a work shift, semen and urine samples were collected for pesticide metabolite analysis. Semen samples were analyzed for sperm concentration, percentage of motility, and percentage of normal structure. Within the exposed group, the mean end-of-shift urinary p-nitrophenol levels were 0.22 and 0.15 mg/L for the high- and low-exposure subgroups, respectively. Linear regression analysis of individual semen parameters revealed a significant reduction of sperm concentration (35.9 x 10(6) vs 62.8 x 10(6), p < 0.01) and percentage of motility (47% vs 57%, p = 0.03) but not percentage of sperm with normal structure (57% vs 61%, p = 0.13). Multivariate modeling showed a significant overall shift in the mean semen parameter. Occupational exposure to ethylparathion and methamidophos seems to have a moderately adverse effect on semen quality.

Adverse effects of chorionic villus sampling: a meta-analysis.

Meta-analysis is a popular tool for combining evidence from several related studies. The technique is usually used to combine randomized clinical trials, case-control studies or prospective studies where each study has its own exposed and unexposed groups. By including separate 'study effects' (either fixed or random), one can combine information about differences between control and exposed groups, while still allowing for study heterogeneity. In this paper, we extend existing methods to combine studies of disparate designs, where some studies do not include concurrent controls. We apply the methods to a meta-analysis of the association of prenatal testing via chorionic villus sampling with the occurrence of terminal transverse limb defects.

Phosphocitrate blocks nitric oxide-induced calcification of cartilage and chondrocyte-derived apoptotic bodies.

OBJECTIVE: To examine whether phosphocitrate (PC) will block nitric oxide-induced calcification of cartilage or chondrocyte-derived apoptotic bodies. DESIGN: Articular cartilage vesicles (ACV) or apoptotic bodies (AB) were isolated from untreated or 1mM sodium nitroprusside (SNP) treated porcine cartilage slices. Mineralization of ACV, AB, control untreated and SNP-treated cartilage were done in the presence or absence of PC (1mM)+/-ATP (1mM). RESULTS: PC [1mM] blocked both the ATP-dependent and -independent mineralization in ACV and AB, untreated and SNP treated cartilage. Moreover, PC had no effect on NTPPPH activity in either ACV or AB fraction in the presence or absence of ATP suggesting that PC did not block the mineralization through the inhibition of NTPPPH activity. CONCLUSIONS: PC inhibits nitric oxide-induced calcification of cartilage and cartilage-derived apoptotic bodies.

The role of crystals in osteoarthritis.

The deposition of calcium-containing crystals in articular tissues is probably an under-recognized event. Clinical observations indicate that an exaggerated and uniquely distributed cartilage degeneration is associated with these deposits. Measurements of putative markers of cartilage breakdown suggest that the presence of these crystals magnifies the degenerative process. In vitro studies indicate two potential mechanisms by which crystals cause degeneration. These involve the stimulation of mitogenesis in synovial fibroblasts and the secretion of proteases by cells that phagocytose these crystals. Approaches that might ameliorate the degenerative process may ensue from new information about how crystals form and how they exert their biologic effects.

Morphology and other prognostic factors of hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma is a malignancy found worldwide that has typically poor prognosis despite treatment. Although several studies have dealt with prognostic factors, just a few detailed analyses of large series correlating the pathology of hepatocellular carcinoma with prognosis are available. The present study was undertaken to address this limitation. PATIENTS AND METHODS: Our prior clinical study described 432 patients, but sufficient tissue was available for evaluation in only 299 patients. Of these, 224 samples contained primary hepatocellular carcinoma, while the remainder contained only metastatic tumor. Characteristics evaluated included degree of tumor differentiation, associated cirrhosis or hepatitis, presence of cytoplasmic inclusion bodies, and blood vessel invasion by the neoplasm. RESULTS: Of the 224 patients, 71% were male, 65% white, and 73% over the age of 45 years. Ninety-one percent were from North America. A total of 42 patients were found to have cirrhosis. Thirty-five percent had cytoplasmic inclusion bodies, and 25% showed evidence of blood vessel invasion. Tumor response rates (tumor shrinkage) were low (8%) regardless of treatment. Presence of cytoplasmic eosinophilic inclusion bodies and blood vessel invasion were not associated with increased survival. Some histopathologies (pelioid, spindle cell, fibrolamellar) were associated with a better prognosis. Patients with a predominant trabecular pattern (43%) did particularly poorly. Although sex was significantly associated with survival using a univariate analysis, this effect disappeared in a multivariate Cox model that adjusted simultaneously for other factors. CONCLUSION: This investigation suggests that histologic subtype and clinical features may provide useful prognostic information in hepatocellular carcinoma. Poorer survival was observed in males, older patients with poorly differentiated tumors, or when associated with cirrhosis. Age younger than 45 years was a good prognostic factor, and presence of cirrhosis had an adverse effect on survival.

Transduction mechanisms of porcine chondrocyte inorganic pyrophosphate elaboration.

OBJECTIVE: To investigate cellular signaling mechanisms that influence chondrocyte production of inorganic pyrophosphate (PPi), which promotes calcium pyrophosphate dihydrate (CPPD) crystal deposition. METHODS: Articular chondrocyte and cartilage cultures were stimulated with protein kinase C (PKC) activator and adenyl cyclase activator. Generation of extracellular PPi was measured. RESULTS: Adenyl cyclase activation resulted in diminished pyrophosphate generation. PKC activation stimulated pyrophosphate elaboration. CONCLUSION: Two signaling pathways, cAMP and PKC, modulate generation of extracellular pyrophosphate by cartilage and chondrocytes. They are novel targets for potentially diminishing extracellular pyrophosphate elaboration that leads to CPPD crystal deposition.

Local EM estimation of the hazard function for interval-censored data.

We propose a smooth hazard estimator for interval-censored survival data using the method of local likelihood. The model is fit using a local EM algorithm. The estimator is more descriptive than traditional empirical estimates in regions of concentrated information and takes on a parametric flavor in regions of sparse information. We derive two different standard error estimates for the smooth curve, one based on asymptotic theory and the other on the bootstrap. We illustrate the local EM method for times to breast cosmesis deterioration (Finkelstein, 1986, Biometrics 42, 845-854) and for times to HIV-1 infection for individuals with hemophilia (Kroner et al., 1994, Journal of AIDS 7, 279-286). Our hazard estimates for each of these data sets show interesting structures that would not be found using a standard parametric hazard model or empirical survivorship estimates.

Phase II protocol for the evaluation of new treatments in patients with advanced gastric carcinoma: results of ECOG 5282.

The study was a Phase II randomized study to evaluate the efficacy of new agents for the treatment of advanced gastric carcinoma. Patients were randomized to receive single agent chemotherapy with mitoxantrone, etoposide, aclacinomycin-A or spirogermanium. The patients were stratified by prior use of chemotherapy, prior doxorubicin use and ECOG performance status. Patients with a history of cardiac disease or prior doxorubicin exceeding a dose of 400 mg/m2 were restrictively randomized to sopirogermanium or etoposide only. One hundred and fourteen patients were registered for the study. Among 98 evaluable patients there were only two partial responses (both in the etoposide arm), and one complete response in the mitoxantrone arm. The median survival on the study was 3.3 months. One hundred and six patients were analyzable for toxicity. There were four treatment-related deaths and four life-threatening toxicities. Because of low response rates and relatively high toxicities the studied compounds were not deemed worth further investigation for advanced gastric cancer.

Serum nucleotide pyrophosphohydrolase activity; elevated levels in osteoarthritis, calcium pyrophosphate crystal deposition disease, scleroderma, and fibromyalgia.

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.

High dose chemoradiotherapy followed by esophagectomy for adenocarcinoma of the esophagus and gastroesophageal junction: results of a phase II study of the Eastern Cooperative Oncology Group.

BACKGROUND: To assess the toxicity, local response, and survival associated with multimodality therapy in a cooperative group setting, patients with biopsy-proven clinical Stage I or II adenocarcinoma of the esophagus (staged according to 1983 American Joint Committee on Cancer criteria) or gastroesophageal junction were treated with concomitant radiation and chemotherapy followed by esophagectomy. METHODS: Radiotherapy was administered in daily 2-gray (Gy) fractions 5 days a week until a total of 60 Gy was reached. 5-fluorouracil (5-FU) was infused continuously at a dose of 1000 mg/m2/day for 96 hours on Days 2-5 and 28-31. On Day 2, a 10 mg/m2 bolus of mitomycin was injected intravenously. Esophagectomy was performed 4-8 weeks following completion of the radiotherapy. RESULTS: During the 18-month study period (August 1991 through January 1993), 46 eligible patients were accrued from 21 institutions. Eight patients were Stage I and 38 Stage II. Eighty-seven percent of patients (40 of 46) received 6000 centigray (cGy), and all received >5000 cGy. Seventy-eight percent of patients (36 of 46) received >90% of the planned 5-FU dose. Follow-up ranged from 11 to 36 months (median, 22 months). There were eight treatment-related deaths; two were preoperative (from adult respiratory distress syndrome) and six were postoperative. Complete or partial response prior to esophagectomy was observed in 63% of cases, stable disease in 15%, and progression in 20%. Thirty-three patients underwent esophagectomy (transhiatal, n=14; Ivor Lewis, n=16; other, n=3). No tumor was found in the specimens resected from 8 of these 33 patients; this represented a pathologic complete response rate of 17% overall and 24% for those who underwent esophagectomy. Overall median survival was 16.6 months, 1-year survival 57%, and 2-year survival 27%. Survival was significantly worse for patients with circumferential cancers (median, 18.1 months vs. 8.3 months; P <0.05). CONCLUSION: High dose radiation therapy with concurrent 5-FU and mitomycin may be administered to patients with esophageal adenocarcinoma with acceptable morbidity. However, in a cooperative group setting, esophagogastrectomy following intensive chemoradiotherapy is associated with excessive morbidity and mortality. Circumferential tumor growth is a significant adverse prognostic factor.

Combined chemoradiotherapy vs. radiotherapy alone for early stage squamous cell carcinoma of the esophagus: a study of the Eastern Cooperative Oncology Group.

Squamous carcinoma of the thoracic esophagus has an extremely poor prognosis. This study, EST-1282, was undertaken by the Eastern Cooperative Oncology Group (ECOG) to determine whether the combined use of 5-fluorouracil (5-FU), mitomycin C, and radiation therapy improved the disease-free survival and overall survival of patients with carcinoma of the esophagus, compared to those who received radiation therapy alone. Two- and 5-year survivals were 12% and 7% in the radiation alone arm and 27% and 9% in the chemoradiation arm. Patients treated with chemoradiation had a longer median survival (14.8 months), compared to patients receiving radiation therapy alone (9.2 months). This difference was statistically significant. The same pattern of survival was noted in almost all subgroups independent of whether surgical resection was performed.

Testing for trend with count data.

Among the tests that can be used to detect dose-related trends in count data from toxicological studies are nonparametric tests such as the Jonckheere-Terpstra and likelihood-based tests, for example, based on a Poisson model. This paper was motivated by a data set of tumor counts in which conflicting conclusions were obtained using these two tests. To define situations where one test may be preferable, we compared the small and large sample performance of these two tests as well as a robust and conditional version of the likelihood-based test in the absence and presence of a dose-related trend for both Poisson and overdispersed Poisson data. Based on our results, we suggest using the Poisson test when little overdispersion is present in the data. For more overdispersed data, we recommend using the robust Poisson test for highly discrete data (response rate lower than 2-3) and the robust Poisson test or the Jonckheere-Terpstra test for moderately discrete or continuous data (average responses larger than 2 or 3). We also studied the effects of dose metameter misspecification. A clear effect on efficiency was seen when the 'wrong' dose metameter was used to compute the test statistic. In general, unless there is strong reason to do otherwise, we recommend the use of equally spaced dose levels when applying the Poisson or robust Poisson test for trend.

Survival implications of enucleation after definitive radiotherapy for choroidal melanoma: an example of regression on time-dependent covariates.

OBJECTIVE: To evaluate whether the removal of the eye after radiotherapy alters the rates of metastatic death in patients with melanoma of the choroid. PATIENTS AND METHODS: Using an extension of the Cox model, we based our analysis on a cohort of 1541 consecutive patients with unilateral choroidal or ciliary body melanoma treated with protons (70 cobalt-gray equivalent in 5 to 7 fractions) at the Harvard University (Boston, Mass) cyclotron between July 1, 1975, through December 31, 1993, and who were observed prospectively up to September 30, 1995. Patient survival and the status of the treated eye were updated annually. RESULTS: By September 1995 (median follow-up among survivors, 8 years), 137 patients underwent enucleation after radiotherapy for complications (n=103) or tumor regrowth (n=34). The overall 10-year rate of eye retention was 89% (95% confidence interval, 87%-91%). Of the 1541 patients, 300 died of tumor metastasis, 38 following enucleation of the affected eye (mean interval from enucleation to death, 25 months). The multivariate rate ratio for metastatic death associated with enucleation (modeled as a time-dependent covariate) was 0.9 (95% confidence interval, 0.6-1.4) for enucleation due to complications and 3.8 (95% confidence interval, 2.3-6.3) for enucleation associated with tumor regrowth. CONCLUSIONS: In the absence of tumor viability, enucleation after primary irradiation for choroidal melanoma has no deleterious effect on patients' survival. Enucleation concurrent with tumor regrowth is associated with high death rates; growth of the tumor in the eye may presage systemic recurrence and death from metastasis.

Tutorial in biostatistics methods for interval-censored data.

In standard time-to-event or survival analysis, occurrence times of the event of interest are observed exactly or are right-censored, meaning that it is only known that the event occurred after the last observation time. There are numerous methods available for estimating the survival curve and for testing and estimation of the effects of covariates in this context. In some situations, however, the times of the events of interest may only be known to have occurred within an interval of time. In clinical trials, for example, patients are often seen at pre-scheduled visits but the event of interest may occur in between visits. These data are interval-censored. Owing to the lack of well-known statistical methodology and available software, a common ad hoc approach is to assume that the event occurred at the end (or beginning or midpoint) of each interval, and then apply methods for standard time-to-event data. However, this approach can lead to invalid inferences, and in particular will tend to underestimate the standard errors of the estimated parameters. The purpose of this tutorial is to illustrate and compare available methods which correctly treat the data as being interval-censored. It is not meant to be a full review of all existing methods, but only those which are available in standard statistical software, or which can be easily programmed. All approaches will be illustrated on two data sets and compared with methods which ignore the interval-censored nature of the data. We hope this tutorial will allow those familiar with the application of standard survival analysis techniques the option of applying appropriate methods when presented with interval-censored data.