Estimating the incidence of medical conditions using claims data often requires constructing a prevalence period that predates an event of interest, for instance the diagnosis of cancer, to exclude those with pre-existing conditions from the incidence risk set. Those conditions missed during the prevalence period may be misclassified as incident conditions (false positives) after the event of interest.
Using Medicare claims, we examined the impact of selecting shorter versus longer prevalence periods on the incidence and misclassification of 12 relatively common conditions in older persons.
The source of data for this study was the National Cancer Institute’s Surveillance, Epidemiology, and End Results cancer registry linked to Medicare claims. Two cohorts of women were included: 33,731 diagnosed with breast cancer between 2000 and 2002, who had ≥ 36 months of Medicare eligibility prior to cancer, the event of interest; and 101,649 without cancer meeting the same Medicare eligibility criterion. Cancer patients were followed from 36 months before cancer diagnosis (prevalence period) up to 3 months after diagnosis (incidence period). Non-cancer patients were followed for up to 39 months after the beginning of Medicare eligibility. A sham date was inserted after 36 months to separate the prevalence and incidence periods. Using 36 months as the gold standard, the prevalence period was then shortened in 6-month increments to examine the impact on the number of conditions first detected during the incidence period.
In the breast cancer cohort, shortening the prevalence period from 36 to 6 months increased the incidence rates (per 1,000 patients) of all conditions; for example: hypertension 196 to 243; diabetes 34 to 76; chronic obstructive pulmonary disease 29 to 46; osteoarthritis 27 to 36; congestive heart failure 20 to 36; osteoporosis 22 to 29; and cerebrovascular disease 13 to 21. Shortening the prevalence period has less impact on those without cancer.
Selecting a short prevalence period to rule out pre-existing conditions can, through misclassification, substantially inflate estimates of incident conditions. In incidence studies based on Medicare claims, selecting a prevalence period of ≥24 months balances the need to exclude pre-existing conditions with retaining the largest possible cohort.
Incidence; Prevalence; Misclassification; Look back; Medical claims; Medicare
In breast cancer, diabetes diagnosed prior to cancer (previously diagnosed) is associated with advanced cancer stage and increased mortality. However, in the general population, 40% of diabetes is undiagnosed until glucose testing, and evidence suggests one consequence of increased evaluation and management around breast cancer diagnosis is the increased detection of previously undiagnosed diabetes. Biological factors – for instance, higher insulin levels due to untreated disease - and others underlying the association between previously diagnosed diabetes and breast cancer could differ in those whose diabetes remains undiagnosed until cancer. Our objectives were to identify factors associated with previously undiagnosed diabetes in breast cancer, and to examine associations between previously undiagnosed diabetes and cancer stage, treatment patterns, and mortality.
Using Surveillance, Epidemiology, and End Results-Medicare, we identified women diagnosed with breast cancer and diabetes between 01/2001 and 12/2005. Diabetes was classified as previously diagnosed if it was identified within Medicare claims between 24 and 4 months before cancer diagnosis, and previously undiagnosed if it was identified from 3 months before to ≤ 3 months after cancer. Patients were followed until 12/2007 or death, whichever came first. Multivariate analyses were performed to examine risk factors for previously undiagnosed diabetes and associations between undiagnosed (compared to previously diagnosed) diabetes, cancer stage, treatment, and mortality.
Of 2,418 patients, 634 (26%) had previously undiagnosed diabetes; the remainder had previously diagnosed diabetes. The mean age was 77.8 years, and 49.4% were diagnosed with in situ or stage I disease. Age > 80 years (40% of the cohort) and limited health system contact (primary care physician and/or preventive services) prior to cancer were associated with higher adjusted odds of previously undiagnosed diabetes. Previously undiagnosed diabetes was associated with higher adjusted odds of advanced stage (III/IV) cancer (Odds Ratio = 1.37: 95% Confidence Interval (CI) 1.05 – 1.80; P = 0.02), and a higher adjusted mortality rate due to causes other than cancer (Hazard Ratio = 1.29; 95% CI 1.02 – 1.63; P = 0.03).
In breast cancer, previously undiagnosed diabetes is associated with advanced stage cancer and increased mortality. Identifying biological factors would require further investigation.
Breast cancer; Diabetes; Previously undiagnosed; Risk factors; Stage; Mortality; Survival
Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities.
Rituximab improves survival in follicular lymphoma (FL), but is considerably more expensive than conventional chemotherapy. We estimated the total direct medical costs, cumulative survival, and cost-effectiveness of adding rituximab to first-line chemotherapy for FL, based on a single source of data representing routine practice in the elderly. Using surveillance, epidemiology, and end results (SEER) registry data plus Medicare claims, we identified 1,117 FL patients who received first-line CHOP (cyclophosphamide (C), doxorubicin, vincristine (V), and prednisone (P)) or CVP +/− rituximab. Multivariate regression was used to estimate adjusted cumulative cost and survival differences between the two groups over four years after beginning treatment. The median age was 73 years (minimum 66 years), 56% had stage III-IV disease, and 67% received rituximab. Adding rituximab to first-line chemotherapy was associated with higher adjusted incremental total cost ($18,695; 95% Confidence Interval (CI) $9,302–$28,643) and longer adjusted cumulative survival (0.18 years; 95% CI 0.10–0.27) over four years of followup. The expected cost-effectiveness was $102,142 (95% CI $34,531–296,337) per life-year gained. In routine clinical practice, adding rituximab to first-line chemotherapy for elderly patients with FL results in higher direct medical costs to Medicare and longer cumulative survival after four years.
Background. Trastuzumab improves survival in HER2-positive women with metastatic breast cancer (MBC). The consequences of longer survival include a higher likelihood of additional metastases, including those in the central nervous system (CNS). The effect of CNS metastases on both trastuzumab discontinuation and survival in older patients has not been described. Patients and Methods. We used the Surveillance Epidemiology and End Results (SEER) Medicare data to identify a cohort of 562 women age 66 or older with MBC who were diagnosed between January 1, 2000 and December 31, 2005, free of CNS metastases, and initiated trastuzumab after MBC diagnosis. Time to discontinuation and time to death were analyzed using proportional hazards models. Results. Newly diagnosed CNS metastases were associated with both higher risk of trastuzumab discontinuation (relative hazard [RH] = 1.78, 95% CI 1.11–2.87) and higher risk of death (RH = 2.49, 95% CI 1.84–3.37). The incidence rate of new CNS metastases was comparable among various sites of metastasis (10.7 to 14.7 per 1,000 patient-months), except for bone which was higher (24.1 per 1,000). Conclusion. The diagnosis of CNS metastases was associated with an increase in both the likelihood of discontinuing trastuzumab therapy as well as the risk of death.
Vascular injury and remodeling are common pathological sequelae of hypertension. Previous studies have suggested that the renin-angiotensin system (RAS) acting through the type I (AT1) angiotensin (AT1)-receptor promotes vascular pathology in hypertension. To study the role of AT1-receptors in this process, we generated mice with cell-specific deletion of AT1-receptors in VSMCs using Cre/Loxp technology. We crossed the SM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Agtr1a gene (Agtr1a flox), encoding the major murine AT1-receptor isoform (AT1A). In SM22α-Cre+Agtr1a flox/flox (SMKO) mice, AT1A-receptors were efficiently deleted from VSMCs in larger vessels, but not from resistance vessels such as pre-glomerular arterioles. Thus, vasoconstrictor responses to angiotensin II were preserved in SMKOs. To induce hypertensive vascular remodeling, mice were continuously infused with angiotensin II for 4 weeks. During infusion of angiotensin II, blood pressures increased significantly and to a similar extent in SMKOs and controls. In control mice, there was evidence of vascular oxidative stress indicated by enhanced nitrated tyrosine residues in segments of aorta; this was significantly attenuated in SMKOs. Despite these differences in oxidative stress, the extent of aortic medial expansion induced by angiotensin II infusion was virtually identical in both groups. Thus, vascular AT1A-receptors promote oxidative stress in the aortic wall but are not required for remodeling in angiotensin II-dependent hypertension.
angiotensin II; hypertrophy; hyperplasia; aorta; smooth muscle; hypertension
Practice guidelines define hemodialysis catheter dysfunction as blood flow rate (BFR) <300 mL/min. We conducted a study using data from DaVita and the United States Renal Data System to evaluate the impact of catheter dysfunction on dialysis and other medical services. Patients were included if they had ≥8 consecutive weeks of catheter dialysis between 8/2004 and 12/2006. Actual BFR <300 mL/min despite planned BFR ≥300 mL/min was used to define catheter dysfunction during each dialysis session. Among 9,707 patients, the average age was 62,53% were female, and 40% were black. The median duration of catheter dialysis was 190 days, and the cohort accounted for 1,075,701 catheter dialysis sessions. There were 70,361 sessions with catheter dysfunction, and 6,33 1 (65.2%) patients had at least one session with catheter dysfunction. In multivariate repeated measures analysis, catheter dysfunction was associated with increased odds of missing a dialysis session due to access problems (Odds ratio [OR] 2.50; P < 0.001), having an access-related procedure (OR 2.10; P < 0.001), and being hospitalized (OR 1.10; P = 0.001). Catheter dysfunction defined according to NKF vascular access guidelines results in disruptions of dialysis treatment and increased use of other medical services.
Performing inference on contemporary samples of DNA sequence data is an important and challenging task. Computationally intensive methods such as importance sampling (IS) are attractive because they make full use of the available data, but in the presence of recombination the large state space of genealogies can be prohibitive. In this article, we make progress by developing an efficient IS proposal distribution for a two-locus model of sequence data. We show that the proposal developed here leads to much greater efficiency, outperforming existing IS methods that could be adapted to this model. Among several possible applications, the algorithm can be used to find maximum likelihood estimates for mutation and crossover rates, and to perform ancestral inference. We illustrate the method on previously reported sequence data covering two loci either side of the well-studied TAP2 recombination hotspot. The two loci are themselves largely non-recombining, so we obtain a gene tree at each locus and are able to infer in detail the effect of the hotspot on their joint ancestry. We summarize this joint ancestry by introducing the gene graph, a summary of the well-known ancestral recombination graph.
coalescence; Monte Carlo likelihood; probability; sequences; stochastic processes
Blood flow rate (BFR) <300 mL/min commonly is used to define hemodialysis catheter dysfunction and the need for interventions to prevent complications. The objective of this study was to describe patterns of unplanned BFR <300 mL/min during catheter hemodialysis using data from DaVita dialysis facilities and the United States Renal Data System. Patients were included if they received at least eight weeks of hemodialysis exclusively through a catheter between 08/04 and 12/06, and catheter hemodialysis was the first treatment modality following diagnosis of end-stage renal disease (first access), or it immediately followed at least one 30-day period of dialysis exclusively through a fistula or graft (replacement access). Actual BFR <300 mL/min despite a planned BFR ≥300 mL/min defined catheter dysfunction during each dialysis session. There were 3,364 patients, 268,363 catheter dialysis sessions, and 19,118 (7.1%) sessions with catheter dysfunction. Almost two-thirds of patients had ≥1 catheter dysfunction session, and 30% had ≥1 catheter dysfunction session per month. Patients with catheter as a replacement access had a higher rate of catheter dysfunction than those with a catheter as first access (hazard ratio: 1.13; P = 0.04). Catheter dysfunction affects almost one-third of catheter dialysis patients each month and two-thirds overall.
We used Surveillance, Epidemiology, and End Results-Medicare data (2000-2006) to describe treatment and survival in women diagnosed with metastatic breast cancer (MBC) who received trastuzumab. There were 610 patients with a mean age of 74 years. Overall, 32% received trastuzumab alone and 47% received trastuzumab plus a taxane. In multivariate analysis, trastuzumab plus chemotherapy was associated with a lower adjusted cancer mortality rate (Hazard Ratio [HR] 0.54; 95% Confidence Interval [CI] 0.39-0.74; p < .001) than trastuzumab alone among patients who received trastuzumab as part of first-line therapy. Adding chemotherapy to first-line trastuzumab for metastatic breast cancer is associated with improved cancer survival.
Breast cancers; Chemotherapy; Outcomes research
Productivity and predation are thought to be crucial drivers of bacterial diversity. We tested how the productivity–diversity of a natural bacterial community is modified by the presence of protist predators with different feeding preferences. In the absence of predators, there was a unimodal relationship between bacterial diversity and productivity. We found that three protist species (Bodo, Spumella and Cyclidium) had widely divergent effects on bacterial diversity across the productivity gradient. Bodo and Cyclidium had little effect on the shape of the productivity–diversity gradient, while Spumella flattened the relationship. We explain these results in terms of the feeding preferences of these predators.
bacteria; productivity; biodiversity; protist; predation
Prostaglandin (PG) E2 has multiple actions that may affect blood pressure. It is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclooxygenases, and PGE synthases. While microsomal PGE synthase 1 (mPGES1) is the only genetically-verified PGE synthase, results of previous studies examining the consequences of mPGES1-deficiency on blood pressure (BP) are conflicting. To determine whether genetic background modifies the impact of mPGES1 on BP, we generated mPGES1−/− mice on two distinct inbred backgrounds, DBA/1lacJ and 129/SvEv. On the DBA/1 background, baseline BP was similar between wild-type (WT) and mPGES1−/− mice. By contrast, on the 129 background, baseline BPs were significantly higher in mPGES1−/− animals than WT controls. During angiotensin II infusion, the DBA/1 mPGES1−/− and WT mice developed mild hypertension of similar magnitude, while 129-mPGES1−/− mice developed more severe hypertension than WT controls. DBA/1 animals developed only minimal albuminuria in response to angiotensin II infusion. By contrast, WT 129 mice had significantly higher levels of albumin excretion than WT DBA/1 and the extent of albuminuria was further augmented in 129 mPGES1−/− animals. In WT mice of both strains, the increase in urinary excretion of PGE2 with angiotensin II was attenuated in mPGES1−/− animals. Urinary excretion of thromboxane was unaffected by angiotensin II in the DBA/1 lines but increased more than 4-fold in 129 mPGES1−/− mice. These data indicate that genetic background significantly modifies the BP response to mPGES1 deficiency. Exaggerated production of thromboxane may contribute to the robust hypertension and albuminuria in 129 mPGES1-deficient mice.
prostanoids; PGE synthase; blood pressure; strain; hypertension
Diffuse large B-cell lymphoma (DLBCL) comprises 31% of lymphomas in the United States. Although it is an aggressive type of lymphoma, 40% to 50% of patients are cured with treatment. The study objectives were to identify patient factors associated with treatment and survival in DLBCL.
Using Surveillance, Epidemiology, and End Results (SEER) registry data linked to Medicare claims, we identified 7,048 patients diagnosed with DLBCL between January 1, 2001 and December 31, 2005. Patients were followed from diagnosis until the end of their claims history (maximum December 31, 2007) or death. Medicare claims were used to characterize the first infused chemo-immunotherapy (C-I therapy) regimen and to identify radiation. Multivariate analyses were performed to identify patient demographic, socioeconomic, and clinical factors associated with treatment and with survival. Outcomes variables in the survival analysis were all-cause mortality, non-Hodgkin's lymphoma (NHL) mortality, and other/unknown cause mortality.
Overall, 84% (n = 5,887) received C-I therapy or radiation treatment during the observation period: both, 26%; C-I therapy alone, 53%; and radiation alone, 5%. Median age at diagnosis was 77 years, 54% were female, 88% were white, and 43% had Stage III or IV disease at diagnosis. The median time to first treatment was 42 days, and 92% of these patients had received their first treatment by day 180 following diagnosis. In multivariate analysis, the treatment rate was significantly lower among patients ≥ 80 years old, blacks versus whites, those living in a census tract with ≥ 12% poverty, and extra-nodal disease. Blacks had a lower treatment rate overall (Hazard Ratio [HR] 0.77; P < 0.001), and were less likely to receive treatment within 180 days of diagnosis (Odds Ratio [OR] 0.63; P = 0.002) than whites. In multivariate survival analysis, black race was associated with higher all-cause mortality (HR 1.24; P = 0.01) and other/unknown cause mortality (HR 1.35; P = 0.01), but not mortality due to NHL (HR 1.16; P = 0.19).
In elderly patients diagnosed with DLBCL, there are large differences in treatment access and survival between blacks and whites.
Drugs and antibodies that interrupt vascular endothelial growth factor (VEGF) signaling pathways improve outcomes in patients with a variety of cancers by inhibiting tumor angiogenesis. A major adverse effect of these treatments is hypertension, suggesting a critical role for VEGF in blood pressure (BP) regulation. However, the physiological mechanisms underlying the control of BP by VEGF are unclear. To address this question, we administered a specific antibody against the major VEGF receptor, VEGFR2, to normal mice and assessed the consequences on BP. Compared to vehicle-treated controls, administration of the anti-VEGFR2 antibody caused a rapid and sustained increase in BP of ≈10 mm Hg. This increase in BP was associated with a significant reduction in renin mRNA expression in the kidney (p=0.019) and in urinary excretion of aldosterone (p<0.05). Treatment with the anti-VEGFR2 antibody also caused marked reduction in expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in the kidney. To examine the role of nitric oxide (NO) in the hypertension caused by blocking VEGFR2, mice were treated with Nω-nitro-L-arginine methyl ester (L-NAME) (20 mg/kg/day), an inhibitor of NO production. L-NAME administration abolished the difference in blood pressure between the vehicle- and anti-VEGFR2-treated groups. Our data suggest that VEGF, acting via VEGFR2, plays a critical role in blood pressure control by promoting NOS expression and NO activity. Interfering with this pathway is likely to be one mechanism underlying hypertension caused by anti-angiogenic agents targeting VEGF.
hypertension; angiogenesis; cancer; vascular endothelial growth factor; nitric oxide
The diffusion-generator approximation technique developed by De Iorio and Griffiths (2004a) is a very useful method of constructing importance sampling proposal distributions. Being based on general mathematical principles, the method can be applied to various models in population genetics. In this paper we extend the technique to the neutral coalescent model with recombination, thus obtaining novel sampling distributions for the two-locus model. We consider the case with subdivided population structure, as well as the classic case with only a single population. In the latter case we also consider the importance sampling proposal distributions suggested by Fearnhead and Donnelly (2001), and show that their two-locus distributions generally differ from ours. In the case of the infinitely-many-alleles model, our approximate sampling distributions are shown to be generally closer to the true distributions than are Fearnhead and Donnelly’s.
Coalescent process; recombination; diffusion process; importance sampling; migration; subdivided population
The carboxypeptidase ACE2 is a homologue of angiotensin-converting enzyme (ACE). To clarify the physiological roles of ACE2, we generated mice with targeted disruption of the Ace2 gene. ACE2-deficient mice were viable, fertile, and lacked any gross structural abnormalities. We found normal cardiac dimensions and function in ACE2-deficient animals with mixed or inbred genetic backgrounds. On the C57BL/6 background, ACE2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ACE2 had no effect on baseline blood pressures in 129/SvEv mice. After acute Ang II infusion, plasma concentrations of Ang II increased almost 3-fold higher in ACE2-deficient mice than in controls. In a model of Ang II–dependent hypertension, blood pressures were substantially higher in the ACE2-deficient mice than in WT. Severe hypertension in ACE2-deficient mice was associated with exaggerated accumulation of Ang II in the kidney, as determined by MALDI-TOF mass spectrometry. Although the absence of functional ACE2 causes enhanced susceptibility to Ang II–induced hypertension, we found no evidence for a role of ACE2 in the regulation of cardiac structure or function. Our data suggest that ACE2 is a functional component of the renin-angiotensin system, metabolizing Ang II and thereby contributing to regulation of blood pressure.
Angiotensin II, acting through type 1 angiotensin (AT1) receptors, has potent effects that alter renal excretory mechanisms. Control of sodium excretion by the kidney has been suggested to be the critical mechanism for blood pressure regulation by the renin-angiotensin system (RAS). However, since AT1 receptors are ubiquitously expressed, precisely dissecting their physiological actions in individual tissue compartments including the kidney with conventional pharmacological or gene targeting experiments has been difficult. Here, we used a cross-transplantation strategy and AT1A receptor–deficient mice to demonstrate distinct and virtually equivalent contributions of AT1 receptor actions in the kidney and in extrarenal tissues to determining the level of blood pressure. We demonstrate that regulation of blood pressure by extrarenal AT1A receptors cannot be explained by altered aldosterone generation, which suggests that AT1 receptor actions in systemic tissues such as the vascular and/or the central nervous systems make nonredundant contributions to blood pressure regulation. We also show that interruption of the AT1 receptor–mediated short-loop feedback in the kidney is not sufficient to explain the marked stimulation of renin production induced by global AT1 receptor deficiency or by receptor blockade. Instead, the renin response seems to be primarily determined by renal baroreceptor mechanisms triggered by reduced blood pressure. Thus, the regulation of blood pressure by the RAS is mediated by AT1 receptors both within and outside the kidney.
The effects of water stress upon the diversity and culturable activity of bacterial communities in the rhizosphere of an established upland grassland soil have been investigated. Intact monoliths were subjected to different watering regimens over a 2-month period to study community adaptation to moisture limitation and subsequent response to stress alleviation following rewetting. Genetic diversity was analyzed with 16S-based denaturing gradient gel electrophoresis (DGGE) of total soil-extracted DNA (rRNA genes) and RNA (rRNA transcripts) in an attempt to discriminate between total and active communities. Physiological response was monitored by plate counts, total counts, and BIOLOG-GN2 substrate utilization analyses. Controlled soil drying decreased the total number of CFU on all the media tested and also decreased the substrate utilization response. Following rewetting of dried soil, culture-based analyses indicated physiological recovery of the microbial population by the end of the experiment. In contrast, DGGE analyses of community 16S rRNA genes, rRNA transcripts and cultured communities did not reveal any changes relating to the moisture regimens, despite the observed physiological effects. We conclude that the imposed moisture regimen modulated the physiological status of the bacterial community and that bacterial communities in this soil are resistant to water stress. Further, we highlight the need for a reexamination of rRNA transcript-based molecular profiling techniques as a means of describing the active component of soil bacterial communities.
Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.
The role played by antigenic peptides bound to major histocompatibility complex (MHC) molecules is evaluated with H2-DMα−/− mice. These mice have predominantly class II–associated invariant chain peptide (CLIP)-, not antigenic peptide–bound, MHC class II. H2-DMα−/− donor heart grafts survived three times longer than wild-type grafts and slightly longer than I-Aβb−/− grafts. Proliferative T cell response was absent, and cytolytic response was reduced against the H2-DMα−/− grafts in vivo. Residual cytolytic T cell and antibody responses against intact MHC class I lead to eventual rejection. Removal of both H2-DMα and β2-microglobulin (β2m) in cardiac grafts lead to greater (8–10 times) graft survival, whereas removal of β2m alone did not have any effect. These results demonstrate the significance of peptide rather than just allogeneic MHC, in eliciting graft rejection.
major histocompatibility complex class II; H2-DM; class II–associated antigenic peptide; alloreactivity; transplantation
A rapid protocol for the extraction of total nucleic acids from environmental samples is described. The method facilitates concomitant assessment of microbial 16S rRNA diversity by PCR and reverse transcription-PCR amplification from a single extraction. Denaturing gradient gel electrophoresis microbial community analysis differentiated the active component (rRNA derived) from the total bacterial diversity (ribosomal DNA derived) down the horizons of an established grassland soil.
Measurements of methane oxidation rates were made in southeastern Bering Sea water samples with [14C]methane. The rate at which 14CO2 evolved from samples exposed to one methane concentration was defined as the relative methane oxidation rate. Rate determinations at three methane concentrations were used to estimate methane oxidation kinetics. The rate constant calculated from the kinetics and the observed methane concentration in the same water sample were used to calculate an in situ methane oxidation rate and the turnover time. First-order kinetics were observed in essentially all experiments in which methane oxidation kinetics were measured. Relative methane oxidation rates were greater in waters collected at inshore stations than at the offshore stations and were greater in bottom samples than in surface samples. In most water samples analyzed, there was essentially no radioactivity associated with the cells. The resulting respiration percentages were therefore very high with a mean of >98%. These data suggest that most of the methane was used by the microflora as an energy source and that very little of it was used in biosynthesis. The relative methane oxidation rates were not closely correlated with methane concentrations and did not appear to be linked to either oxygen or dissolved inorganic nitrogen concentrations. However, there was a significant correlation with relative microbial activity. Our data suggest that the methane oxidizers were associated with the general microbial heterotrophic community. Since these organisms did not appear to be using methane as a carbon source, it is unlikely that they have been isolated and identified as methane oxidizers in the past.
The effects of crude oil on uptake and respiration (mineralization) of glucose and glutamate in marine sediments were investigated. After the sediments were treated with crude oil, they were replaced at or near the collection site by scuba divers. These sediments remained in situ until they were retrieved for analysis. Glucose and glutamate uptake rates were found to decrease, and the percent respired was found to increase in Arctic and subarctic marine sediments that had been exposed to fresh crude oil. These same changes were also observed when “weathered” crude oil was used and when untreated sediments were overlaid with oiled sediments. When the kinetics of glutamate uptake were determined, both the maximum potential uptake rate and the turnover time were significantly affected. A comparison between the proportion of glucose taken into the cells and that respired as CO2 indicated that crude oil affected biosynthetic mechanisms. A study of sediments that had been exposed to crude oil for at least 5 months showed that glutamate transport into the cells was affected more extensively than biosynthetic mechanisms. In the initial months of exposure, bacterial concentrations and total adenylate concentrations were found to decrease in the presence of crude oil. Our data suggest that secondary productivity in the marine environment could be adversely affected by the presence of crude oil in marine sediments.
The acute effects of crude oil on glucose uptake rates by marine microorganisms were studied in 215 water and 162 sediment samples collected from both arctic and subarctic marine waters. The mean percentage reduction of glucose uptake rates ranged from 37 to 58 in the water samples exposed to crude oil and from 14 to 36 in the sediment samples. Substrate uptake kinetic studies indicated that the observed reductions by microbial populations exposed to crude oil were caused by metabolic inhibition. The effect of crude oil was less in sediments than in the water samples, with the difference being significant at the P < 0.0002 level. With the exception of one sediment study, all of the differences observed in the uptake rates between treated and nontreated samples were statistically significant. A high degree of variability was observed in the degree which glucose and glutamate uptake rates were altered in water samples exposed to crude oil. In some cases, uptake rates were greater in the samples exposed to crude oil. Data on samples collected in Cook Inlet suggested that areas where pelagic microorganisms are most probably chronically exposed to crude oil are also the areas where the effects of crude oil on glucose uptake are the lowest. Two studies indicated that after pelagic populations are exposed to crude oil for several days, the heterotrophic population adjusts to the presence of crude oil.
Rates of nitrogen fixation and denitrification were measured in Alaskan continental shelf sediments. In some regions, rates of nitrogen fixation and denitrification appeared to be equal; in other areas, rates were significantly different. Potential rates of denitrification were found to be limited primarily by the available nitrate substrate. Major regional differences in rates of denitrification were not statistically significant, but significant differences were found for nitrogen fixation rates in different regions of the Alaskan continental shelf. Estimated net losses of nitrogen from Bering Sea sediments were calculated as 1.8 × 1012 g of N/yr. Experimental exposure of continental shelf sediments to petroleum hydrocarbons reduced rates of nitrogen fixation and denitrification in some cases but not others. Long-term exposure was necessary before a reduction in nitrogen fixation rates was observed; unamended rates of denitrification but not potential denitrification rates (NO3− added) were depressed after exposure to hydrocarbons.