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The differential performance of polygenic risk scores (PRSs) by group is one of the major ethical barriers to their clinical use. It is also one of the main practical challenges for any implementation effort. The social repercussions of how people are grouped in PRS research must be considered in communications with research participants, including return of results. Here, we outline the decisions faced and choices made by a large multi-site clinical implementation study returning PRSs to diverse participants in handling this issue of differential performance. Our approach to managing the complexities associated with the differential performance of PRSs serves as a case study that can help future implementers of PRSs to plot an anticipatory course in response to this issue., Competing Interests: Declaration of interests A.C.F.L. owns some stock in Fabric Genomics; E.D.E. is an employee and stockholder of Invitae, advisor and stockholder of Taproot Health, and advisor and stockholder of Exir; R.C.G. receives compensation for advising the following companies: Allelica, Atria, Fabric, Genomic Life, and Juniper Genomics; and is co-founder of Genome Medical and Nurture Genomics; E.E.K. has received personal fees from Regeneron Pharmaceuticals, 23&Me, Allelica, and Illumina; has received research funding from Allelica; and serves on the advisory boards for Encompass Biosciences, Overtone, and Galatea Bio; N.L. received personal fees from Illumina Inc; E.P. is a paid consultant for Allelica Inc.; M.S. is a member of the Institutional Review Board of the All of Us Research Program; J.W.S. is a member of the Scientific Advisory Board of Sensorium Therapeutics (with equity), has received grant support from Biogen, Inc., and is PI of a collaborative study of the genetics of depression and bipolar disorder sponsored by 23andMe for which 23andMe provides analysis time as in-kind support but no payments., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Polygenic risk scores (PRSs) have improved in predictive performance, but several challenges remain to be addressed before PRSs can be implemented in the clinic, including reduced predictive performance of PRSs in diverse populations, and the interpretation and communication of genetic results to both providers and patients. To address these challenges, the National Human Genome Research Institute-funded Electronic Medical Records and Genomics (eMERGE) Network has developed a framework and pipeline for return of a PRS-based genome-informed risk assessment to 25,000 diverse adults and children as part of a clinical study. From an initial list of 23 conditions, ten were selected for implementation based on PRS performance, medical actionability and potential clinical utility, including cardiometabolic diseases and cancer. Standardized metrics were considered in the selection process, with additional consideration given to strength of evidence in African and Hispanic populations. We then developed a pipeline for clinical PRS implementation (score transfer to a clinical laboratory, validation and verification of score performance), and used genetic ancestry to calibrate PRS mean and variance, utilizing genetically diverse data from 13,475 participants of the All of Us Research Program cohort to train and test model parameters. Finally, we created a framework for regulatory compliance and developed a PRS clinical report for return to providers and for inclusion in an additional genome-informed risk assessment. The initial experience from eMERGE can inform the approach needed to implement PRS-based testing in diverse clinical settings., (© 2024. The Author(s).)

As large-scale genomic screening becomes increasingly prevalent, understanding the influence of actionable results on healthcare utilization is key to estimating the potential long-term clinical impact. The eMERGE network sequenced individuals for actionable genes in multiple genetic conditions and returned results to individuals, providers, and the electronic health record. Differences in recommended health services (laboratory, imaging, and procedural testing) delivered within 12 months of return were compared among individuals with pathogenic or likely pathogenic (P/LP) findings to matched individuals with negative findings before and after return of results. Of 16,218 adults, 477 unselected individuals were found to have a monogenic risk for arrhythmia (n = 95), breast cancer (n = 96), cardiomyopathy (n = 95), colorectal cancer (n = 105), or familial hypercholesterolemia (n = 86). Individuals with P/LP results more frequently received services after return (43.8%) compared to before return (25.6%) of results and compared to individuals with negative findings (24.9%; p < 0.0001). The annual cost of qualifying healthcare services increased from an average of $162 before return to $343 after return of results among the P/LP group (p < 0.0001); differences in the negative group were non-significant. The mean difference-in-differences was $149 (p < 0.0001), which describes the increased cost within the P/LP group corrected for cost changes in the negative group. When stratified by individual conditions, significant cost differences were observed for arrhythmia, breast cancer, and cardiomyopathy. In conclusion, less than half of individuals received billed health services after monogenic return, which modestly increased healthcare costs for payors in the year following return., Competing Interests: Declaration of interests W.K.C. is on the board of directors of Prime Medicine and D.L.V. is a consultant for Illumina and has a funded project from GeneDx., (Copyright © 2023 American Society of Human Genetics. All rights reserved.)

Polygenic risk scores (PRS) have potential to improve health care by identifying individuals that have elevated risk for common complex conditions. Use of PRS in clinical practice, however, requires careful assessment of the needs and capabilities of patients, providers, and health care systems. The electronic Medical Records and Genomics (eMERGE) network is conducting a collaborative study which will return PRS to 25,000 pediatric and adult participants. All participants will receive a risk report, potentially classifying them as high risk (∼2-10% per condition) for 1 or more of 10 conditions based on PRS. The study population is enriched by participants from racial and ethnic minority populations, underserved populations, and populations who experience poorer medical outcomes. All 10 eMERGE clinical sites conducted focus groups, interviews, and/or surveys to understand educational needs among key stakeholders-participants, providers, and/or study staff. Together, these studies highlighted the need for tools that address the perceived benefit/value of PRS, types of education/support needed, accessibility, and PRS-related knowledge and understanding. Based on findings from these preliminary studies, the network harmonized training initiatives and formal/informal educational resources. This paper summarizes eMERGE's collective approach to assessing educational needs and developing educational approaches for primary stakeholders. It discusses challenges encountered and solutions provided., Competing Interests: Conflict of Interest Emma Perez is a paid consultant for Allelica Inc. Lori Orlando and Tejinder Rakhra-Burris are founders of a company developing MeTree. Maureen Smith is a Section Editor for the Journal of Genetic Counseling. Maya Sabatello serves as IRB member of the All of Us Research Program. All other authors declare no conflicts of interest., (Copyright © 2023 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Purpose: Assessing the risk of common, complex diseases requires consideration of clinical risk factors as well as monogenic and polygenic risks, which in turn may be reflected in family history. Returning risks to individuals and providers may influence preventive care or use of prophylactic therapies for those individuals at high genetic risk., Methods: To enable integrated genetic risk assessment, the eMERGE (electronic MEdical Records and GEnomics) network is enrolling 25,000 diverse individuals in a prospective cohort study across 10 sites. The network developed methods to return cross-ancestry polygenic risk scores, monogenic risks, family history, and clinical risk assessments via a genome-informed risk assessment (GIRA) report and will assess uptake of care recommendations after return of results., Results: GIRAs include summary care recommendations for 11 conditions, education pages, and clinical laboratory reports. The return of high-risk GIRA to individuals and providers includes guidelines for care and lifestyle recommendations. Assembling the GIRA required infrastructure and workflows for ingesting and presenting content from multiple sources. Recruitment began in February 2022., Conclusion: Return of a novel report for communicating monogenic, polygenic, and family history-based risk factors will inform the benefits of integrated genetic risk assessment for routine health care., Competing Interests: Conflict of Interest N.S.A.-H. is an employee and equity holder of 23andMe; serves as a scientific advisory board member for Allelica, Inc; received personal fees from Genentech Inc, Allelica Inc, and 23andMe; received research funding from Akcea Therapeutics; and was previously employed by Regeneron Pharmaceuticals. T.W. has grant funding from Gilead Sciences, Inc. L.O. and T.R.-B are founders of a company developing MeTree. T.S., E.D.E., and E.H. are employees and stockholders of Invitae Corporation. E.M.M. has been a consultant for Avidity Bioscience, Amgen Inc, AstraZeneca, Cytokinetics, Invitae Corporation, Janssen Pharmaceuticals, Pfizer Inc, PepGen Inc, Tenaya Therapeutics, and Stealth BioTherapeutics Inc; she is also the founder of Ikaika Therapeutics. E.E.K. received personal fees from Illumina Inc, 23andMe, and Regeneron Pharmaceuticals and serves as a scientific advisory board member for Encompass Bioscience, Foresite Labs, and Galateo Bio. B.K. is an advisory board member and stockholder of Genome Medical. M.S. is a member of the Institutional Review Board of the All of Us Research Program. E.F.P. is a paid consultant for Allecia Inc. J.F.P. is a paid consultant for Natera Inc. All other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The implications of secondary findings detected in large-scale sequencing projects remain uncertain. We assessed prevalence and penetrance of pathogenic familial hypercholesterolemia (FH) variants, their association with coronary heart disease (CHD), and 1-year outcomes following return of results in phase III of the electronic medical records and genomics network., Methods: Adult participants (n=18 544) at 7 sites were enrolled in a prospective cohort study to assess the clinical impact of returning results from targeted sequencing of 68 actionable genes, including LDLR , APOB , and PCSK9 . FH variant prevalence and penetrance (defined as low-density lipoprotein cholesterol >155 mg/dL) were estimated after excluding participants enrolled on the basis of hypercholesterolemia. Multivariable logistic regression was used to estimate the odds of CHD compared to age- and sex-matched controls without FH-associated variants. Process (eg, referral to a specialist or ordering new tests), intermediate (eg, new diagnosis of FH), and clinical (eg, treatment modification) outcomes within 1 year after return of results were ascertained by electronic health record review., Results: The prevalence of FH-associated pathogenic variants was 1 in 188 (69 of 13,019 unselected participants). Penetrance was 87.5%. The presence of an FH variant was associated with CHD (odds ratio, 3.02 [2.00-4.53]) and premature CHD (odds ratio, 3.68 [2.34-5.78]). At least 1 outcome occurred in 92% of participants; 44% received a new diagnosis of FH and 26% had treatment modified following return of results., Conclusions: In a multisite cohort of electronic health record-linked biobanks, monogenic FH was prevalent, penetrant, and associated with presence of CHD. Nearly half of participants with an FH-associated variant received a new diagnosis of FH and a quarter had treatment modified after return of results. These results highlight the potential utility of sequencing electronic health record-linked biobanks to detect FH.

The electronic Medical Records and Genomics (eMERGE) Network assessed the feasibility of deploying portable phenotype rule-based algorithms with natural language processing (NLP) components added to improve performance of existing algorithms using electronic health records (EHRs). Based on scientific merit and predicted difficulty, eMERGE selected six existing phenotypes to enhance with NLP. We assessed performance, portability, and ease of use. We summarized lessons learned by: (1) challenges; (2) best practices to address challenges based on existing evidence and/or eMERGE experience; and (3) opportunities for future research. Adding NLP resulted in improved, or the same, precision and/or recall for all but one algorithm. Portability, phenotyping workflow/process, and technology were major themes. With NLP, development and validation took longer. Besides portability of NLP technology and algorithm replicability, factors to ensure success include privacy protection, technical infrastructure setup, intellectual property agreement, and efficient communication. Workflow improvements can improve communication and reduce implementation time. NLP performance varied mainly due to clinical document heterogeneity; therefore, we suggest using semi-structured notes, comprehensive documentation, and customization options. NLP portability is possible with improved phenotype algorithm performance, but careful planning and architecture of the algorithms is essential to support local customizations., (© 2023. The Author(s).)

Dogs with thoracolumbar intervertebral disc extrusion (TL-IVDE) can exhibit variable neurologic deficits after decompressive surgery. The objectives of this study were to quantify changes in static weight distribution (SWD) and limb and body circumference over time in dogs recovering from surgery for TL-IVDE. Dogs with acute TL-IVDE were prospectively evaluated at baseline (48-72 h post-operatively), 2, 4, 8, and 12 weeks post-operatively. Commercially-available digital scales were used to measure weight distributed to the pelvic limbs (PL%) and asymmetry between left and right pelvic limbs (LRA), each expressed as a percentage of total body weight. Trunk and thigh circumference measurements were performed using a spring-loaded tape measurement device. Measurements were performed in triplicate, compared to neurologically normal small breed control dogs and analyzed for changes over time. P <0.05 was significant. Twenty-one dogs were enrolled; 18 regained ambulation and 3 did not by study completion. PL% increased from 27.6% at baseline to 30.7% at 12 weeks but remained lower than in control dogs (37%) at all time points ( p < 0.0001), even excluding dogs still non-ambulatory at 12 weeks ( p < 0.025). LRA was similar to the control dogs, and did not have an association with surgical side. Caudal trunk girth decreased over time to 95% of baseline ( p = 0.0002), but this was no longer significant after accounting for reductions in body weight ( p = 0.30). Forward shifting of body weight persisted in dogs with TL-IVDE 12 weeks after surgery even among ambulatory dogs. SWD and circumference measurements could provide additional objective measures to monitor recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amaral Marrero, Thomovsky, Linder, Bowditch, Lind, Kazmierczak, Moore and Lewis.)

Recent advances in genomic technology and widespread adoption of electronic health records (EHRs) have accelerated the development of genomic medicine, bringing promising research findings from genome science into clinical practice. Genomic and phenomic data, accrued across large populations through biobanks linked to EHRs, have enabled the study of genetic variation at a phenome-wide scale. Through new quantitative techniques, pleiotropy can be explored with phenome-wide association studies, the occurrence of common complex diseases can be predicted using the cumulative influence of many genetic variants (polygenic risk scores), and undiagnosed Mendelian syndromes can be identified using EHR-based phenotypic signatures (phenotype risk scores). In this review, we trace the role of EHRs from the development of genome-wide analytic techniques to translational efforts to test these new interventions to the clinic. Throughout, we describe the challenges that remain when combining EHRs with genetics to improve clinical care.

Background: Objective outcome measures capable of tracking different aspects of functional recovery in dogs with acute intervertebral disc herniation are needed to optimize physical rehabilitation protocols. Normal, pre-injury distribution of body weight in this population is unknown. The aims of this study were to quantify static weight distribution (SWD) using digital scales and to establish the feasibility of different scale methods in neurologically normal, mature, chondrodystrophic small breed dogs predisposed to intervertebral disc herniation., Results: Twenty-five healthy, mature dogs were enrolled with a mean age of 4.6 years (SD 2.7) and a mean total body weight of 11.5 kg (SD 3.6). SWD for the thoracic and pelvic limbs and between individual limbs was acquired in triplicate and expressed as a percentage of total body weight using commercially available digital scales in four combinations: two bathroom, two kitchen (with thoracic and pelvic limbs combined), four bathroom and four kitchen (with limbs measured individually). SWD was also obtained using a pressure sensing walkway for comparison to scale data. Feasibility for each method was determined and coefficients of variation were used to calculate inter-trial variability. Mean SWD values were compared between methods using an ANOVA. The two bathroom scales method had the highest feasibility and lowest inter-trial variability and resulted in mean thoracic and pelvic limb SWD of 63 % (SD 3 %) and 37 % (SD 3 %), respectively. Thoracic limb mean SWD was higher for the PSW compared to any of the scale methods (p < 0.0001)., Conclusions: SWD in a population of healthy chondrodystrophic dogs was simple to obtain using inexpensive and readily available digital scales. This study generated SWD data for subsequent comparison to dogs recovering from acute intervertebral disc herniation.

Problem: GDM has been associated with disturbances in iron homeostasis and exaggerated immune activation. We sought to investigate the extent to which placental iron storage and macrophage accumulations were altered in GDM., Method of Study: We conducted a retrospective, case-control study of archived placental tissues obtained from 22 pregnancies complicated by GDM and 22 unaffected controls. Controls were matched to cases based on maternal age, gestational age at birth, and method of delivery. Placental tissues were assessed for altered histology and CD68 and CD163 staining. Tissue iron was assessed using Prussian blue staining., Results: Maternal hematocrit levels were higher in GDM participants compared to controls (P = 0.02). The presence of meconium-laden macrophages was significantly greater within the amnion of GDM cases (adjusted odds ratio (OR) 12.51). Although the total abundance of CD68-expressing macrophages was not significantly different between groups, we detected a significantly greater abundance of CD163 expression within the chorion and decidua of cases. The total area staining positive for iron was 24% (95% confidence intervals of 2%-46%) greater in GDM placentae versus controls., Conclusion: GDM is associated with altered placental histology and increases in meconium-laden macrophages. Greater iron stores within the placentae of women with GDM is consistent with reports that iron excess is associated with an increased risk for GDM. The higher level of expression of CD163 on macrophage-like cells of the chorion and decidua in GDM suggests an increase in M2-like macrophages. Overall, our results add to growing evidence that GDM has direct effects on placental structure., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Delineate risk factors associated with severe hypoxemia (O 2 sat ≤87%) in infants and children younger than 2 years hospitalized with single pathogen HRV infection., Study Design: Prospective study in a yearly catchment population of 56 560 children <2 years old between 2011 and 2013 in Argentina. All children with respiratory signs and O 2 sat <93% on admission were included. HRV infections were identified by reverse transcriptase-polymerase chain reaction. Epidemiologic, clinical, viral, and immunological risk factors were assessed., Results: Among 5012 hospitalized patients, HRV was detected as a single pathogen in 347 (6.92%) subjects. Thirty-two (9.2%) had life-threatening disease. Traditional risk factors for severe bronchiolitis did not affect severity of illness. HRV viral load, HRV groups, and type II and III interferons did not associate with severe hypoxemia. Interleukin-13 Levels in respiratory secretions at the time of admission (OR = 7.43 (3-18.4); P < 0.001 for IL-13 >10 pg/mL) predisposed to life-threatening disease., Conclusions: Targeted interventions against IL-13 should be evaluated to decrease severity of HRV illness in infancy and early childhood., (© 2018 Wiley Periodicals, Inc.)

Background: The Vanderbilt Institute for Clinical and Translational Research piloted the development of Project PLACENTA (PathLink Acquired gEstatioNal Tissue bAnk). This project investigated the feasibility of a fresh gestational tissue biobank, which provides tissue linked to electronic medical records for investigators interested in maternal-fetal health., Methods: We developed a pipeline for collection of placental tissue from Labor and Delivery within approximately 30 minutes of delivery. An email alert was developed, to signal delivery, with the ability to specifically flag patients with certain phenotypic traits. Once collected, 4 to 8 mm punch biopsy cores were snap frozen and subsequently used for DNA, RNA and protein extraction. Tissue was also collected for Formalin Fixed Paraffin Embedded (FFPE) histology, flow cytometry, and quality control measures., Results: Of 60 deliveries using the email notification system, 25 (42%) were sent to Pathology or assigned to other research protocols and were not available for collection, 10 (16%) were discarded prior to arrival at Labor and Delivery, and 25 (42%) were available for collection. Twenty placentas were collected and averaged 38 minutes per collection. DNA extraction yielded an average of 53 µg/µl per sample and RNA extraction yielded 679 ng/µl on average per sample. Proteomic studies showed no degradation of protein, abundant and similar quantities of protein across samples and differentiation between the amnion, decidua, and villi. Histological studies showed good quality for interpretation and occasional pathology including multifocal chronic villitis, meconium laden macrophages, and Stage 2 acute chorioamnionitis. Flow cytometry demonstrated good cell viability after isolation.

Salmonella Enteritidis (SE) is one of the most common causes of bacterial food-borne illnesses in the world. Despite the SE's ability to colonize and infect a wide-range of host, the most common source of infection continues to be the consumption of contaminated shell eggs and egg-based products. To date, the role of the source of SE infection has not been studied as it relates to SE pathogenesis and resulting disease. Using a streptomycin-treated mouse model of human colitis, this study examined the virulence of SE grown in egg yolk and Luria Bertani (LB) broth, and mouse feces collected from mice experimentally infected with SEE1 (SEE1 passed through mice). Primary observations revealed that the mice infected with SE grown in egg yolk displayed greater illness and disease markers than those infected with SE passed through mice or grown in LB broth. Furthermore, the SE grown in egg yolk achieved higher rates of colonization in the mouse intestines and extra-intestinal organs of infected mice than the SE from LB broth or mouse feces. Our results here indicate that the source of SE infection may contribute to the overall pathogenesis of SE in a second host. These results also suggest that reservoir-pathogen dynamics may be critical for SE's ability to establish colonization and priming for virulence potential.

Human rhinoviruses (RV) are associated with the majority of viral respiratory illnesses in infants, children and adults. Over the last several years, researchers have begun to sequence the many different species and strains of RV in order to determine if certain species were associated with increased disease severity. There are a variety of techniques employed to prepare samples for sequencing. One method utilizes plasmid-cloning, which is expensive and takes several hours to complete. Recently, some investigators have instead used direct sequencing to sequence RV strains, allowing for omission of the time- and labor-intensive cloning step. This study formally compares and contrasts the sequencing results obtained from plasmid-cloning and direct Sanger sequencing of a 500 base pair PCR product covering the VP4/VP2 region of RV. A slightly longer sequence (by 65 base pairs on average) was obtained when specimens were plasmid-cloned, and the sequences were 86% similar. After trimming the extra base pairs from the cloned sequences, the sequences were 99.7% identical. Overall success of directly sequencing samples was similar to that of cloning, 5% on average failed for each technique. Therefore, in many instances, directly sequencing samples may be considered in lieu of the more expensive and time-consuming plasmid-cloning technique., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Asthma and wheezing account for a substantial disease burden around the world. Very low birth weight (VLBW, <1500 grams) infants are at an increased risk for the development of severe acute respiratory illness (ARI) and recurrent wheeze/asthma. The role of respiratory viruses in asthma predisposition in premature infants is not well understood. Preliminary evidence suggests that infection with human rhinovirus (RV) early in life may contribute to greater burden of asthma later in life., Methods: A prospective cohort study of premature VLBW infants from Buenos Aires, Argentina, was enrolled year-round during a three-year period in the neonatal intensive care unit and followed during every ARI and with monthly well visits during the first year of life. Longitudinal follow-up up until age five years is ongoing., Results: This report describes the objectives, design, and recruitment results of this prospective cohort. Two hundred and five patients were enrolled from August 2011 through January 2014, and follow-up is ongoing. A total of 319 ARI episodes were observed from August 2011 to July 2014, and 910 well visits occurred during this time period., Conclusions: The Argentina Premature Asthma and Respiratory Team (APART) is a unique cohort consisting of over 200 patients and over 1200 specimens who have been and will continue to be followed intensively from NICU discharge to capture baseline risk factors and every ARI, with interceding well visits during the first year of life, as well as longitudinal follow-up to age 5 years for asthma and atopy outcomes., Competing Interests: Competing interests: The authors have declared that no competing interests exist.

Background: Human rhinoviruses (HRVs) cause common colds, and the recently discovered HRV-C is increasingly associated with lower respiratory illness among populations such as children and asthmatic patients., Objective: To determine how HRV-C is associated with respiratory illness and to evaluate changes in prevalence and species over 2 decades., Methods: A prospective study of children younger than 5 years was performed at the Vanderbilt Vaccine Clinic over a 21-year period. Nasal-wash specimens from children presenting with upper or lower respiratory illness at acute care visits were tested for HRV and HRV-positives genotyped. Demographic and clinical features were compared between children with or without HRV, and with different HRV species., Results: HRV was detected in 190 of 527 (36%) specimens from a population of 2009 children from 1982 through 2003. Of these, 36% were HRV-C. Age (P = .039) and month of illness (P < .001) were associated with HRV infection and HRV species. HRV-C was significantly associated with lower respiratory illness, compared with HRV-A (P = .014). HRV-A and HRV-C prevalence fluctuated throughout the 21-year period; HRV-C was more prevalent during winter (P = .058)., Conclusions: HRV-C is not a new virus but has been significantly associated with childhood lower respiratory illness in this population for several decades. Temporal changes in virus prevalence occur, and season may predict virus species. Our findings have implications for diagnostic, preventive, and treatment strategies due to the variation in disease season and severity based on species of HRV infection., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Activation of the immune system is beneficial in defending against pathogens, but may also have costly side effects on an organism's fitness. In this study we examine the fitness consequences of immune challenge in female Drosophila melanogaster by examining both direct (within generation) and indirect (between generations) costs and benefits of immune challenge. Though passing immunity to offspring has been studied in mammals for many years, only recently have researchers found evidence for a cross-generational priming response in invertebrates. By examining both potential fitness costs and benefits in the next generation, we were able to determine what effect immune challenge has on fitness. In agreement with other studies, we found a direct cost to infection, where immune challenged females laid fewer eggs than unchallenged females in two of the three lines we examined. In addition, we found some evidence for indirect costs. Offspring from immune challenged mothers had shorter lifespans than those from unchallenged mothers in two of the three lines. Interestingly, we do not see any effect of maternal immune challenge on offspring's ability to overcome an infection, nor do we see an effect on other fitness traits measured, including egg size, egg-adult viability and offspring resistance to oxidative stress. While previous studies in bumblebees and beetles have demonstrated cross-generation priming, our results suggest that it may not be a general phenomenon, and more work is needed to determine how widespread it is.

Drosophila melanogaster is widely used to study immune system function in insects. However, little work has been done in D. melanogaster on the effect of temperature on the immune system. Here we describe experiments that demonstrate that cooler temperatures enhance survival after infection and alter expression of immune-related genes in flies. This effect appears to be due not only to the fact that colder temperatures slow down bacterial growth, but also to the beneficial effects of cooler temperature on immune function. We explore the possibility that heat shock proteins, and in particular, Hsp83, may improve immune function at cool temperatures. We have long known that temperature can alter immune responses against microbial pathogens in insects. The approach described here allows us to determine whether this effect is due primarily to temperature-specific effects on the host or on its pathogen. These results suggest that both may be important.

Reproductive costs are an essential component of evolutionary theory. For instance, an increase in reproduction is generally coupled with a decrease in immunocompetence shortly after mating. However, recent work in Drosophila melanogaster suggests that the potential to mount an immune response, as measured by the levels of immune gene expression, increases after mating. These data are in contrast to previous studies, which suggest that mating can reduce a fly's ability to survive an actual bacterial challenge (realized immunity). This pattern may be driven by some aspect of mating, independent of resource limitation, which reduces immune function by inhibiting the effective deployment of immune gene products. Though several studies have examined both the potential and the realized immunity after mating, none have examined these immune measures simultaneously. Here, we examined the link between the potential and the realized immunity in a sterile mutant of D. melanogaster. Shortly after mating, we found that female immune gene expression was high, but survival against infection was low. Surprisingly, this pattern was reversed within 24 h. Thus, estimates of immunity based on gene expression do not appear to reflect an actual ability to defend against pathogens in the hours following copulation. We discuss the possible mechanisms that may account for this pattern.

We describe a graphical model of interlocus coevolution used to distinguish between the interlocus sexual conflict that leads to sexually antagonistic coevolution, and the intrinsic conflict over mating rate that is an integral part of traditional models of sexual selection. We next distinguish the 'laboratory island' approach from the study of both inbred lines and laboratory populations that are newly derived from nature, discuss why we consider it to be one of the most fitting forms of laboratory analysis to study interlocus sexual conflict, and then describe four experiments using this approach with Drosophila melanogaster. The first experiment evaluates the efficacy of the laboratory model system to study interlocus sexual conflict by comparing remating rates of females when they are, or are not, provided with a spatial refuge from persistent male courtship. The second experiment tests for a lag-load in males that is due to adaptations that have accumulated in females, which diminish male-induced harm while simultaneously interfering with a male's ability to compete in the context of sexual selection. The third and fourth experiments test for a lag-load in females owing to direct costs from their interactions with males, and for the capacity for indirect benefits to compensate for these direct costs.

The empirical foundation for sexual conflict theory is the data from many different taxa demonstrating that females are harmed while interacting with males. However, the interpretation of this keystone evidence has been challenged because females may more than counterbalance the direct costs of interacting with males by the indirect benefits of obtaining higher quality genes for their offspring. A quantification of this trade-off is critical to resolve the controversy and is presented here. A multi-generation fitness assay in the Drosophila melanogaster laboratory model system was used to quantify both the direct costs to females due to interactions with males and indirect benefits via sexy sons. We specifically focus on the interactions that occur between males and nonvirgin females. In the laboratory environment of our base population, females mate soon after eclosion and store sufficient sperm for their entire lifetime, yet males persistently court these nonvirgin females and frequently succeed in re-mating them. Females may benefit from these interactions despite direct costs to their lifetime fecundity if re-mating allows them to trade-up to mates of higher genetic quality and thereby secure indirect benefits for their offspring. We found that direct costs of interactions between males and nonvirgin females substantially exceeded indirect benefits through sexy sons. These data, in combination with past studies of the good genes route of indirect benefits, demonstrate that inter-sexual interactions drive sexually antagonistic co-evolution in this model system.

One of Ernst Mayr's legacies is the consensus that the allopatry model is the predominant mode of speciation in most sexually reproducing lineages. In this model, reproductive isolation develops as a pleiotropic byproduct of the genetic divergence that develops among physically isolated populations. Presently, there is no consensus concerning which, if any, evolutionary process is primarily responsible for driving the specific genetic divergence that leads to reproductive isolation. Here, we focus on the hypothesis that inter-locus antagonistic coevolution drives rapid genetic divergence among allopatric populations and thereby acts as an important "engine" of speciation. We assert that only data from studies of experimental evolution, rather than descriptive patterns of molecular evolution, can provide definitive evidence for this hypothesis. We describe and use an experimental approach, called hemiclonal analysis, that can be used in the Drosophila melanogaster laboratory model system to simultaneously screen nearly the entire genome for both standing genetic variation within a population and the net-selection gradient acting on the variation. Hemiclonal analysis has four stages: (i) creation of a laboratory "island population"; (ii) cytogenetic cloning of nearly genome-wide haplotypes to construct hemiclones; (iii) measurement of additive genetic variation among hemiclones; and (iv) measurement of the selection gradient acting on phenotypic variation among hemiclones. We apply hemiclonal analysis to test the hypothesis that there is ongoing antagonistic coevolution between the sexes in the D. melanogaster laboratory model system and then discuss the relevance of this analysis to natural systems.

The sexual conflict hypothesis predicts that males evolve traits that exploit the higher parental investment of females, which generates selection for females to counter-evolve resistance. In Drosophila melanogaster it is now established that males harm females and that there is genetic variation among males for the degree of this harm. Genetic variation among females for resistance to harm from males, and the corresponding strength of selection on this variation, however, have not been quantified previously. Here we carryout a genome-wide screen for female resistance to harm from males. We estimate that the cost of interactions with males depresses lifetime fecundity of females by 15% (95% CI: 8.2-22.0), that genetic variation for female resistance constitutes 17% of total genetic variation for female adult fitness, and that propensity to remate in response to persistent male courtship is a major factor contributing to genetic variation for female resistance.

The significance of blood monocytes as a source of osteoclast precursors was investigated during 1 alpha-hydroxycholecalciferol-stimulated bone resorption in mice. Animals were given three injections of tritiated thymidine at 8 hourly intervals in order to label blood monocytes. The proportion of labelled monocytes was then compared with the proportion of labelled nuclei in osteoclasts, the formation of which was provoked by daily injections of 1 alpha-hydroxycholecalciferol, beginning 48 hours after the first injection of isotope. Although more than 60% of blood monocytes were labelled during the period of peak osteoclast formation, labelling of osteoclast nuclei in the metaphyseal endosteum of the femur never exceeded 8%. These results suggest strongly that the majority of osteoclast nuclei were derived from a source of unlabelled precursors, and that very few osteoclasts were derived directly from blood monocytes. Although few labelled osteoclasts were found, the proportion of labelled spindle shaped cells in the metaphyseal endosteum rose gradually to 16%. It is likely, therefore, that labelled mononuclear cells, possibly blood monocytes, were attracted to the endosteum where they formed a population of tissue macrophages. At this site they may participate in bone resorption, either individually or by providing a local pool of osteoclast precursors.

Changes in neutrophil precursor populations in the bone marrow and their alkaline phosphatase reactivity following an inflammatory stimulus were studied in the rat using an osmiophilic method. Seven groups each of 3 Sprague-Dawley rats received subcutaneous injections of turpentine, and femoral marrow was examined at intervals up to 72 hr. Depletion of mature neutrophils resulted in an increased first in the myeloblast-promyelocyte compartment and at 48-72 hr in the myelocyte-metamyelocyte population. By 72 hr replenishment of the mature neutrophil marrow population had begun. Within 6 hr marked acceleration of cytoplasmic maturation was evident, together with accelerated synthetic activity, manifested by marked dilatation of the rough endoplasmic reticulum and an enlarged Golgi. Together with these changes there was an increase in the number of alkaline phosphatase reactive cells, which was evident first in the myeloblast-promyelocyte population at 2 hr. An absolute increase in the amount of enzyme reaction product associated with individual cells was also seen.

Mammalian epidermis and oral epithelial possess an intercellular permeability barrier which is located in the superficial region of the tissue. This study reports a staining reaction which appears to demonstrate a histological correlate of this functional property. Specimens of ear skin, palate, buccal and oesophageal mucosa and of cornea and bladder were obtained from adult rabbits and rats, bisected and either incubated in vitro with 2.5% horseradish peroxidase as a tracer or fixed and processed for light microscopy and stained with a modification of Hart's elastin stain. Examination of specimens prepared by each procedure showed a complementary staining pattern in the intercellular spaces of the stratum corneum or in the superficial region of the non-keratinized tissue. In the epidermis and oral and oesophageal epithelia, the region which excluded the tracer stained with the modified elastin stain. In contrast, the corneal and bladder epithelia neither excluded the tracer nor showed intercellular staining. This relationship between staining of the intercellular space and the exclusion of tracer suggests that the intercellular material in the superficial region of epithelia may be chemically altered to form a barrier substance, possibly as the result of the discharge of the contents of the membrane-coating granules which are present in all the epithelia examined except the cornea and bladder.

The ultrastructural localization of alkaline phosphatase in eosinophil leucocytes, obtained from experimentally-induced peritoneal exudates in rats, has been studied using an osmiophilic technique with 2-naphthylthiolphosphoryl dichloride as substrate, fast Blue BBN as diazonium salt and postosmication with 1% aqueous osmium tetroxide. With this method identical incubation procedures could be used for both light and electron microscope examination. Eosinophils were the only cells which contained alkaline phosphatase. The enzyme was predominantly associated with the outer surface of the plasma membrane, being present in much lower concentrations in cytoplasmic cisternae. Eosinophil granules only rarely showed reaction product. The plasma membrane location of alkaline phosphatase in eosinophil leucocytes is identical to that recently demonstrated in the human neutrophil.

Demineralization of cartilage with alcoholic EDTA provides cartilage staining that is no better, as measured by scanning microdensitometry, than that of adequately fixed specimens demineralized with aqueous EDTA. Aqueous EDTA is a faster demineralizing agent than alcoholic EDTA. Certain fixatives can preserve maximal proteoglycan staining in articular cartilage even with subsequent rapid demineralization in formate buffer at pH 3.3. Although alcoholic formalin fixation provided optimum quantitative cartilage staining, cetylpyridinium chloride (CPC) in aqueous buffered formalin improved cellular detail, but CPC partially suppressed matrix staining.

A study of emergency medicine residency training graduates was conducted to determine their perceptions of the quality of their graduate training. A sample of 300 individuals was randomly selected from a population of 1,000 persons graduating from 1982 through 1984. Respondents were asked to use a scale of 1 to 5 (with 1 being highest) to rate the adequacy of their residency training relative to 20 major core content areas. A 50% response rate (N = 151) was achieved. Mean ratings of residents' perceptions of the adequacy of their training relative to the core content ranged from 1.7 to 3.24. Training in resuscitation and stabilization, principles of emergency care, and general assessment were among the most highly rated, while training in physician interpersonal skills, disorders related to the immune system, and cutaneous disorders were rated the lowest. Overall, residents were quite positive in their perceptions regarding the quality of their training. They indicated plans to attend continuing medical education programs to reinforce some of their training and to address some of the deficiencies they perceived in residency training. Programs are encouraged to conduct similar surveys with their own graduates to assess particular strengths and weaknesses.

Methyl green-pyronin is a notoriously difficult stain to reproduce. Although very useful in detecting cells containing substantial amounts of RNA, it is of limited use in broader problems of cell identification. By careful standardization of the proportions of methyl green to pyronin and combination of these stains with hematoxylin to enhance nuclear contrast and with orange G to improve connective tissue staining, it was possible to produce a consistently reliable staining preparation in which it is possible to identify all the component cells of a mixed inflammatory infiltrate in routine paraffin sections.

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.

In order to investigate a monocyte origin for osteoclasts, tritiated thymidine labelled blood monocytes, harvested from the blood of donor mice, were injected intravenously into syngeneic recipient animals in which osteoclast formation was being stimulated by concomitant intraperitoneal injections of 1 alpha-hydroxycholecalciferol. Labelled osteoclasts were found in autoradiographs prepared from the femurs of recipient mice, demonstrating for the first time that, during hormonally stimulated osteoclast formation, blood monocytes form one source of osteoclasts.