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OBJECTIVES: The purpose of this paper is to describe the student body of a culturally diverse nurse anesthesia program and to identify implications for professional socialization as well as nurse anesthesiology education and practice. METHODS: A five year study (1997-2002) of Barry University's Master of Science Program in Anesthesiology was conducted. Findings were compared with available statistics published by the American Association of Nurse Anesthetists as well as a large national sample in a study by Waugaman and Lu (1999). RESULTS: Findings revealed a diverse student body with respect to ethnicity, gender, and past career experiences. Despite students' varied backgrounds, the median grade point average for the program graduates was 3.8 on a 4.0 scale. Retention rate for the program has averaged about 90% per year. The program also attained a 100% board certification passage rate in 2003. CONCLUSIONS: Diverse students need to be recruited and retained in nurse anesthesiology to be able to deliver culturally sensitive care to multicultural, underserved, and various other populations. [ABSTRACT FROM AUTHOR]

Competing Interests: Conflicts of interest I have no conflicts of interest.

Background: Based on records dating from 1859 to 2021, we provide an overview of the marine animal diversity reported for Galiano Island, British Columbia, Canada. More than 650 taxa are represented by 20,000 species occurrence records in this curated dataset, which includes dive records documented through the Pacific Marine Life Surveys, museum voucher specimens, ecological data and crowd-sourced observations from the BC Cetacean Sightings Network and iNaturalist., New Information: We describe Galiano Island's marine animal diversity in relation to the Salish Sea's overall biodiversity and quantify the proportional contributions of different types of sampling effort to our current local knowledge. Overviews are provided for each taxonomic group in a format intended to be accessible to amateur naturalists interested in furthering research into the region's marine biodiversity. In summary, we find that the Pacific Marine Life Surveys, a regional community science diving initiative, account for 60% of novel records reported for Galiano Island. Voucher specimens account for 19% and crowd-sourced biodiversity data 18% of novel records, respectively, with the remaining 3% of reports coming from other sources. These findings shed light on the complementarity of different types of sampling effort and demonstrate the potential for community science to contribute to the global biodiversity research community. We present a biodiversity informatics framework that is designed to enable these practices by supporting collaboration among researchers and communities in the collection, curation and dissemination of biodiversity data.

Background: Surgical site infections (SSIs) continue to represent a substantial source of morbidity, mortality, and healthcare costs. The purpose of this study was to determine the effect of implementing a protocol using home pre-operative surgical preparation on the SSI rate at a large, urban safety-net medical center. Patients and Methods: From July through December 2020, Nose-to-Toes ® (N2T; Sage Products-Stryker Corporation, Cary, IL) full-body preparation was applied by patients at home on the morning of scheduled surgical procedures. This study was a single-institution, retrospective observational analysis to determine the rates of SSI ≤30 days after an operation. Patients having skin preparation during 2020 (post-N2T) were compared with patients having the same operation during 2019 without having skin preparation (pre-N2T). Results: For gynecology, 10 (7.4%) of 135 pre-N2T and three (2.2%) of 135 post-N2T patients had SSIs. For surgical and gynecologic oncology, 13 (15.1%) of 86 pre-N2T and four (4.7%) of 86 post-N2T patients had SSIs. For orthopedics, four (4.3%) of 94 pre-N2T and zerp of 94 post-N2T patients had SSIs. Overall, 27 (8.6%) of 315 pre-N2T and seven (2.2%) of 315 post-N2T patients had SSIs (p = 0.0004). Conclusions: The implementation of pre-operative full-body preparation was associated with a substantial reduction in the incidence of SSI.

Introduction: Amyloid-related imaging abnormalities (ARIA) are a common, dose-dependent effect of amyloid-targeting antibodies, strongly associated with the apolipoprotein E (APOE) ε4 allele., Methods: We describe the clinical course and management of a 66-year-old white male (APOE ε4/ε4) enrolled in an observational study that included amyloid and tau positron emission tomography (PET), who received aducanumab through the ENGAGE clinical trial., Results: Acute symptoms included headache and encephalopathy, and magnetic resonance imaging revealed ARIA-E and ARIA-H. Malignant hypertension and epileptiform activity were treated with nicardipine and levetiracetam. Subsequent clinical/imaging worsening prompted a course of methylprednisolone. Symptoms and ARIA-E resolved over 6 months, while ARIA-H persisted. Quantitative analysis of interval amyloid PET showed reduced signal in pre-existing areas but increased signal posteriorly; while tau PET showed increased signal overall., Discussion: In an APOE ε4/ε4 patient, ARIA symptoms were accompanied by malignant hypertension and epileptiform activity, and pulsed steroids reversed edema. Studies from larger cohorts may clarify the optimal treatment and pathophysiology of ARIA., Competing Interests: Adam L. Boxer has received research support from NIH, the Tau Research Consortium, the Association for Frontotemporal Degeneration, Bluefield Project to Cure Frontotemporal Dementia, Corticobasal Degeneration Solutions, the Alzheimer's Drug Discovery Foundation, and the Alzheimer's Association. He has served as a consultant for Aeton, Abbvie, Alector, Amgen, Arkuda, Arvinas, Asceneuron, Ionis, Lundbeck, Novartis, Passage BIO, Samumed, Third Rock, Toyama, and UCB, and received research support from Avid, Biogen, BMS, C2N, Cortice, Eli Lilly, Forum, Genentech, Janssen, Novartis, Pfizer, Roche, and TauRx. Other authors report no conflict of interest. Biogen, Incorporated, funded the ENGAGE trial, and Avid Radiopharmaceuticals enabled use of the precursor for 18F‐Flortaucipir, but neither Biogen nor Avid provided direct funding to the authors and were not involved in data analysis or interpretation. Biogen and Avid were allowed to review and comment on the manuscript prior to submission., (© 2020 The Authors. Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring published by Wiley Periodicals, LLC on behalf of Alzheimer's Association.)

Background: Animal-assisted intervention (AAI) programs are increasing in popularity, but it is unknown to what extent therapy animal organizations that provide AAI and the hospitals and eldercare facilities they work with implement effective animal health and safety policies to ensure safety of both animals and humans. Our study objective was to survey hospitals, eldercare facilities, and therapy animal organizations on their AAI policies and procedures., Methods: A survey of United States hospitals, eldercare facilities, and therapy animal organizations was administered to assess existing health and safety policies related to AAI programs., Results: Forty-five eldercare facilities, 45 hospitals, and 27 therapy animal organizations were surveyed. Health and safety policies varied widely and potentially compromised human and animal safety. For example, 70% of therapy animal organizations potentially put patients at risk by allowing therapy animals eating raw meat diets to visit facilities. In general, hospitals had stricter requirements than eldercare facilities., Discussion: This information suggests that there are gaps between the policies of facilities and therapy animal organizations compared with recent guidelines for animal visitation in hospitals., Conclusions: Facilities with AAI programs need to review their policies to address recent AAI guidelines to ensure the safety of animals and humans involved., (Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Animal-assisted activities (AAA) and animal-assisted therapy (AAT) programs are increasing in popularity, but current programs vary in their safety and health policies. Veterinarians can have an important role in ensuring the safety of both the animals and humans involved, but it is unclear how best to educate veterinary students to serve effectively in this role. Therefore, the goal of this study was to assess the knowledge gaps and perceptions of first-year veterinary students on health and safety aspects of AAA/AAT programs by administering a survey. This information could then guide future educational training in veterinary schools to address the knowledge gaps in this area. Formal education during the veterinary curriculum had not yet been provided to these students on AAA/AAT before the survey. Of 98 first-year veterinary students, 91 completed the survey. When asked about policies on visiting animals, 58% of students responded that nursing homes are required to have a policy and 67% responded that hospitals are required to have one. Three quarters of students reported that veterinarians, animal handlers, and facilities should share the responsibility for ensuring safe human-animal interaction in AAA/AAT programs. Most (82%) of the students responded that all or most national and local therapy animal groups prohibit animals that consume raw meat diets from participating in AAA/AAT programs. The results of this survey will help veterinary schools better identify knowledge gaps that can be addressed in veterinary curricula so future veterinarians will be equipped to provide appropriate public health information regarding AAA/AAT programs.

Bone Morphogenic Protein 2 (BMP2) can induce ectopic bone. This ability, which first motivated the widespread application of BMP2 in fracture healing and spinal arthrodesis has, more recently, been indicated as one of several serious adverse effects associated with the supra-physiological doses of BMP2 relied upon for clinical efficacy. Key to harnessing BMPs and other agents safely and effectively will be the ability to localize activity at a target site at substantially reduced doses. Clay (Laponite) nanoparticles can self assemble into gels under physiological conditions and bind growth factors for enhanced and localized efficacy. Here we show the ability to localize and enhance the activity of BMP2 to achieve ectopic bone formation at doses within the sub-microgram per ml range of concentrations sufficient to induce differentiation of responsive cell populations in vitro and at approximately 3000 fold lower than those employed in clinical practice., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Addition of bioactive materials such as calcium phosphates or Bioglass, and incorporation of porosity into polyetheretherketone (PEEK) has been identified as an effective approach to improve bone-implant interfaces and osseointegration of PEEK-based devices. In this paper, a novel production technique based on the extrusion freeforming method is proposed that yields a bioactive PEEK/hydroxyapatite (PEEK/HA) composite with a unique configuration in which the bioactive phase (i.e., HA) distribution is computer-controlled within a PEEK matrix. The 100% interconnectivity of the HA network in the biocomposite confers an advantage over alternative forms of other microstructural configurations. Moreover, the technique can be employed to produce porous PEEK structures with controlled pore size and distribution, facilitating greater cellular infiltration and biological integration of PEEK composites within patient tissue. The results of unconfined, uniaxial compressive tests on these new PEEK/HA biocomposites with 40% HA under both static and cyclic mode were promising, showing the composites possess yield and compressive strength within the range of human cortical bone suitable for load bearing applications. In addition, preliminary evidence supporting initial biological safety of the new technique developed is demonstrated in this paper. Sufficient cell attachment, sustained viability in contact with the sample over a seven-day period, evidence of cell bridging and matrix deposition all confirmed excellent biocompatibility.

This review explores the application of hydrogels in orthopaedic clinical situations which may benefit from enhanced growth factor delivery and improved osteogenesis of bone graft material. Hydrogels are defined, and in vivo evidence supporting their application in these clinical areas is explored. Our focus is on clinically pertinent properties, such as the chemistry of formation, biocompatibility, efficacy of cell and growth factor delivery, ability to withstand mechanical loading and potential to be delivered via an injection. Naturally derived hydrogels, such as gelatin, hyaluronic acid and fibroin, together with a number of synthetic polyethylene glycol-based gels combined with protease-sensitive domains, have shown excellent biocompatibility. There is significant literature evidence supporting the ability of hydrogels to facilitate growth factor and cell delivery. Burst release of the selected growth factor remains a consistent challenge, which has been overcome in some studies with chemical modifications of the hydrogel. Interestingly, a number of studies detail percutaneous delivery with hydrogels combined with calcium-based minerals to enhance osteogenicity, with mixed results. Few of the studies explored the biomechanical properties of the materials, and none of the studies reviewed demonstrated the ability of a hydrogel/graft material to withstand mechanical loading in a clinically relevant segmental bone defect model., (Copyright © 2014 John Wiley & Sons, Ltd.)

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca(2+) late during activation of CD4 cells. Increased levels of mitochondrial Ca(2+) in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca(2+) is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases.

Additive manufacturing (AM) is a broad term encompassing 3D printing and several other varieties of material processing, which involve computer-directed layer-by-layer synthesis of materials. As the popularity of AM increases, so to do expectations of the medical therapies this process may offer. Clinical requirements and limitations of current treatment strategies in bone grafting, spinal arthrodesis, osteochondral injury and treatment of periprosthetic joint infection are discussed. The various approaches to AM are described, and the current state of clinical translation of AM across these orthopedic clinical scenarios is assessed. Finally, we attempt to distinguish between what AM may offer orthopedic surgery from the hype of what has been promised by AM.

Controversy remains regarding the optimal post-operative analgesic regimen following total knee replacement. A delicate balance is required between the provision of adequate pain relief and early mobilisation. By reviewing 29 randomised trials we sought to establish whether local infiltration of analgesia directly into the knee during surgery provides better pain relief and a more rapid rehabilitation. Although we were able to conclude that local infiltration can provide improved post-operative pain relief, and to suggest the most promising technique of administration, there is no evidence that it reduces hospital stay.

Background: To determine if patients presenting with an infrarenal aorta of 25-30 mm in diameter benefit from continued ultrasound surveillance, and if so to identify the frequency required., Methods: All patients in the Leicestershire abdominal aortic aneurysm (AAA) screening program with an initial AAA diameter of 25-30 mm and who had undergone two or more surveillance scans were identified (345 patients). The primary endpoint was death from AAA rupture, referral for elective repair, or presentation with rupture. This information together with the duration of surveillance was recorded., Results: A total of 345 patients were followed up for mean of 4.25 years (1-11). At 5 years surveillance, there was a 97% freedom from referral for repair or death from rupture., Conclusion: Patients presenting with an AAA diameter 25-30 mm may be safely deferred any further surveillance for a period of 5 years., (Copyright © 2010. Published by Elsevier Inc.)

Cementoplasty is a well-established intervention for symptomatic control of localized skeletal metastases; however, its use has never been reported for metastases in the foot. We report on a patient with symptomatic skeletal metastases throughout the foot in which cementoplasty, in conjunction with internal fixation, was used to alleviate pain and enable comfortable ambulation., (Copyright 2010 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.)

After respiratory virus infections, memory CD8+ T cells are maintained in the lung airways by a process of continual recruitment. Previous studies have suggested that this process is controlled, at least in the initial weeks after virus clearance, by residual antigen in the lung-draining mediastinal lymph nodes (MLNs). We used mouse models of influenza and parainfluenza virus infection to show that intranasally (i.n.) primed memory CD8+ T cells possess a unique ability to be reactivated by residual antigen in the MLN compared with intraperitoneally (i.p.) primed CD8+ T cells, resulting in the preferential recruitment of i.n.-primed memory CD8+ T cells to the lung airways. Furthermore, we demonstrate that the inability of i.p.-primed memory CD8+ T cells to access residual antigen can be corrected by a subsequent i.n. virus infection. Thus, two independent factors, initial CD8+ T cell priming in the MLN and prolonged presentation of residual antigen in the MLN, are required to maintain large numbers of antigen-specific memory CD8+ T cells in the lung airways.

In the face of emerging infectious diseases caused by rapidly evolving and highly virulent pathogens, such as influenza, we are challenged to develop innovative vaccine strategies that can induce lasting protection. Since CD4 T cells are needed to generate and maintain protective B-cell and CD8 T-cell immunity, and can also mediate additional protective mechanisms, vaccines should ideally elicit efficient CD4 T cell, in addition to CD8 T and B-cell responses. We outline here the process of CD4 T-cell differentiation from naïve to effector and from effector to memory with an emphasis on how exposure to microbial products and variables in antigen presentation can impact the functional quality and heterogeneity of activation-based CD4 T-cell subsets in vitro and in vivo. We discuss the impact of different phases of antigen recognition, the inflammatory milieu, acute versus chronic antigen presentation, and the contribution of residual antigen depots on CD4 T-cell effector differentiation and the formation and maintenance of CD4 T-cell memory. We propose that novel vaccine strategies, which incorporate both microbial products and antigen targeting, may provide a flexible and long-lived memory CD4 T-cell pool.

Encounter with Ag during chronic infections results in the generation of phenotypically and functionally heterogeneous subsets of Ag-specific CD8 T cells. Influenza, an acute infection, results in the generation of similar CD8 T cell heterogeneity, which may be attributed to long-lived depots of flu Ags that stimulate T cell proliferation well after virus clearance. We hypothesized that the heterogeneity of flu-specific CD8 T cells and maintenance of T cell memory required the recruitment of new CD8 T cells to persistent depots of flu Ag, as was the case for flu-specific CD4 T cell responses. However, robust expansion and generation of highly differentiated cytolytic effectors and memory T cells only occurred when naive CD8 T cells were primed during the first week of flu infection. Priming of new naive CD8 T cells after the first week of infection resulted in low numbers of poorly functional effectors, with little to no cytolytic activity, and a negligible contribution to the memory pool. Therefore, although the presentation of flu Ag during the late stages of infection may provide a mechanism for maintaining an activated population of CD8 T cells in the lung, few latecomer CD8 T cells are recruited into the functional memory T cell pool.

We present the results of a technique of dynamic hip screw insertion through a very small incision, typically 2-2.5 cm. The technique is performed using a standard dynamic hip screw set and requires no additional equipment. We compared the results to those of an age and sex-matched group who had undergone the operation through a traditional approach. We compared the time spent in theatre, the pre- and post-operative haemoglobin concentration, haematocrit and prevalence of wound infection. Thirteen consecutive cases were performed by one surgeon using the percutaneous technique. There were nine females and four males with a mean age of 84 years (range 62-96 years). Each had a 135 degrees four-hole plate. The mean post-operative drop in haemoglobin concentration in the percutaneous group was 2.2 g/dl (range 0-4.4 g/dl) compared to 3.5 g/dl (range 1.2-5.4 g/dl) in the control group (p = 0.014). The mean haematocrit drop was 0.07 (range 0-0.12) in the percutaneous group compared to 0.10 (range 0.03-0.17) in the control group (p = 0.017). The mean theatre time with the percutaneous technique was 57 min (range 40-75 min) and in the control group, 60 min (range 30-95 min). There were no wound problems. It is likely that this minimally invasive technique offers a better clinical outcome at no extra expense and warrants further evaluation in a larger study.

Migration of dendritic cells (DCs) to the draining lymph node (DLN) is required for the activation of naive T cells. We show here that migration of DCs from the lung to the DLN after Mycobacterium tuberculosis (Mtb) exposure is defective in mice lacking interleukin (IL)-12p40. This defect compromises the ability of IL-12p40-deficient DCs to activate naive T cells in vivo; however, DCs that express IL-12p40 alone can activate naive T cells. Treatment of IL-12p40-deficient DCs with IL-12p40 homodimer (IL-12(p40)(2)) restores Mtb-induced DC migration and the ability of IL-12p40-deficient DCs to activate naive T cells. These data define a novel and fundamental role for IL-12p40 in the pathogen-induced activation of pulmonary DCs.

We have outlined the carefully orchestrated process of CD4+ T-cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T-cell interaction with antigen-presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4+ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multifaceted roles of CD4+ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4+ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.

The kinetics of presentation of influenza virus-derived antigens (Ags), resulting in CD4 T cell effector and memory generation, remains undefined. Naive influenza-specific CD4 T cells were transferred into mice at various times after influenza infection to determine the duration and impact of virus-derived Ag presentation. Ag-specific T cell responses were generated even when the donor T cells were transferred 3-4 wk after viral clearance. Transfer of naive CD4 T cells during early phases of infection resulted in a robust expansion of highly differentiated effectors, which then contracted to a small number of memory T cells. Importantly, T cell transfer during later phases of infection resulted in a modest expansion of effectors with intermediate phenotypes, which were capable of persisting as memory with high efficiency. Thus, distinct stages of pathogen-derived Ag presentation may provide a mechanism by which T cell heterogeneity is generated and diverse memory subsets are maintained.

IL-12p70 induced IFN-gamma is required to control Mycobacterium tuberculosis growth; however, in the absence of IL-12p70, an IL-12p40-dependent pathway mediates induction of IFN-gamma and initial bacteriostatic activity. IL-23 is an IL-12p40-dependent cytokine containing an IL-12p40 subunit covalently bound to a p19 subunit that is implicated in the induction of CD4 T cells associated with autoimmunity and inflammation. We show that in IL-23 p19-deficient mice, mycobacterial growth is controlled, and there is no diminution in either the number of IFN-gamma-producing Ag-specific CD4 T cells or local IFN-gamma mRNA expression. Conversely, there is an almost total loss of both IL-17-producing Ag-specific CD4 T cells and local production of IL-17 mRNA in these mice. The absence of IL-17 does not alter expression of the antimycobacterial genes, NO synthase 2 and LRG-47, and the absence of IL-23 or IL-17, both of which are implicated in mediating inflammation, fails to substantially affect the granulomatous response to M. tuberculosis infection of the lung. Despite this redundancy, IL-23 is required to provide a moderate level of protection in the absence of IL-12p70, and this protection correlates with a requirement for IL-23 in the IL-12p70-independent induction of Ag-specific, IFN-gamma-producing CD4 T cells. We also show that IL-23 is required for the induction of an IL-17-producing Ag-specific phenotype in naive CD4 T cells in vitro and that absence of IL-12p70 promotes an increase in the number of IL-17-producing Ag-specific CD4 T cells both in vitro and in vivo.

Chronic infections often result in CD8 T-cell deletion or functional nonresponsiveness. However, to date no definitive studies have attempted to determine the impact of repeated T cell receptor stimulation on CD4 effector T cell generation. We have determined that when antigen presentation is limited to 2 d, optimum in vitro CD4 effector generation is achieved. Alternatively, repeated stimulation results in decreased CD4 effector expansion, decreased cytokine production, and altered migration. Similarly, functionally impaired effectors develop in vivo when antigen-pulsed antigen-presenting cells are replenished every 24 h during a primary immune response. CD4 effectors that are generated with repeated stimulation provide no protection during influenza infection, and have an impaired ability to provide cognate help to B cells. These results suggest that duration of antigen presentation dictates CD4 effector function, and repeated T cell receptor stimulation in vitro and in vivo that exceeds an optimal threshold results in effectors with impaired function.

Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4(+) T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIP(L), receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4(+) T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling.

We studied MHC class II (MHC-II)-restricted antigen processing of viable Streptococcus pyogenes by murine macrophages for presentation of two CD4 T cell epitopes of the surface M5 protein. We show that presentation of both epitopes was prevented if actin polymerization was inhibited by cytochalasin D, but not if clathrin-dependent receptor-mediated endocytosis was prevented, suggesting uptake of streptococci by phagocytosis or macropinocytosis was required for presentation of the surface M protein. However, treatment of macrophages with amiloride, which selectively blocks membrane ruffling and subsequent macropinocytosis, inhibited the response to one epitope (M5(308-319)), but had no effect on presentation of the other (M5(17-31)). The effect of the inhibitors on uptake of streptococci was analyzed by electron microscopy. Cytochalasin D completely blocked uptake of streptococci, while dimethyl-amiloride only inhibited uptake into spacious compartments. Neither of the inhibitors altered the cell-surface expression of MHC-II and costimulatory molecules analyzed by flow cytometry. The data suggest that distinct epitopes of a protein associated with viable bacteria may be presented optimally following different uptake mechanisms in the same antigen-presenting cells.

CD23 is constitutively and atypically expressed on malignant B-cells in patients with chronic lymphocytic leukemia. It exists in two isoforms that differ only in a short amino acid sequence at the N-terminus. The CD23a isoform exhibits an endocytosis signal, that renders it more efficient in antigen uptake than CD23b. Therefore, we analyzed the regulation of CD23 isoforms and tested the ability to stimulate T-cell clones by targeting antigen to CD23 on CLL B-cells. Investigation of several stimulators to promote CD23a expression on CLL versus normal B-cells confirmed a different CD23 regulation in B-CLL. We did not find any evidence for a differential regulation of the two CD23 isoforms in B-CLL. However, CD23a is always predominantly expressed with a constant ratio of CD23a:CD23b. We show that antigen targeted to CD23 on CLL B-cells is very efficiently presented. Therefore, CD23 is likely to provide a suitable target for receptor-mediated antigen presentation in B-CLL which can be used to activate a T-cell response.

Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved.

Efficient peptide presentation by professional APC to naive and effector CD4 T cells in vitro is limited to the first 1-2 days of culture, but is nonetheless optimum for effector expansion and cytokine production. In fact, prolonging Ag presentation leads to high levels of T cell death, decreased effector expansion, and decreased cytokine production by recovered effectors. Despite the absence of Ag presentation beyond day 2, T cell division continues at a constant rate throughout the 4-day culture. The Ag-independent later stage depends on the presence of IL-2, and we conclude optimum effector generation depends on an initial 2 days of TCR stimulation followed by an additional 2 days of Ag-independent, cytokine driven T cell expansion and differentiation.

Ag presentation to CD4 T cells is a critical event in the generation of protective immunity. IgG, in the form of IgG-pathogen (Ag) complexes, is capable of mediating FcgammaR-dependent Ag presentation, and thereby enhanced T cell activation. Therefore, it is important to understand the ability of the individual human IgG subclasses to function in enhanced T cell activation. We hypothesized that increased delivery of Ag to monocyte FcgammaR by high affinity human IgG subclasses, IgG1 and IgG3, would lead to increased Ag presentation, as compared to low affinity IgG subclasses, IgG2 and IgG4. To create immune complexes, we linked biotinylated IgG subclasses to biotinylated Ag via an avidin bridge, and examined T cell responses to them. Although IgG2- and IgG4-Ag complexes bound to monocytes at significantly lower levels than those made with IgG1 and IgG3, we observed no significant difference in the ability of the four human IgG subclasses to mediate enhanced T cell activation. Studies suggest the explanation for this dichotomy lies within the first 24 h of Ag processing, and that processing efficiency may vary with IgG subclass. They also suggest the existence of a highly efficient, and selective processing pathway, which is dependent on IgG subclass, and can compensate for low level production and FcgammaR binding of IgG2- and IgG4-Ag complexes.

Generation of an effective cellular immune response is key to the successful development of both humoral and cellular immune defences against most pathogens. However, while the type of cellular immune response elicited by any given pathogen is dictated by the entire array of antigens and molecules which comprise that pathogen, most studies of human immune responses to bacterial pathogens tend to focus on selected antigens. This is a result, in part, of a desire to find those antigens that will generate a desired immune response, as well as limited technology for monitoring the complex array of responses generated by an intact organism. Utilizing Streptococcus mutans as a model Gram-positive organism, a novel flow cytometric assay that permits the identification of individual cells within a responding population, and highly sensitive cytokine assays, we show for the first time that CD8 T cells and natural killer (NK) cells comprise a significant component of the response to this organism in humans. This is despite the fact that CD8 T cells are traditionally thought to respond to endogenously derived antigens only. In addition, we provide the first evidence that a Gram-positive organism can actively inhibit interleukin-2 (IL-2), an important autocrine growth factor for T cells. The latter observation could represent an additional mechanism by which Gram-positive organisms evade host defences.

Basilar artery blood flow was measured by transcranial Doppler ultrasonography before and during hyperventilation in nine patients with panic disorder and nine normal comparison subjects. The hyperventilation-induced decrease in basilar artery blood flow was significantly greater in patients with panic attacks than in comparison subjects. Two patients with decreases in basilar flow greater than 80% were successfully treated with nimodipine, a centrally active calcium channel blocker.

Hypophysial protal blood was collected from pentobarbital anesthetized male rats, and the content of corticotropin releasing factor-like immunoreactivity (CRF-LI) was measured using an RIA developed for synthetic ovine CRF. The concentration of CRF-LI in portal plasma was 104.9 +/- 9.7 pM. This concentration is in the range which has been shown to stimulate ACTH and beta-endorphin secretion in vitro. These data support the hypothesis that a molecule with the same or similar structure as synthetic ovine CRF is a physiologically significant hypothalamic releasing factor.

To determine atrial natriuretic factor (ANF) concentrations in the circulation and body fluids of adult pregnant sheep and their fetuses, pregnant ewes were anesthetized with pentobarbital sodium, and the fetuses were exteriorized for sampling. ANF concentration, as measured by radioimmunoassay, was 47 +/- 6 (SE) pg/ml in maternal plasma, which was significantly higher than the 15 +/- 3 pg/ml in maternal urine. In the fetus, plasma ANF concentration was 265 +/- 49 pg/ml, 5.6 times that in maternal plasma. No umbilical arterial and venous difference in ANF concentration was observed. Fetal urine ANF concentration (13 +/- 2 pg/ml) was significantly lower than that in fetal plasma, and was similar to that measured in amniotic and allantoic fluid. In chronically catheterized maternal and fetal sheep, fetal plasma ANF was again 5.1 times that in maternal plasma, and these levels were not different from those measured in acutely anesthetized animals. These results demonstrate that immunoreactive ANF is present in the fetal circulation at levels higher than those found in the mother. The low concentration of ANF in fetal urine suggests that ANF is probably metabolized and/or reabsorbed by the fetal kidney.

Depressed patients exhibit an abnormal "supersensitive" increase in the plasma concentration of several pituitary hormones following intravenous injection of the acetyl cholinesterase inhibitor physostigmine (PHY). In the present study, we examined the effects of PHY treatments on the plasma concentrations of prolactin (PRL) and adrenocorticotrophic hormone (ACTH) in the rat. Physostigmine (0-0.6 mg/kg, s.c.) produced a dose-dependent increase in PRL and ACTH immunoreactivity in unoperated animals. Neurotoxin-induced depletion of brain dopamine (DA) or norepinephrine (NE) did not significantly alter baseline plasma PRL or ACTH values. Following depletion of brain DA, but not NE, animals exhibited a "supersensitive" increase in plasma ACTH values, which was evidenced by a sixfold left shift in the dose-response properties of PHY. These results suggest that there are intriguing parallels between the abnormal endocrine response to PHY demonstrated by depressed patients and that demonstrated by rats following depletion of central nervous system (CNS) DA levels.

This study was designed to determine if active secretion of epinephrine (EPI) and/or 5-hydroxytryptamine (5-HT) from the hypothalamus into the hypophysial portal vasculature takes place, in addition to the well-known secretion of dopamine (DA). Hypophysial portal plasma was collected from urethane-anesthetized male rats by stalk cannulation (Porter method) or by periodic aspiration of portal blood (Worthington and Fink method). Portal and peripheral plasma concentrations of EPI, 5-HT, DA and 5-hydroxyindole-3-acetic acid (5-HIAA) were concurrently measured by high performance liquid chromatography with electrochemical detection. Significantly higher concentrations of EPI were found in hypophysial portal than in peripheral plasma. After adrenalectomy (ADX), peripheral plasma levels of EPI were undetectable, whereas portal plasma EPI levels were only slightly attenuated. Although 5-HT levels in portal and peripheral plasma were not different, 5-HIAA levels were 3-fold higher in portal plasma. DA was 10-15 fold higher in portal plasma. All the above differences were found independent of the collection method. The high level of 5-HIAA in portal plasma was not due to conversion of 5-HT to 5-HIAA by monoamine oxidase in plasma. The results indicate that in addition to DA, another amine (EPI) and an amine metabolite (5-HIAA) have a concentration gradient in portal vs peripheral plasma. Moreover, the presence of EPI in portal plasma after ADX is a strong indication that EPI is primarily derived from a central source, suggesting that the amine may have a direct physiological role in the regulation of anterior pituitary function.

The distribution of atrial natriuretic factor (ANF)-like reactivity was examined in rat brain and heart by immunohistochemistry. Immunostaining in heart was confined to atrial myocytes. In the hypothalamus, ANF-absorbable immunoreactivity was observed in magnocellular perikarya of the paraventricular and supraoptic nuclei, and in their projections to the neural lobe of the pituitary gland. No staining was seen in the preoptic or arcuate hypothalamic nuclei or in brain stem nuclei as previously reported by other investigators. The patterns of reactivity for ANF reported here is similar to that observed for neurophysins (NPs). Comparison of sequence data between rat ANF-28 and bovine NPs revealed three regions of 3 amino acid homology between these hypothalamic peptides. Preabsorption of the ANF antiserum with Affigel-coupled bovine NP I also resulted in complete elimination of all "ANF-immunoreactivity" in both atrium and hypothalamus. Cross-reactivity of the ANF antiserum with bovine NP I and II was further confirmed by Western blot analysis. Our findings suggest that ANF antisera can cross-react with NPs if they are directed against the shared antigenic epitopes; complete elimination of staining by preabsorption of the antibody with the immunogen, therefore, does not guarantee authenticity of localization. These observations may have relevance to an earlier study which reported on the existence of ANF-immunoreactivity in oxytocin neurons of the hypothalamus.

Recent reports have described modulation of atrial natriuretic factor (ANF) secretion by atrial stretch, increased Na+ concentration, and a variety of hormones and neurotransmitters. One problem in the study of ANF secretion has been the isolation of stimulatory effects from interference due to rhythmic myocardial contraction. To avoid this problem, rat atria were dispersed, the myocytes were suspended in a polyacrylamide gel matrix, and the resulting cell column was perifused with a physiological buffer. The secretion of ANF immunoactivity was markedly stimulated by increases in extracellular osmolality, regardless of the solute (NaCl, KCl, or glucose). This effect did not require extracellular Ca2+, nor could it be mimicked by depolarizing concentrations of K+ in an isotonic medium. Functional viability of this model was demonstrated by significant dose-related increases in ANF release in response to as little as 1 nM epinephrine. The dissociation of ANF secretion from depolarization-induced changes in Ca2+ flux is unusual and may represent an adaptation to the dual roles of the atrial myocyte, contraction and secretion.

Using rhesus monkeys, we studied the effects of a behavioral stress on plasma concentrations of adrenocorticotropin, oxytocin, and vasopressin. The stress resulted in significant increases in adrenocorticotropin and significant decreases in oxytocin concentrations. No significant changes were seen in vasopressin concentrations. To further explore the relationship between plasma oxytocin and pituitary-adrenal function, dexamethasone was administered to rhesus monkeys. This resulted in significant increases in plasma oxytocin concentrations, while adrenocorticotropin decreased.

The release of oxytocin (OT) into pituitary portal blood during the estrous cycle was studied in female rats under Althesin anesthesia. On each afternoon of the 4-day estrous cycle, animals were anesthetized with Althesin, and pituitary portal blood samples were collected. The concentration of OT in portal plasma collected between 14.00 and 16.00 h of proestrus (1,079 +/- 185 pg/ml) was significantly higher than that at any other time studied. There were no appreciable differences between the mean OT concentration on the days of estrus (198 +/- 29 pg/ml), metestrus (283 +/- 55 pg/ml), and diestrus (267 +/- 49 pg/ml). The concentration of OT in pituitary portal plasma was also greater than that in blood withdrawn from the external jugular vein (less than 41-67 pg/ml). Comparison of the portal plasma OT with peripheral plasma prolactin in Althesin-anesthetized rats indicated an association between the release of these hormones during the estrous cycle.

Oxytocin (OT) generally has a stimulatory effect on ACTH secretion both in vitro and in vivo. As part of a study of ACTH-releasing factors in hypophysial portal blood, the effects of i.v. OT administration on plasma ACTH levels were tested in urethane-anesthetized rats. Surprisingly, i.v. injection of 10 micrograms OT lowered plasma ACTH levels by about 35% (P less than 0.01). It was reasoned that this paradoxical inhibition of ACTH secretion by OT might be mediated by inhibition of the unusually high rate of peripheral catecholamine secretion in this model. Measurement of plasma catecholamines before and after i.v. administration of 10 micrograms OT revealed a 53% inhibition of EPI (P less than 0.01) and 43% inhibition of NE (P less than 0.05). Administration of the beta-adrenergic antagonist propranolol (400 micrograms) 15 min before the beginning of the experiment completely blocked the inhibitory effects of OT on ACTH secretion and in fact unmasked the stimulatory effects of OT normally seen in conscious animals and in vitro. Superfused bisected adrenal glands exposed to 10(-6) M OT for 10 min secreted more than 30% less EPI and NE than control adrenals suggesting that the inhibition of EPI and NE secretion by OT in vivo occurs, at least in part, directly at the level of the adrenal. The data support the hypothesis that peripheral catecholamines may at times be directly involved in the control of ACTH secretion and also suggest that OT, which has recently been identified in the adrenal medulla, may have important paracrine functions in the regulation of adrenal catecholamine secretion.

Pituitary portal blood was collected from urethane-anesthetized rats and examined for the presence of neuropeptide Y (NPY) using high-performance liquid chromatography and radioimmunoassay. Other rats were perfused with fixative, and coronal sections through the hypothalamus and median eminence were processed for immunohistochemical localization of NPY. Combined high-performance liquid chromatography and radioimmunoassay analysis of pituitary portal plasma and systemic plasma revealed a single peak of NPY immunoreactivity which corresponded in retention time to synthetic porcine NPY. Increasing amounts of portal or systemic plasma produced displacement curves which were parallel to the NPY standard curve. The concentration of NPY immunoreactivity in portal plasma (52.0 +/- 4.0 ng/ml, mean +/- SEM) was three times greater (p less than 0.005) than in systemic plasma (16 +/- 4.5 ng/ml). NPY-labeled fibers were observed in the external zone of the median eminence in the vicinity of hypothalamo-hypophyseal portal vessels. The observation of significantly higher concentrations of NPY immunoreactivity in the portal plasma supports the hypothesis that NPY may be released from the hypothalamus to affect pituitary function.

ACTH secretion is primarily controlled by hypothalamic secretion of corticotropin releasing factor (CRF) into pituitary portal blood. However arginine vasopressin (AVP) and oxytocin (OT) can modulate the actions of CRF and at times may be important mediators of stress-induced ACTH secretion. The relative contributions of CRF, AVP, and OT to the control of ACTH secretion vary with different types of stress. In general, AVP stimulates ACTH secretion in all species studied. OT also stimulates ACTH release in rats but is inhibitory in primates. The involvement of AVP and OT in the control of ACTH secretion may have important implications for physiological and pathological conditions associated with activation of the hypothalamo--hypophysial--adrenal cortical axis.

Two methods for the collection of pituitary portal blood were compared. The portal blood aspiration method developed by Worthington and Fink consists of periodically aspirating portal blood as it collects around the transected pituitary stalk. The stalk cannulation method developed by Porter eliminates possible contamination of the sample by extraneous blood by collecting the portal blood into a polyethylene cannula which fits tightly around the stalk. In this study, contamination of portal blood collected by the portal blood aspiration method was identified and quantitated by measuring flow rates and LH concentration of blood from the various sources contributing to the collection. Blood collected by the portal blood aspiration method contained 68% pure portal blood, 21% extraneous peripheral blood from damaged tissues, and 11% pituitary blood from the distal end of the transected stalk. The flow rate of pure portal blood was the same in the two methods. Despite the problem of contamination and dilution, the portal blood aspiration method is well suited for measurement of labile substances because the blood can be added to appropriate preservatives within seconds. For the measurement of more stable substances, the stalk cannulation method is preferred.

The role of oxytocin (OT) in regulating stress-induced ACTH secretion was investigated by immunoneutralization of endogenous OT with an antiserum raised against synthetic OT. Rats were subjected to one of three stresses: novel environment, tail-hang, or ether. In otherwise untreated rats, ACTH levels rose at least 3-fold in response to all three stresses whereas OT levels increased only in response to tail-hang and ether. Injection of a highly specific antiserum to OT 60 min before the experiment inhibited the ACTH response to tail-hang and ether by 64 and 56%, respectively, but had no effect on the ACTH response to novel environment stress. The data support a physiologic role for OT in the control of ACTH secretion but suggest that it is not until OT levels rise in response to stress that the effects of OT are expressed.

The effects of CRF(41), oxytocin (OT), and arginine vasopressin (AVP) on ACTH secretion were studied alone and in combination in an in vitro system of superfused rat hemipituitaries. CRF(41) (10(-9)M) and AVP (10(-8)M) alone produced a significant increase in ACTH secretion while OT (10(-8)M) alone had no effect. However the same concentration of OT markedly potentiated the ACTH response to CRF(41) while having no effect on the ACTH response to AVP. The data support a physiologic role for OT in the regulation of ACTH secretion.

To investigate the mechanism by which ACTH secretion is inhibited during hypothermia, hypophysial portal blood was collected from euthermic and hypothermic rats, and the concentrations of corticotropin-releasing factor (CRF), vasopressin (AVP), and oxytocin (OT) were measured by RIA. Whereas CRF levels in portal plasma were not different in the two groups, AVP and OT levels were significantly lower in hypothermic rats. The concentration of AVP and OT in peripheral plasma was also significantly lower in hypothermic rats compared with euthermic controls. The pituitary responsiveness to CRF during hypothermia was tested in vivo and in vitro. In pentobarbital-anesthetized male rats injected iv with 0.1 or 1.0 nmol CRF, the ACTH response was significantly smaller in hypothermic compared with euthermic animals. However, hemipituitaries superfused at 31 C released the same amount of ACTH in response to 1 nM CRF as hemipituitaries superfused at 37 C (31 C, 541 +/- 90 pg; 37 C, 563 +/- 29 pg) despite reduced baseline secretion (31 C, 77 +/- 10 pg/10 min; 37 C, 114 +/- 14 pg/10 min; P less than 0.05). The data suggest that the inhibition of ACTH secretion during hypothermia is mediated by decreased hypothalamic secretion of AVP and OT which in turn decreases the pituitary responsiveness to CRF.