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In five lines of cultured human squamous carcinoma cells, transglutaminase activity and envelope competence were highly sensitive to retinoic acid and calcium levels in the growth medium. In cells grown in low calcium medium, these measures of keratinocyte differentiation were reduced. Retinoic acid suppressed envelope competence but total transglutaminase activity was markedly reduced, slightly affected, or greatly stimulated depending upon the cell line and whether the cells were grown in low calcium or 1.8 mM calcium-containing medium. Examination by anion exchange chromatography of the transglutaminase activity in SCC-12B2 cultures showed that expression of the particulate form (type I) of the enzyme was greatly stimulated by calcium. The increase in this activity to high levels that occurs at confluence could be almost completely suppressed by retinoic acid in the medium. The soluble form (type II) in the SCC-12B2 cells was induced in growing or confluent cultures by retinoic acid independent of the calcium concentration in the medium, but the 50% effective concentration (100 nM) for its stimulation was approximately 50-fold higher than the 50% effective concentration for suppression of the type I enzyme (2 nM). Thus, these enzymes appear to be distinct and independently regulated. This conclusion is supported by the finding that SCC-4 and SCC-9 almost exclusively expressed types II and I forms, respectively. In contrast to the results with neoplastic cells, in cultured normal epidermal cells type I enzyme comprised the overwhelming majority of activity and was only partially (75-90%) suppressible by retinoic acid, while type II enzyme seemed poorly if at all stimulable. Thus, the SCC lines appear appropriate for studying biochemical mechanisms of action of certain physiological agents, the molecular basis for altered regulation of differentiated function in neoplastic cells, and the origin of diversity within tumors.

3-Methylcholanthrene (3-MC) greatly inhibits the growth of two lines of human squamous carcinoma cells, SCC-9 and SCC-12B2. Exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin alone was much less effective and, in the presence of 3-MC, did not alter the sensitivity (EC50 = 0.3 microM) or extent of growth inhibition by the latter. The degree of 3-MC-mediated inhibition, however, was markedly alleviated by inclusion of retinoic acid (EC50 greater than or equal to 0.7 microM) and hydrocortisone (EC50 = 40 nM) or dexamethasone (EC50 = 3 nM) in the culture medium. These physiological effectors, which are known to have opposing actions on keratinocyte character in SCC cells, did not significantly alter either aryl hydrocarbon hydroxylase activity or macromolecular adduct formation. Further analysis of the cellular responses indicated that hydrocortisone and, in some experiments, retinoids increased the growth rate in 3-MC-exposed cultures, while 3-MC increased the saturation density in retinoic acid-exposed cultures, an example of interference with a physiological response of the cells. These results indicate that alteration of the differentiated state, regardless of the direction of the change, can alter the sensitivity of the cells to toxic stimuli. Further investigation of the bases of such toxic responses and their modulation by the microenvironment may enhance our understanding of the target cell specificity of polycyclic aromatic hydrocarbons.

Exploiting the sensitivity of neoplastic keratinocytes to physiological effectors, this work analyzes the degree of coordination among differentiation markers in the established human epidermal squamous carcinoma cell line SCC-13 in comparison to normal human epidermal cells. This analysis showed that overall keratin content was modulated substantially and in parallel with particulate transglutaminase activity in response to variation of calcium, retinoic acid, and hydrocortisone concentrations in the medium. The changes in keratin expression were evident primarily in the striking stimulation by hydrocortisone or calcium and the virtual suppression by retinoic acid of species in the 56-58 kd region, which have not previously been reported subject to such physiological modulation. In contrast, involucrin levels were coordinated only to a limited degree with particulate transglutaminase activity and keratin content. The very low involucrin levels observed in low calcium medium were increased 5- to 10-fold in high calcium medium. However, they were also increased 5- to 30-fold in low calcium medium by retinoic acid, a clear example of uncoupling. Activities of the tissue transglutaminase were altered considerably by the various culture conditions but were not obviously coordinated to keratinocyte markers. In normal epidermal cells, the suppressive effect of retinoic acid was much more evident with particulate transglutaminase than involucrin levels. While calcium had a large stimulatory effect on both markers, hydrocortisone had little or no influence. These results emphasize the potential importance of quantitative analysis of differentiation markers for resolving the contribution of physiological elements in coordination of cellular programming.

Retinoic acid and retinyl acetate induce tissue transglutaminase to high levels in cultured SCC-4 keratinocytes, increasing the enzyme specific activity over 50-fold under optimal conditions. Pretreatment of the cells for a day with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC) or benzo[a]pyrene almost completely prevented the induction observed upon subsequent treatment with retinoic acid for 2 days. Similar aromatic compounds that do not induce aryl hydrocarbon hydroxylase (pyrene, dibenzofuran) did not exhibit this suppressive effect. The concentration dependence on TCDD for induction of aryl hydrocarbon hydroxylase was nearly identical to that for its suppression of transglutaminase induction, with half-maximal effects observed at approximately 20 pM in each instance. Similarly, the concentrations of 3-MC giving half-maximal stimulation of the hydroxylase and suppression of the transglutaminase were comparable (0.9 and 0.3 microM, respectively), although this agent was almost five orders of magnitude less potent than TCDD. These observations reveal a loss of cellular sensitivity to vitamin A mediated by the Ah receptor.

In five lines of cultured human squamous carcinoma cells, transglutaminase activity and envelope competence were highly sensitive to retinoic acid and calcium levels in the growth medium. In cells grown in low calcium medium, these measures of keratinocyte differentiation were reduced. Retinoic acid suppressed envelope competence but total transglutaminase activity was markedly reduced, slightly affected, or greatly stimulated depending upon the cell line and whether the cells were grown in low calcium or 1.8 mM calcium-containing medium. Examination by anion exchange chromatography of the transglutaminase activity in SCC-12B2 cultures showed that expression of the particulate form (type I) of the enzyme was greatly stimulated by calcium. The increase in this activity to high levels that occurs at confluence could be almost completely suppressed by retinoic acid in the medium. The soluble form (type II) in the SCC-12B2 cells was induced in growing or confluent cultures by retinoic acid independent of the calcium concentration in the medium, but the 50% effective concentration (100 nM) for its stimulation was approximately 50-fold higher than the 50% effective concentration for suppression of the type I enzyme (2 nM). Thus, these enzymes appear to be distinct and independently regulated. This conclusion is supported by the finding that SCC-4 and SCC-9 almost exclusively expressed types II and I forms, respectively. In contrast to the results with neoplastic cells, in cultured normal epidermal cells type I enzyme comprised the overwhelming majority of activity and was only partially (75-90%) suppressible by retinoic acid, while type II enzyme seemed poorly if at all stimulable. Thus, the SCC lines appear appropriate for studying biochemical mechanisms of action of certain physiological agents, the molecular basis for altered regulation of differentiated function in neoplastic cells, and the origin of diversity within tumors.

Sleep is essential for nervous system functioning and sleep disorders are associated with several neurodegenerative diseases. However, the macroscale connectivity changes in brain networking during different sleep states are poorly understood. One of the hindering factors is the difficulty to combine functional connectivity investigation methods with spontaneously sleeping animals, which prevents the use of numerous preclinical animal models. Recent studies, however, have implicated that urethane anesthesia can uniquely induce different sleep-like brain states, resembling rapid eye movement (REM) and non-REM (NREM) sleep, in rodents. Therefore, the aim of this study was to assess changes in global connectivity and topology between sleep-like states in urethane anesthetized rats, using blood oxygenation level dependent (BOLD) functional magnetic resonance imaging. We detected significant changes in corticocortical (increased in NREM-like state) and corticothalamic connectivity (increased in REM-like state). Additionally, in graph analysis the modularity, the measure of functional integration in the brain, was higher in NREM-like state than in REM-like state, indicating a decrease in arousal level, as in normal sleep. The fMRI findings were supported by the supplementary electrophysiological measurements. Taken together, our results show that macroscale functional connectivity changes between sleep states can be detected robustly with resting-state fMRI in urethane anesthetized rats. Our findings pave the way for studies in animal models of neurodegenerative diseases where sleep abnormalities are often one of the first markers for the disorder development.

Background: The emergency department is a critical juncture in the trajectory of care of patients with serious, life-limiting illness. Implementation of a clinical decision support (CDS) tool automates identification of older adults who may benefit from palliative care instead of relying upon providers to identify such patients, thus improving quality of care by assisting providers with adhering to guidelines. The Primary Palliative Care for Emergency Medicine (PRIM-ER) study aims to optimize the use of the electronic health record by creating a CDS tool to identify high risk patients most likely to benefit from primary palliative care and provide point-of-care clinical recommendations., Methods: A clinical decision support tool entitled Emergency Department Supportive Care Clinical Decision Support (Support-ED) was developed as part of an institutionally-sponsored value based medicine initiative at the Ronald O. Perelman Department of Emergency Medicine at NYU Langone Health. A multidisciplinary approach was used to develop Support-ED including: a scoping review of ED palliative care screening tools; launch of a workgroup to identify patient screening criteria and appropriate referral services; initial design and usability testing via the standard System Usability Scale questionnaire, education of the ED workforce on the Support-ED background, purpose and use, and; creation of a dashboard for monitoring and feedback., Results: The scoping review identified the Palliative Care and Rapid Emergency Screening (P-CaRES) survey as a validated instrument in which to adapt and apply for the creation of the CDS tool. The multidisciplinary workshops identified two primary objectives of the CDS: to identify patients with indicators of serious life limiting illness, and to assist with referrals to services such as palliative care or social work. Additionally, the iterative design process yielded three specific patient scenarios that trigger a clinical alert to fire, including: 1) when an advance care planning document was present, 2) when a patient had a previous disposition to hospice, and 3) when historical and/or current clinical data points identify a serious life-limiting illness without an advance care planning document present. Monitoring and feedback indicated a need for several modifications to improve CDS functionality., Conclusions: CDS can be an effective tool in the implementation of primary palliative care quality improvement best practices. Health systems should thoughtfully consider tailoring their CDSs in order to adapt to their unique workflows and environments. The findings of this research can assist health systems in effectively integrating a primary palliative care CDS system seamlessly into their processes of care., Trial Registration: ClinicalTrials.gov Identifier: NCT03424109. Registered 6 February 2018, Grant Number: AT009844-01.

Introduction: Emergency departments (ED) care for society's most vulnerable older adults who present with exacerbations of chronic disease at the end of life, yet the clinical paradigm focuses on treatment of acute pathologies. Palliative care interventions in the ED capture high-risk patients at a time of crisis and can dramatically improve patient-centred outcomes. This study aims to implement and evaluate Primary Palliative Care for Emergency Medicine (PRIM-ER) on ED disposition, healthcare utilisation and survival in older adults with serious illness., Methods and Analysis: This is the protocol for a pragmatic, cluster-randomised stepped wedge trial to test the effectiveness of PRIM-ER in 35 EDs across the USA. The intervention includes four core components: (1) evidence-based, multidisciplinary primary palliative care education; (2) simulation-based workshops; (3) clinical decision support; and (4) audit and feedback. The study is divided into two phases: a pilot phase, to ensure feasibility in two sites, and an implementation and evaluation phase, where we implement the intervention and test the effectiveness in 33 EDs over 2 years. Using Centers for Medicare and Medicaid Services (CMS) data, we will assess the primary outcomes in approximately 300 000 patients: ED disposition to an acute care setting, healthcare utilisation in the 6 months following the ED visit and survival following the index ED visit. Analysis will also determine the site, provider and patient-level characteristics that are associated with variation in impact of PRIM-ER., Ethics and Dissemination: Institutional Review Board approval was obtained at New York University School of Medicine to evaluate the CMS data. Oversight will also be provided by the National Institutes of Health, an Independent Monitoring Committee and a Clinical Informatics Advisory Board. Trial results will be submitted for publication in a peer-reviewed journal., Trial Registration Number: NCT03424109; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Pancreatic cancer is a deadly disease for which available biomarkers, such as CA19-9, lack the desired sensitivity and specificity for early detection. Additional biomarkers are needed to improve both its sensitivity and specificity., Methods: Multiplex immunoassays were developed for selected biomarkers using a Bio-Plex 200 system, and analytical performance was optimized. All proteins were analyzed in sera of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC; n = 188) or benign pancreatic conditions (131) and healthy controls (89). The clinical performance of these markers was evaluated individually or in combination for their ability to complement CA19-9 for the early detection of pancreatic cancer., Results: A 6-plex immunoassay was developed with negligible cross-reactivity, wide dynamic range, recovery of 89% to 104%, and intra-assay and interassay precision of 10.2% to 19.6% and 13.7% to 29.3%, respectively. Individually, the best biomarkers to separate PDAC early stage from chronic pancreatitis or intraductal papillary mucinous neoplasm (IPMN) were CA19-9 and MIA or CA19-9 and MIC-1. Logistic regression modeling selected the two-marker panels that significantly improved the individual biomarker performance in discriminating PDAC early stage from chronic pancreatitis (AUC CA19-9+MIA = 0.86 vs. AUC CA19-9 = 0.81 or AUC MIA = 0.75 only, P < 0.05) or IPMN (AUC CA19-9+MIC-1 = 0.81 vs. AUC CA19-9 = 0.75 or AUC MIC-1 = 0.73 only, P < 0.05). It was observed that osteopontin (OPN) outperformed CA19-9 in separating IPMN from chronic pancreatitis (AUC OPN = 0.80 vs. AUC CA19-9 = 0.70, P < 0.01)., Conclusions: The biomarker panels evaluated by assays with high analytical performance demonstrated potential complementary values to CA19-9, warranting additional clinical validation to determine their role in early detection of pancreatic cancer., Impact: The validated biomarker panels could lead to earlier intervention and better outcomes., (©2018 American Association for Cancer Research.)

Exposure of certain cell lines to methylcholanthrene, X-rays, or physiological growth constraint leads to preneoplastic transformation in all or most of the treated cells. After attaining confluence, a fraction in those cells progress to full transformation, as evidenced by their ability to form discrete foci distinguishable from the surrounding cells by virtue of their higher density. Transformation induced by suspension in agar, an even stronger growth-selective condition than confluence, is reminiscent of all but the final differentiated stage of a normal developmental process, epithelial-mesenchymal transition. Changes associated with transformation are not restricted to focus-forming cells, as the permissiveness for focus formation provided by confluent cells surrounding transformed foci is greater than that of nonselected cells. The neoplastic process can also be reversed in culture. Transformed cells passaged at low density in high serum revert to normal morphology and growth behavior in vitro and lose the capacity for tumor formation in vivo. We propose that transformation and its reversal are driven by a process of phenotypic selection that involves entire heterogeneous populations of cells responding to microenvironmental changes. Because of the involvement of whole cell populations, we view this process as fundamentally adaptive and epigenetic in nature., Competing Interests: The authors declare no conflict of interest.

Experiential and associative learning are essential to optimal decision making. However, research shows that, even when exposed to repeated trials, people often fail to learn probabilities and cause/effect covariations. Consistent with the counterfactual inflation hypothesis, it is proposed that counterfactuals can interfere with memory of repeated exposures and therefore inhibit learning. Five experimental studies tested counterfactual thinking as a potential mechanism underlying this learning deficit using a simple, biased coin flipping paradigm. Participants were instructed to either simply observe or to predict and observe outcomes of a biased coin being flipped in multiple trials (Experiments 1-4). In all four experiments, counterfactual thought frequency mediated the relationship between task instructions and the extent of bias detection (i.e., learning). Experiment 5 showed that mental simulations of alternative outcomes were especially deleterious to learning and decision making. Findings are discussed in light of experiential learning theory and applied implications., (© 2016 by the Society for Personality and Social Psychology, Inc.)

The Special Olympics World Games (SOWG) were held in Los Angeles, CA, during the summer of 2015. Medical care for 26 sporting events spread over six major venues across the city was provided to more than 6,000 athletes and 3,000 delegates from 170 countries. Education on care for individuals with intellectual and developmental disabilities and athletes with additional medical issues was provided in addition to the usual sports medicine care. This required coordination between major medical providers as well as law enforcement, fire rescue, transportation, public health, and the organizers of the games. This article reviews the planning, training, and outcomes of the medical care and the Healthy Athletes program for the SOWG 2015.

Cancer cells and their associated tumors have long been considered to exhibit unregulated proliferation or growth. However, a substantial body of evidence indicates that tumor growth is subject to both positive and negative regulatory controls. Here, we describe a novel property of tumor growth regulation that is neither species nor tumor-type specific. This property, functionally a type of feedback control, is triggered by the encapsulation of neoplastic cells in a growth-restricting hydrogel composed of an agarose matrix with a second coating of agarose to form 6- to 8-mm diameter macrobeads. In a mouse cell model of renal adenocarcinoma (RENCA cells), this process resulted in selection for a stem cell-like subpopulation which together with at least one other cell subpopulation drove colony formation in the macrobeads. Cells in these colonies produced diffusible substances that markedly inhibited in vitro and in vivo proliferation of epithelial-derived tumor cells outside the macrobeads. RENCA cells in monolayer culture that were exposed to RENCA macrobead-conditioned media exhibited cell-cycle accumulation in S phase due to activation of a G(2)/M checkpoint. At least 10 proteins with known tumor suppression functions were identified by analysis of RENCA macrobead-conditioned media, the properties of which offer opportunities to further dissect the molecular basis for tumor growth control. More generally, macrobead culture may permit the isolation of cancer stem cells and other cells of the stem cell niche, perhaps providing strategies to define more effective biologically based clinical approaches to treat neoplastic disease.

The culture of tumor cell lines in three-dimensional scaffolds is considered to more closely replicate the in vivo tumor microenvironment than the standard method of two-dimensional cell culture. We hypothesized that our method of encapsulating and maintaining viable and functional pancreatic islets in agarose-agarose macrobeads (diameter 6-8 mm) might provide a novel method for the culture of tumor cell lines. In this report we describe and characterize tumor colonies that form within macrobeads seeded with mouse renal adenocarcinoma cells. Approximately 1% of seeded tumor cells survive in the macrobead and over several months form discrete elliptical colonies appearing as tumor cell niches with increasing metabolic activity in parallel to colony size. The tumor colonies demonstrate ongoing cell turnover as shown by BrdU incorporation and activated caspase-3 and TUNEL staining. Genes upregulated in the tumor colonies of the macrobead are likely adaptations to this novel environment, as well as an amplification of G(1)/S cell-cycle checkpoints. The data presented, including SCA-1 and Oct4 positivity and the upregulation of stem cell-like genes such as those associated with the Wnt pathway, support the notion that the macrobead selects for a subpopulation of cells with cancer stem cell or cancer progenitor properties.

The team physician may be asked to travel with the team to away venues. This takes special planning to provide for travel advice, proper immunizations, treatment of travel related diseases, appropriate planning for supplies, and care not to disturb any local laws and regulations. Increased security screening and limitations of what is allowed on the airplane in the cabin and in checked luggage have particularly affected air travel. Local laws, be they state or national, take precedence over the laws and regulations we are all used to practicing under. Close relationships with the hosting team's medical staff can help mitigate many of the problems that may arise.

Islet xenografts from porcine donors can reverse diabetes in experimental animal models and may be an alternative to human islet transplantation. We have recently reported the ability of porcine islets encapsulated in a double layer of hydrophilic agarose to maintain in vitro functional ability for >6 months. Although beta-cells are capable of adapting their secretory capacity in response to glucose levels, evidence has shown that prolonged hyperglycemia can compromise this ability. The aim of the present study was to determine the effects of diet manipulation on the long-term regulation of blood glucose levels, and the preservation of functional islet in the macrobeads. Twenty-one streptozotocin-induced diabetic Wistar-Furth male rats were randomly assigned to two diets containing 64% carbohydrate (CHO) or 20% CHO. Groups of five to six animals assigned to either diet were implanted with either empty (EM) or porcine islet-containing macrobeads (PIM) and followed for 333 days. Observations included general condition, body weight, blood glucose, and food and water intakes. Monthly blood samples were collected for insulin and C-peptide analysis. The 20% CHO diet significantly lowered blood glucose values when compared with those of the 64% CHO groups for both the empty (14.94 +/- 0.41 vs. 16.26 +/- 0.42 mmol/L, respectively, p < 0.001) and islet macrobead recipients (12.88 +/- 0.39 vs. 15.57 +/-0.85 mmol/L, respectively, p <0.001). The different diets, however, had no statistically significant effects on the preservation of islet mass in the macrobead. Serum porcine C-peptide was detected throughout the experiment in animals receiving porcine islet macrobeads, regardless of diet. Diabetic rats fed a low carbohydrate level diet and transplanted with porcine islet macrobeads had improved total tissue glucose disposal and improved clinical parameters associated with diabetes.

Carbaryl is an agricultural and garden insecticide that controls a broad spectrum of insects. Although moderately water soluble, it neither vaporizes nor volatilizes readily. However, upon spray application the insecticide is susceptible to drift. It is unstable under alkaline conditions, thus easily hydrolyzed. Carbaryl has been detected in water at ppb concentrations but degradation is relatively rapid, with 1-naphthol identified as the major degradation product. Indirect and direct photolysis of carbaryl produces different naphthoquinones as well as some hydroxyl substituted naphthoquinones. Sorption of the insecticide to soil is kinetically rapid. However, although both the mineral and organic fractions contribute, because of its moderate water solubility it is only minimally sorbed. Also, sorption to soil minerals strongly depends on the presence of specific exchangeable cations and increases with organic matter aromaticity and age. Soil microbes (bacteria and fungi) are capable of degrading carbaryl; the process is more rapid in anoxic than aerobic systems and with increased temperature and moisture. Carbaryl presents a significant problem to pregnant dogs and their offspring, but some have questioned the applicability of these data to humans. In addition, for toxicokinetic and/or physiological reasons, it has been argued that dogs are more sensitive than humans to carbaryl-induced reproductive or developmental toxicity. However, these arguments are based on either older pharmacokinetic studies or on speculation about possible reproductive differences between dogs on the one hand and rats and humans on the other. In view of the wider evidence from both human epidemiological and laboratory animal studies, the question of the possible developmental and reproductive toxicity of carbaryl should be considered open and requiring further study.

The ability to culture porcine islets for extended times allows for both their functional assessment and the assurance of their microbiological safety prior to transplantation. We have previously shown that agarose-encapsulated porcine islets can be cultured for at least 24 weeks. In the current study, porcine islet agarose macrobeads cultured for up to 67 weeks were assessed for their ability to restore normoglycemia, respond to an intraperitoneal glucose challenge, maintain spontaneously diabetic BB rats free of insulin therapy for more than 6 months, and for their biocompatibility. Porcine islets were encapsulated in agarose macrobeads and subjected to weekly static perifusion assays for the assessment of insulin production. After in vitro culture for either 9, 40, or 67 weeks, 56-60 macrobeads were transplanted to each spontaneously diabetic BB rat. Transplanted rats were monitored daily for blood glucose levels. Glucose tolerance tests and assessments for porcine C-peptide were conducted at various intervals throughout the study. Normoglycemia (100-200 mg/dl) was initially restored in all islet transplanted rats. Moderate hyperglycemia (200-400 mg/dl) developed at around 30 days posttransplantation and continued throughout the study period of 201-202 days. Importantly, all rats that received encapsulated porcine islets continued to gain weight and were free of exogenous insulin therapy for the entire study. Porcine C-peptide (0.2-0.9 ng/ml) was detected in the serum of islet recipients throughout the study period. No differences were detected between recipient animals receiving islet macrobeads of various ages. These results demonstrate that the encapsulation of porcine islets in agarose macrobeads allows for extended culture periods and is an appropriate strategy for functional and microbiological assessment prior to clinical use.

There is an art and a science to determining the contents of an appropriate medical bag for sports and event medicine. Sports and event medicine encompass a broad range of activities and venues, and the medical bag's contents must be adapted accordingly. We discuss relevant considerations as well as general principles and recommendations accompanied by a checklist, using coverage of football games as a model.

Background: An approach to endotoxin (lipopolysaccharide [LPS]) blockade makes use of the ability of lipoproteins, via surface phospholipids, to bind and neutralize LPS. The aim of the present study was to determine whether the intravenous administration of a protein-free, phospholipid-rich emulsion is an effective method for neutralizing the effects of LPS in healthy persons., Methods: This was a double-blind, placebo-controlled study in 20 volunteers. Volunteers received Escherichia coli endotoxin (2 ng/kg) intravenously 2 h into a 6-h infusion of either emulsion (210 mg/kg) or placebo (Intralipid diluted 1 : 64)., Results: The volunteers who received emulsion had a lower mean clinical score (P<.01), temperature (P<.05), pulse rate (P<.05), neutrophil count (P<.05), tumor necrosis factor- alpha level (P<.05), and interleukin-6 level (P<.05) than did the volunteers who received placebo. Response was related to serum phospholipid level. The greatest effects were observed in the volunteers achieving phospholipid levels of approximately 500 mg/dL or higher., Conclusion: Phospholipid emulsion attenuates the clinical and laboratory effects associated with the administration of LPS in humans, suggesting a novel approach to the treatment of endotoxemia.

For clinical xenogenic islet transplantation to be successful, several requirements must be met. Among them is a sizeable and reliable source of fully functional and microbiologically safe islets. The inherent variability among porcine pancreases, with respect to islet yield, prompted us to develop a Biopsy Score technique to determine the suitability of each pancreas for islet isolation processing. The Biopsy Score consists of an assessment of five variables: warm ischemia time, pancreas color, fat content, islet size, and islet demarcation, each of which is assigned a value of -1 or +1, depending on whether or not the established criteria is met. For determination of islet size and demarcation, fresh biopsies of porcine pancreases are stained with dithizone (DTZ) solution and examined under a dissecting microscope. Based on the scoring of such biopsies in pancreases from 26-56-month-old sows, we report here that the presence of large (>100 microm diameter), well-demarcated islets in the pancreas biopsy is a reliable predictor of isolation success. Encapsulation of the isolated porcine islets within the inner layer of a 1.5% agarose and an outer layer of 5.0% agarose macrobead, containing 500 equivalent islet number (EIN), provides for extended in vitro functional viability (>6 months of insulin production in response to glucose), as well as for comprehensive microbiological testing and at least partial isolation of the xenogeneic islets from the host immune system. All microbiological testing to date has been negative, except for the presence of porcine endogenous retrovirus (PERV). Taken together, we believe that the Biopsy Score enhancement of our islet isolation technique and our agarose-agarose macroencapsulation methodology bring us significantly closer to realizing clinical porcine islet xenotransplantation for the treatment of insulin-dependent diabetic patients.

There is a heightened awareness of terrorism in this country. There always remains the possibility of nonterrorist disasters at sporting venues. The team physician will be among the first medical responders to a disaster at a sporting venue. By being involved in the creation of an emergency action plan, learning the incident command system, understanding triage, and obtaining basic trauma life support skills, the team physician can be prepared to respond to mass casualty incidents at sporting events.

Endotoxemia is associated with rapid and marked declines in serum levels of LDL and HDL by unknown mechanisms. Six normal volunteers received a single, small intravenous (iv) dose of endotoxin (Escherichia coli 0113, 2 ng/kg) or saline in a random order, cross-over design. After endotoxin treatment, volunteers had mild, transient flu-like symptoms and markedly increased serum levels of tumor necrosis factor and its soluble receptors, interleukin-6, cortisol, serum amyloid A, and C-reactive protein. Triglyceride (TG), VLDL-TG, and nonesterified fatty acid increased (peak at 3-4 h), then TG declined (nadir at 9 h), and then cholesterol, LDL cholesterol, apolipoprotein B (apoB), and phospholipid declined (nadirs at 12-24 h). HDL cholesterol and apoA-I levels were not affected, but half of the decrease in phospholipid was HDL phospholipid. Lipopolysaccharide binding protein (LBP) rose 3-fold (peak at 12 h), with smaller and later decreases in the activities of phospholipid transfer protein and cholesteryl ester transfer protein. In conclusion, a decline in LDL was rapidly induced in normal volunteers with a single iv dose of endotoxin. The selective loss of phospholipid from HDL may have been mediated by LBP and, after more intense or prolonged inflammation, could result in increased HDL clearance and reduced HDL levels.

Background: Lipids and lipoproteins have been shown to bind and neutralize endotoxin and to improve outcomes in animal models of sepsis., Objective: To provide safety and pharmacokinetic data for a protein-free, phospholipid-rich emulsion developed as an agent to neutralize endotoxin, and to study the changes in lipids and lipoproteins following emulsion administration., Methods: Thirty healthy male volunteers (aged 18-45 y) were given an emulsion containing 92.5% soy phospholipid, 7.5% soy triglyceride, and 18 mM sodium cholate using a double-blind, placebo-controlled crossover protocol. Emulsion at 3 escalating doses (75, 150, 300 mg/kg) based on phospholipid content was administered by intravenous infusion over 2 hours in the low- and mid-dose groups and 6 hours in the high-dose group., Results: All subjects completed the protocol without significant toxicities. A slight dose-dependent increase in indirect bilirubin at the 24-hour time point was observed in the emulsion treatment period, with a maximum difference between placebo and emulsion of 0.9 mg/dL. Mean +/- SD peak phospholipid levels were 316 +/- 30, 533 +/- 53, and 709 +/- 86 mg/dL, and phospholipid half-lives were 5.4 +/- 0.6, 5.4 +/- 0.5, and 8.0 +/- 0.8 hours for the low, mid, and high doses, respectively. Increases in total cholesterol, low-density lipoprotein cholesterol and apolipoprotein A-I and B levels were observed. High-density lipoprotein cholesterol decreased immediately following emulsion infusion, but rebounded to above placebo levels by 24 hours., Conclusions: A unique phospholipid-rich emulsion was shown to have a favorable safety profile and to expand the blood lipid and lipoprotein pool without the use of human-derived blood products. Lipid levels expected to protect against the physiologic effects of bacterial endotoxin were achieved.

Emergent and nonemergent splinting, bracing, and casting are effective ways to safely remove an injured athlete from the playing field, allow immediate return to play, and permit an athlete to return to play before an injury has completely healed. Sideline providers should be familiar with the materials and resources currently available, and be aware of the sport-specific guidelines regarding their use. Properly applied splints, casts, braces, and padding should function to prevent further injury, promote comfort, and not pose a risk of injury to other participants.

Lipoprotein phospholipid (PL) plays a major role in neutralization of endotoxin. This study tested the hypothesis that prophylactic administration of a PL-enriched emulsion (PRE), which augments PL content of serum lipoproteins and neutralizes endotoxin in vitro, would preserve cardiovascular function and improve survival in porcine septic peritonitis. A control group was compared with low-, mid-, and high-dose treatment groups that received PRE by primed continuous infusion for 48 h. A fibrin clot containing live Escherichia coli 0111.B4 was implanted intraperitoneally 30 min after the priming dose. Survival increased in a dose-dependent manner and was correlated with serum PL. Infused PL was associated with high-density lipoprotein in the low-dose group and all serum lipoproteins at higher doses. Treatment significantly lowered serum endotoxin and tumor necrosis factor (TNF)-alpha, preserved cardiac output and ejection fraction, and attenuated increases in systemic and pulmonary vascular resistances. This study demonstrated that augmentation of lipoprotein PL via administration of PRE improved survival and offered a novel therapeutic approach to sepsis.

Through the efforts of Edward H. Ahrens, LDL apheresis became available for the treatment of patients, often with familial hypercholesterolemia, who have no alternative therapy for severely elevated LDL cholesterol levels. In the U.S., the FDA has approved this treatment for individuals on maximum diet and drugs with an LDL cholesterol greater than 300 mg/dL or greater than 200 mg/dL with coronary artery disease. Unlike plasmapheresis, apolipoprotein B-containing lipoproteins (LDL, Lp(a), and VLDL) are selectively removed by heparin precipitation or columns containing dextran sulfate cellulose or antibodies to apolipoprotein B. The acute lowering of LDL-cholesterol by a typical 2 - 3 h treatment is up to 80%, and the time-averaged lowering in the 1 to 2 week interval between treatments is up to 50%, with very few side effects. The lowering of LDL-cholesterol and other cardioprotective effects of LDL apheresis have reduced chest pain, prevented new disability and prolonged life. Whole blood compatible columns in development offer the possibility of simpler and less expensive treatments.

Objective: To determine the relationship of hypolipidemia to cytokine concentrations and clinical outcomes in critically ill surgical patients., Design: Consecutive, prospective case series., Setting: Surgical intensive care unit of an urban university hospital., Patients: Subjects were 111 patients with a variety of critical illnesses, for whom serum lipid, lipoprotein, and cytokine concentrations were determined within 24 hrs of admission to a surgical intensive care unit. Controls were 32 healthy men and women for whom serum lipid, lipoprotein, and cytokine concentrations were determined., Interventions: Blood samples were drawn on admission to the intensive care unit. Predetermined clinical outcomes including death, infection subsequent to intensive care unit admission, length of intensive care unit stay, and magnitude of organ dysfunction were monitored prospectively., Measurements and Main Results: Measurements included total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, apolipoproteins A-I and B, phospholipid, triglyceride, interleukin-6, interleukin-10, soluble interleukin-2 receptor, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptors p55 and p75. Mean serum lipid concentrations were extremely low: total cholesterol, 127 +/- 52 mg/dL; low-density lipoprotein cholesterol, 75 +/- 41 mg/dL; high-density lipoprotein cholesterol, 29 +/- 15 mg/dL. Total, low-density lipoprotein, and high-density lipoprotein cholesterol concentrations and apolipoprotein concentrations inversely correlated with interleukin-6, soluble interleukin-2 receptor, and interleukin-10 concentrations, whereas the triglyceride concentration correlated positively with tumor necrosis factor soluble receptors p55 and p75. Clinical outcomes were related to whether the admission cholesterol concentration was above (n = 56) or below (n = 55) the median concentration of 120 mg/dL. Each of the clinical end points occurred between 1.9- and 3.5-fold more frequently in the very low cholesterol (<120 mg/dL) group. Nine patients (8%) died during the hospitalization. Seven of the nine patients who died had total cholesterol concentrations below the median concentration of 120 mg/dL., Conclusions: Low cholesterol and lipoprotein concentrations found in critically ill surgical patients correlate with interleukin-6, soluble interleukin-2 receptor, and interleukin-10 concentrations and predict clinical outcomes.

Background: We investigated the effectiveness of implanted macrobeads containing porcine islets as long-term therapy for type I diabetes mellitus in Biobreeding/Worcester (BB/Wor) rats, an animal model of spontaneous type I human diabetes. End points included acute control of glucose, weight gain, survival time, and the renal changes associated with diabetes., Materials and Methods: Eighteen chronic spontaneously diabetic BB/Wor rats were each implanted with 56-150 porcine islet macrobeads secreting 1.3-5.2 U of insulin/24 hr in culture medium at 37 degrees C. Their clinical courses and selective histological observations were compared with those of animals maintained on Linplant insulin-release implants (6 rats) or protamine zinc insulin alone (10 rats)., Results: The rats that underwent porcine islet macrobead implantation (PIMI) survived for a mean of 171 days (range, 79-288) after implantation without exogenous insulin, immunosuppressive treatment, or lactated Ringer's therapy. All appeared healthy and maintained their body weights (mean 356+/-21 g) throughout this period, even though their nonfasting blood glucose levels fluctuated significantly, with the mean for the group being 245+/-102 mg/dl (range, 157-320 mg/dl). There was mild glucosuria in some animals. In comparison, the 10 BB/Wor rats maintained on exogenous protamine zinc insulin had a mean survival time of 53 days (range, 10-217), a "last entry" mean body weight of 283+/-23 g, and a mean nonfasting glucose level of 340+/-90 mg/dl. The six Linplant implant animals had a mean survival time of 164 days (range, 1-264 days), a "last entry" mean body weight of 374+/-21 g, and a mean nonfasting glucose level of 189+/-91 mg/dl (range, 135-219). Episodes of ketonuria, abrupt loss of body weight, dehydration, and symptomatic hypoglycemia were more common in both these groups than in the PIMI animals. Glucose tolerance tests comparing diabetic animals treated with porcine islet macrobead implants, exogenous insulin-treated diabetic BB/Wor rats, and normal nondiabetic Wistar-Furth rats showed that the responses of those with the macrobead implants were similar to those of the normal rats, while the exogenous insulin-treated diabetic BB/Wor rats had the expected abnormal responses. Light microscopic examination of the PIMI and Linplant animals' kidney sections appeared normal, whereas those of the exogenous insulin-injected BB rats showed moderate focal tubular atrophy and an increased mesangial matrix. Macrobeads retrieved from the peritoneal cavity at necropsy were found to secrete insulin, C-peptide, and glucagon, indicating that they were still functional after 199 or more days in the peritoneal cavity., Conclusions: Our results indicate that macrobeads containing porcine islets implanted intraperitoneally in natural insulin-dependent diabetic BB/Wor rats are capable of normalizing glucose control, permitting a normal life span, and preventing the renal changes normally associated with diabetes. Therefore, further short- and long-term studies of porcine islet macrobead implantation in chemically induced and naturally occurring diabetes in rodents, as well as larger animals including dogs, monkeys and possibly humans, are merited.

Riedel's thyroiditis is a rare fibro-inflammatory process originating in the thyroid gland with progressive extension and invasion of surrounding tissues. Patients frequently present with a stony hard thyroid mass suggestive of anaplastic carcinoma. We report a striking case of Riedel's thyroiditis associated with hypothyroidism, hypoparathyroidism and bilateral vocal cord paralysis. A dramatic response to high dose prednisone and levothyroxine therapy was seen, with recovery of parathyroid and vocal cord function. Our case suggests that early initiation of combination therapy may be important, particularly in the presence of severe disease.

We previously reported that a calcium channel blocker supplemented immunosuppression produced excellent patient and graft survival rates in cadaveric kidney transplantation. We report here the long term outcome of patients treated with nifedipine-supplemented triple immunosuppression as compared with those of historical controls who were treated similarly without nifedipine. Study subjects included 111 patients transplanted in 1990-1994, treated with nifedipine and triple immunosuppression and with functioning grafts for more than one year (Nifedipine group). The results of cyclosporine (CyA) dose, blood pressure (BP), serum creatinine (Cr), and actuarial graft survival rate (GSR) up to 5 years posttransplant in these patients were compared with those of 52 patients transplanted in 1985-1990, treated similarly without calcium channel blockers (Control group). Donor sources, gender ratio, age distribution, causes of end stage renal disease, incidence of hypertension prior to transplantation and incidence of rejection in the first year between the groups were comparable. Throughout the study period the Nifedipine group had significantly lower serum Cr (1.5 +/- 0.7 vs. 1.8 +/- 0.7 mg/dl) and higher GSR (93.8% vs. 88% at 5 years) than the Control group. BP was comparable despite higher CyA doses in the Nifedipine group (4.3 +/- 1.1 vs. 3.3 +/- 1.1 mg/kg/day). We conclude that nifedipine is beneficial in improving long-term graft function and survival in kidney transplant recipients by mitigating CyA associated renal injury.

Objectives: To determine the prevalence and clinical significance of hypolipidemia found in critically ill patients, and whether the addition of a reconstituted lipoprotein preparation could inhibit the generation of tumor necrosis factor-alpha (TNF-alpha) in acute-phase blood taken from these patients., Setting: Surgical intensive care unit (ICU) of a large urban university hospital., Design: Prospective case series., Patients: A total of 32 patients with a variety of critical illnesses had lipid and lipoprotein concentrations determined. Six patients and six age- and gender-matched control subjects had whole blood in vitro studies of the effect of lipoprotein on lipopolysaccharide mediated TNF-alpha production., Interventions: Blood samples were drawn on admission to the ICU and over a subsequent 8-day period., Measurements and Main Results: Mean serum lipid and lipoprotein values obtained from patients within 24 hrs of transfer to the surgical ICU were extremely low: mean total cholesterol was 117 mg/dL (3.03 mmol/L), low-density lipoprotein cholesterol 71 mg/dL (1.84 mmol/L), and high-density lipoprotein cholesterol 25 mg/dL (0.65 mmol/L). Only the mean triglyceride concentration of 105 mg/dL (1.19 mmol/L), and the mean lipoprotein(a) concentration of 25 mg/dL (0.25 g/L) were within the normal range. During the first 8 days following surgical ICU admission, there were trends toward increasing lipid and lipoprotein concentrations that were significant for triglycerides and apolipoprotein B. Survival did not correlate with the lipid or lipoprotein concentrations, but patients with infections had significantly lower (p = .008) high-density lipoprotein cholesterol concentrations compared with noninfected patients. Lipopolysaccharide-stimulated production of TNF-alpha in patient and control blood samples was completely suppressed by the addition of 2 mg/mL of a reconstituted high-density lipoprotein preparation., Conclusions: Patients who are critically ill from a variety of causes have extremely low cholesterol and lipoprotein concentrations. Correction of the hypolipidemia by a reconstituted high-density lipoprotein preparation offers a new strategy for the prevention and treatment of endotoxemia.

Several obstacles have hindered the successful transplantation of islets of Langerhans to human patients in efforts to cure type I diabetes mellitus. One problem is the necessity for short- and long-term storage of islets after isolation and before transplantation. Current long-term storage methods, such as incubation in a physiological medium and cryopreservation, are suboptimal, resulting in significant loss of viable islet mass or function. Better storage methods are needed. In this study we examined the long-term storage of rat islets in macrobeads composed of agarose and collagen. Islets isolated from Wistar-Furth rats were placed into macrobeads (1000 islets/macrobead) and maintained in culture for periods of up to 189 days at 37 degrees C. Insulin released from the cultured macrobeads remained constant for periods of at least 154 days. In one group, insulin release was 1050 mU/24 hr/4 beads on day 3 and 1040 mU/24 hr/4 beads on day 154. In another group, insuling release was 1305 Xenotransplantation of Wistar Furth islet macrobeads, stored for 10 to 112 days at 37 degrees C, degrees C into 42 B6AF/1 mice with streptozotocin-induced diabetes resulted in a return to euglycemia in the recipients within 24 hr. Thereafter, euglycemia was maintained for more than 100 days in 32/42 of the recipients, and removal of the macrobeads caused a return to hyperglycemia within 48 hr in all animals. In addition, a group of 7 mice receiving macrobeads containing 1000 islets stored for 84 days had normal glucose tolerance tests (compared with those of 7 nontreated, nontransplanted mice with streptozotocin-induced diabetes and 7 normal mice), demonstrating that the islets in the macrobeads were functioning as they would in an intact pancreas. Finally, 5 macrobeads transplanted after initial storage of 112 days, removed from the first recipient after 100 days or more, stored again for 4 days in vitro, and retransplanted into 5 other diabetic mice also restored and maintained euglycemia for at least 45 days. Our results indicate that collagen-agarose macrobeads are capable of preserving rat pancreatic islets for extended periods without loss of in vitro insulin release capability or ability to achieve and maintain euglycemia in vivo. As such they should be useful for human islet transplantation efforts.

Depending on the precise conditions and cellular starting material, phorbol-13-myristate-12-acetate (PMA) can induce or suppress the transformation of NIH 3T3 cells. In sublines that do not undergo rapid transformation, exposure to PMA over the course of several weeks accelerated the process, while sublines that are primed for density-mediated transformation respond to PMA with a suppression of the process. This study examines the latter phenomenon. Within 1 h of exposure to 0.02 microgram/ml PMA, sparse cultures had undergone a morphological transition after which the cells appeared smaller and the processes thinner. These sublines exhibited a two- to sixfold increase in the saturation density achieved in 2% calf serum (CS). Phorbol ester analogs with hydrocarbon substitutions of 4 or more carbons at positions 12 and 13 of the phorbol nucleus had a similar effect as PMA on the saturation density. High concentrations of PMA (1 microgram/ml) induced the formation of cell aggregates (pseudofoci) that resembled transformed foci in their high local density, but unlike transformed foci, did not reinitiate focus formation if the cells were diluted and replated without PMA as secondary cultures. PMA inhibited the processes of neoplastic transformation and progression that occur readily in these NIH 3T3 sublines when they reach high cell density. I suggest that such changes occur because PMA abolishes the selection pressure at high densities that favors the transformation of some cells in heterogeneous populations. Induction of transformation by PMA (reported previously) occurs after much longer exposures in sublines that are relatively resistant to rapid density-mediated transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood.

This is a retrospective, clinical study evaluating the long-term outcome of subtotal parathyroidectomy (PTX) in 60 patients with chronic renal failure and severe secondary hyperparathyroidism. Patients were 41 +/- 2 years old (mean +/- SE) at the time of PTX, and followed for 69 +/- 6 months since the procedure. At the time of PTX, three patients had chronic renal failure, 53 had been on chronic hemodialysis, and four had received successful kidney transplants. In more than 80 per cent of patients, symptoms of hyperparathyroidism (bone pain and muscle weakness) resolved within weeks, and biochemical signs (hypercalcemia, and high plasma alkaline phosphatase and parathyroid hormone concentrations) returned to normal ranges within a year. Subperiosteal resorption, bone fractures, and soft tissue calcification frequently improved. Osteosclerosis (rugger-jersey spine), cystic bone changes, osteopenia, and vascular calcifications were, however, often unchanged or progressive. Five patients (8%) who had either persistent or recurrent hyperparathyroidism required additional surgical procedures, and two had subsequent improvement. Twelve patients who had aluminum associated bone disease diagnosed later continued to progress with a high incidence of bone fractures and severe osteopenia. Cystic bone changes, especially of the carpal bones, in association with carpal tunnel syndrome, probably representing amyloid bone disease, also did not respond to PTX. In conclusion, PTX is an effective surgical procedure to reverse complications of hyperparathyroidism in patients with end-stage renal disease, provided that other causes of osteodystrophy, such as aluminum or amyloid-associated bone diseases, are adequately excluded. We feel that subtotal PTX, leaving a small remnant in place, is the procedure of choice.

Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans.

The ability of phorbol 12-myristate 13-acetate (PMA) at 0.02 microgram/ml to induce neoplastic transformation in NIH 3T3 cells is highly dependent on the culture conditions. Optimal transformation, indicated by the saturation density and extent of focus formation in transferred cultures raised under standard conditions, was observed when the original cells were grown in 2% calf serum (CS) and exposed continually to PMA for at least 4 weeks before transfer into the assay. Transformation of stationary cultures in 10% CS occurred later and to a lesser degree than in 2% CS. The same cells subjected to thrice-weekly transfer in 2% or 10% CS at low cell density so that they were in a constant state of exponential growth exhibited no evidence of transformation in response to PMA. This strong condition-dependence of PMA-enhanced transformation is indicative of a selection process similar to that described for spontaneous transformation. In both cases, transformation is promoted by inhibiting multiplication and prevented by maximizing multiplication. Therefore, it has the earmarks of an epigenetic rather than a mutational process and requires phenotypic rather than genotypic variation to supply the states for selection. The concept of "progressive state selection," originally proposed to account for spontaneous transformation, can also account for PMA-enhanced transformation.

Overwhelming bacterial infection is accompanied by fever, hypotension, disseminated intravascular coagulation, and multiple organ failure leading to death in 30-80% of cases. These classical symptoms of septic shock are caused by potent cytokines that are produced in response to endotoxin released from Gram-negative bacteria. Treatments with antibodies and receptor antagonists to block endotoxin or cytokine mediators have given mixed results in clinical trials. High density lipoprotein (HDL) is a natural component of plasma that is known to neutralize endotoxin in vitro. We report here that raising the plasma HDL concentration protects mice against endotoxin in vivo. Transgenic mice with 2-fold-elevated plasma HDL levels had more endotoxin bound to HDL, lower plasma cytokine levels, and improved survival rates compared with low-HDL mice. Intravenous infusion of HDL also protected mice, but only when given as reconstituted HDL prepared from phospholipid and either HDL apoprotein or an 18-amino acid peptide synthesized to mimic the structure of apolipoprotein A-I of HDL. Intact plasma HDL was mildly toxic, and HDL apoprotein was ineffective. The effectiveness of the reconstituted peptide renders very unlikely any significant contribution to protection by trace proteins in apo-HDL. These data suggest a simple leaflet insertion model for binding and neutralization of lipopolysaccharide by phospholipid on the surface of HDL. Plasma HDL may normally act to protect against endotoxin; this protection may be augmented by administration of reconstituted HDL or reconstituted peptides.