Modeling the physiological roles of the heart and kidney in heart failure with preserved ejection fraction during baroreflex activation therapy.

Heart failure (HF) is a leading cause of death and is increasing in prevalence. Unfortunately, therapies that have been efficacious in patients with HF with reduced ejection fraction (HFrEF) have not convincingly shown a reduction in cardiovascular mortality in patients with HF with preserved ejection fraction (HFpEF). It is thought that high sympathetic nerve activity (SNA) in the heart plays a role in HF progression. Clinical trials demonstrate that baroreflex activation therapy reduces left ventricular (LV) mass and blood pressure (BP) in patients with HFpEF and hypertension; however, the mechanisms are unclear. In the present study, we used HumMod, a large physiology model to simulate HFpEF and predict the time-dependent changes in systemic and cardiac hemodynamics, SNA, and cardiac stresses during baroreflex activation. The baseline HFpEF model was associated with elevations in systolic BP, diastolic dysfunction, and LV hypertrophy and stiffness similar to clinical HFpEF. Simulating 12 mo of baroreflex activation resulted in reduced systolic BP (-25 mmHg) and LV mass (-15%) similar to clinical evidence. Baroreflex activation also resulted in sustained decreases in cardiac and renal SNA (-22%) and improvement in LV ß1-adrenergic function. However, the baroreflex-induced reductions in BP and improvements in cardiac stresses, mass, and function were mostly attenuated when renal SNA was clamped at baseline levels. These simulations suggest that the suppression of renal SNA could be a primary determinant of the cardioprotective effects from baroreflex activation in HFpEF.NEW & NOTEWORTHY Treatments that are efficacious in patients with HFrEF have not shown a significant impact on cardiovascular mortality in patients with HFpEF. We believe these simulations offer novel insight into the important roles of the cardiac and renal nerves in HFpEF and the potential mechanisms of how baroreflex activation alleviates HFpEF disease progression.

In silico trial of baroreflex activation therapy for the treatment of obesity-induced hypertension.

Clinical trials evaluating the efficacy of chronic electrical stimulation of the carotid baroreflex for the treatment of hypertension (HTN) are ongoing. However, the mechanisms by which this device lowers blood pressure (BP) are unclear, and it is uncertain which patients are most likely to receive clinical benefit. Mathematical modeling provides the ability to analyze complicated interrelated effects across multiple physiological systems. Our current model HumMod is a large physiological simulator that has been used previously to investigate mechanisms responsible for BP lowering during baroreflex activation therapy (BAT). First, we used HumMod to create a virtual population in which model parameters (n = 335) were randomly varied, resulting in unique models (n = 6092) that we define as a virtual population. This population was calibrated using data from hypertensive obese dogs (n = 6) subjected to BAT. The resultant calibrated virtual population (n = 60) was based on tuning model parameters to match the experimental population in 3 key variables: BP, glomerular filtration rate, and plasma renin activity, both before and after BAT. In the calibrated population, responses of these 3 key variables to chronic BAT were statistically similar to experimental findings. Moreover, blocking suppression of renal sympathetic nerve activity (RSNA) and/or increased secretion of atrial natriuretic peptide (ANP) during BAT markedly blunted the antihypertensive response in the virtual population. These data suggest that in obesity-mediated HTN, RSNA and ANP responses are key factors that contribute to BP lowering during BAT. This modeling approach may be of value in predicting BAT responses in future clinical studies.

Physiological Modeling and Simulation-Validation, Credibility, and Application.

In this review, we discuss the science of model validation as it applies to physiological modeling. There is widespread disagreement and ambiguity about what constitutes model validity. In areas in which models affect real-world decision-making, including within the clinic, in regulatory science, or in the design and engineering of novel therapeutics, this question is of critical importance. Without an answer, it impairs the usefulness of models and casts a shadow over model credibility in all domains. To address this question, we examine the use of nonmathematical models in physiological research, in medical practice, and in engineering to see how models in other domains are used and accepted. We reflect on historic physiological models and how they have been presented to the scientific community. Finally, we look at various validation frameworks that have been proposed as potential solutions during the past decade.

Corrigendum: Racial and Sex Differences in the Response to First-Line Antihypertensive Therapy.

[This corrects the article DOI: 10.3389/fcvm.2020.608037.].

Racial and Sex Differences in the Response to First-Line Antihypertensive Therapy.

Objective: As compared to whites, the black population develops hypertension (HTN) at an earlier age, has a greater frequency and severity of HTN, and has poorer control of blood pressure (BP). Traditional practices and treatment efforts have had minor impact on these disparities, with over a 2-fold higher death rate currently for blacks as compared to whites. The University of Mississippi Medical Center (UMC) is located in the southeastern US and the Stroke Belt, which has higher rates of HTN and related diseases as compared to the rest of the country. Methods: We retrospectively analyzed the UMC's Research Data Warehouse, containing >30 million electronic health records from >900,000 patients to determine the initial BP response following the first prescribed antihypertensive drug. Results: There were 5,973 white (45% overall HTN prevalence) and 10,731 black (57% overall HTN prevalence) patients who met criteria for the study. After controlling for age, BMI, and drug dosage, black males were overall less likely to have controlled BP (defined as < 140/90 mmHg) and were associated with smaller falls in BP as compared to whites and black females. Blockers of the renin-angiotensin system (RAS) failed to significantly improve odds of HTN control vs. the untreated group in black patients. However, our data suggests that these drugs do provide significant benefit in blacks when combined with THZ, as compared to untreated and as compared to THZ alone. Conclusion: These data support the use of a single-pill formulation with ARB or ACE inhibitor with a thiazide in blacks for initial first-line HTN therapy and suggests that HTN treatment strategies should consider both race and gender. Our study gives a unique insight into initial antihypertensive responses in actual clinical practice and could have an impact in BP control efficiency in a state with prevalent socioeconomic and racial disparities.

Simulation of integrative physiology for medical education.

Medical education is founded on the understanding of physiology. While lecture materials and reading contribute to the learning of physiology, the richness and complexity of the subject suggest that more active learning methods may provide a richer introduction to the science as it applies to the practice of medicine. Simulation has been previously used in basic science to better understand the interaction of physiological systems. In the current context, simulation generally refers to interactive case studies performed with a manikin or anatomic device. More recently, simulation has grown to encompass computational simulation: virtual models of physiology and pathophysiology where students can see in a mechanistic setting how tissues and organs interact with one another to respond to changes in their environment. In this manuscript, we discuss how simulation fits into the overall history of medical education, and detail two computational simulation products designed for medical education. The first of these is an acute simulator, JustPhysiology, which reduces the scope of a large model, HumMod, down to a more focused interface. The second is Sycamore, an electronic health record-delivered, real time simulator of patients designed to teach chronic patient care to students. These products represent a new type of tool for medical and allied health students to encourage active learning and integration of basic science knowledge into clinical situations.

Preeminent role of the cardiorenal axis in the antihypertensive response to an arteriovenous fistula: an in silico analysis.

Percutaneous creation of a small central arteriovenous (AV) fistula is currently being evaluated for the treatment of uncontrolled hypertension (HT). Although the mechanisms that contribute to the antihypertensive effects of the fistula are unclear, investigators have speculated that chronic blood pressure (BP) lowering may be due to 1) reduced total peripheral resistance (TPR), 2) increased secretion of atrial natriuretic peptide (ANP), and/or 3) suppression of renal sympathetic nerve activity (RSNA). We used an established integrative mathematical model of human physiology to investigate these possibilities from baseline conditions that mimic sympathetic overactivity and impaired renal function in patients with resistant HT. After a small fistula was stimulated, there were sustained increases in cardiac output, atrial pressures, and plasma ANP concentration (3-fold), without suppression of RSNA; at 8 wk, BP was reduced 14 mmHg along with a 32% fall in TPR. In contrast, when this simulation was repeated while clamping ANP at baseline BP decreased only 4 mmHg, despite a comparable fall in TPR. Furthermore, when chronic resetting of atrial mechanoreceptors was prevented during the fistula, RSNA decreased 7%, and along with the same threefold increase in ANP, BP fell 19 mmHg. This exaggerated fall in BP occurred with a similar decrease in TPR when compared with the above simulations. These findings suggest that ANP, but not TPR, is a key determinant of long-term BP lowering after the creation of an AV fistula and support a contribution of suppressed RSNA if resetting of the atrial-renal reflex is truly incomplete.NEW & NOTEWORTHY The mechanisms that contribute to the antihypertensive effects of a small arteriovenous (AV) fistula comparable to the size used by the ROX coupler currently in clinical trials are unclear and not readily testable in clinical or experimental studies. The integrative mathematical model of human physiology used in the current study provides a tool for understanding key causal relationships that account for blood pressure (BP) lowering and for testing competing hypotheses. The findings from the simulations suggest that after creation of a small AV fistula increased ANP secretion plays a critical role in mediating long-term reductions in BP. Measurement of natriuretic peptide levels in hypertensive patients implanted with the ROX coupler would provide one critical test of this hypothesis.

Role of the heart in blood pressure lowering during chronic baroreflex activation: insight from an in silico analysis.

Electrical stimulation of the baroreflex chronically suppresses sympathetic activity and arterial pressure and is currently being evaluated for the treatment of resistant hypertension. The antihypertensive effects of baroreflex activation are often attributed to renal sympathoinhibition. However, baroreflex activation also decreases heart rate, and robust blood pressure lowering occurs even after renal denervation. Because controlling renal sympathetic nerve activity (RSNA) and cardiac autonomic activity cannot be achieved experimentally, we used an established mathematical model of human physiology (HumMod) to provide mechanistic insights into their relative and combined contributions to the cardiovascular responses during baroreflex activation. Three-week responses to baroreflex activation closely mimicked experimental observations in dogs including decreases in blood pressure, heart rate, and plasma norepinephrine and increases in plasma atrial natriuretic peptide (ANP), providing validation of the model. Simulations showed that baroreflex-induced alterations in cardiac sympathetic and parasympathetic activity lead to a sustained depression of cardiac function and increased secretion of ANP. Increased ANP and suppression of RSNA both enhanced renal excretory function and accounted for most of the chronic blood pressure lowering during baroreflex activation. However, when suppression of RSNA was blocked, the blood pressure response to baroreflex activation was not appreciably impaired due to inordinate fluid accumulation and further increases in atrial pressure and ANP secretion. These simulations provide a mechanistic understanding of experimental and clinical observations showing that baroreflex activation effectively lowers blood pressure in subjects with previous renal denervation. NEW & NOTEWORTHY Both experimental and clinical studies have shown that the presence of renal nerves is not an obligate requirement for sustained reductions in blood pressure during chronic electrical stimulation of the carotid baroreflex. Simulations using HumMod, a mathematical model of integrative human physiology, indicated that both increased secretion of atrial natriuretic peptide and suppressed renal sympathetic nerve activity play key roles in mediating long-term reductions in blood pressure during chronic baroreflex activation.

Validating the Physiologic Model HumMod as a Substitute for Clinical Trials Involving Acute Normovolemic Hemodilution.

BACKGROUND: Blood conservation strategies and transfusion guidelines remain a heavily debated clinical topic. Previous investigational trials have shown that acute isovolemic hemodilution does not limit adequate oxygen delivery; however, a true critical hemoglobin level has never been investigated or defined due to safety concerns for human volunteers. Validated physiologic modeling may be useful to investigate hemodilution at critical hemoglobin levels without the ethical or safety hazards of clinical trials. Our hypothesis is that HumMod, an integrative physiological model, can replicate the cardiovascular and metabolic findings of previous clinical studies of acute isovolemic hemodilution and use coronary blood flow and coronary oxygen delivery in extreme hemodilution to predict a safety threshold. METHODS: By varying cardiovascular and sizing parameters, unique individuals were generated to simulate a population using HumMod, an integrative mathematical model of human physiology. Hemodilution was performed by simultaneously hemorrhaging 500 mL aliquots of blood while infusing equal volumes of hetastarch, 5% albumin balanced salt solution, or triple volumes of lactated Ringer's solution over 10 minutes. Five hemodilution protocols reported over 3 studies were directly replicated with HumMod to compare and validate essential cardiovascular and metabolic responses to hemodilution in moderately healthy, awake adults. Cardiovascular parameters, mental status, arterial and mixed venous oxygen content, and oxyhemoglobin saturation were recorded after the removal of each aliquot. The outputs of this simulation were considered independent variables and were stratified by hemoglobin concentration at the time of measurement to assess hemoglobin as an independent predictor of hemodynamic and metabolic behavior. RESULTS: The published reports exhibited discrepancies: Weiskopf saw increased heart rate and cardiac index, while Jones and Ickx saw no change in these variables. In HumMod, arterial pressure was maintained during moderate hemodilution due to decreases in peripheral resistance opposing increases in cardiac index. HumMod showed preserved ventilation through moderate hemodilution, compensated for by an increased oxygen extraction similar to the studies of Jones and Ickx. The simulation results qualitatively followed the clinical studies, but there were statistical differences. In more extreme hemodilution, HumMod had a lesser increase in cardiac index, which led to deficiencies in oxygen delivery and low venous saturation. In the simulations, coronary blood flow and oxygen delivery increase up to a critical hemoglobin threshold of 55-75 g/L in HumMod. In this range, coronary blood flow and oxygen delivery fell, leading to cardiac injury. The allowable amount of hemodilution before reaching the critical point is most closely correlated with nonmuscle mass (r = 0.69) and resting cardiac output (r = 0.67). CONCLUSIONS: There were significant statistical differences in the model population and the clinical populations, but overall, the model responses lay within the clinical findings. This suggests our model is an effective replication of hemodilution in conscious, healthy adults. A critical hemoglobin range of 5.5-7.5 g/L was predicted and found to be highly correlated with nonmuscle mass and resting cardiac output.

Simulating a virtual population's sensitivity to salt and uninephrectomy.

Salt sensitivity, with or without concomitant hypertension, is associated with increased mortality. Reduced functional renal mass plays an important role in causing salt-sensitive hypertension for many individuals. Factors that are important in the condition of decreased renal mass and how they affect blood pressure (BP) or salt sensitivity are unclear. We used HumMod, an integrative mathematical model of human physiology, to create a heterogeneous population of 1000 virtual patients by randomly varying physiological parameters. We examined potential physiological mechanisms responsible for the change in BP in response to high-salt diet (8× change in salt intake for three weeks) with full kidney mass and again after the removal of one kidney in the same group of virtual patients. We used topological data analysis (TDA), a clustering algorithm tool, to analyse the large dataset and separate patient subpopulations. TDA distinguished five unique clusters of salt-sensitive individuals (more than 15 mmHg change in BP with increased salt). While these clusters had similar BP responses to salt, different collections of variables were responsible for their salt sensitivity, e.g. greater reductions in glomerular filtration rate (GFR) or impairments in the renin-angiotensin system. After simulating uninephrectomy in these virtual patients, the three most salt-sensitive clusters were associated with a blunted increase in renal blood flow (RBF) and higher increase in loop and distal sodium reabsorption when compared with the salt-resistant population. These data suggest that the suppression of sodium reabsorption and renin-angiotensin system is key for salt resistance, and RBF in addition to GFR may be an important factor when considering criteria for kidney donors. Here, we show that in our model of human physiology, different derangements result in the same phenotype. While these concepts are known in the experimental community, they were derived here by considering only the data obtained from our virtual experiments. These methodologies could potentially be used to discover patterns in patient sensitivity to dietary change or interventions and could be a revolutionary tool in personalizing medicine.

Physiologic Mechanisms of Water and Electrolyte Disturbances After Transsphenoidal Pituitary Surgery.

BACKGROUND: Disturbances in water and electrolyte homeostasis are common after transsphenoidal surgery. These disorders are variable and unpredictable, increasing patient risk and complicating postsurgical treatment. Clinically, it is generally accepted that damage to the pituitary is the cause, but the mechanisms behind the response variability and underlying pathophysiology remain unknown. OBJECTIVE: To test the hypothesis that changing the degree of damage to the pituitary stalk produces a spectrum of water and electrolyte disturbance along which all presentations of postsurgical water and electrolyte disturbances can be identified. METHODS: We used HumMod, a large mathematical model of physiology, to simulate pituitary stalk damage at differing fractions: 20%, 40%, 60%, and 80%. The damaged neurons were modeled to undergo a 5-day countdown to degeneration and release stored antidiuretic hormone as they die, as is proposed to occur. RESULTS: Lower pituitary damage (20%) resulted in transient polyuria and intermediate damage (40%) was associated with delayed polyuria and diabetes insipidus. Higher levels of damage (60% and 80%) showed a triphasic pattern of diabetes insipidus. CONCLUSIONS: We postulate that our model provides a plausible mechanistic explanation for some varieties of postsurgical water and electrolyte disturbances, in which increasing damage to the pituitary potentiates the likelihood of a full triphasic response. However, our simulation shows that merely modifying the level of damage does not produce every presentation of water and electrolyte imbalance. This theory suggests that other mechanisms, which are still unclear and not a part of this model, may be responsible for postoperative hyponatremia and require further investigation.

Mechanisms of blood pressure salt sensitivity: new insights from mathematical modeling.

Mathematical modeling is an important tool for understanding quantitative relationships among components of complex physiological systems and for testing competing hypotheses. We used HumMod, a large physiological model, to test hypotheses of blood pressure (BP) salt sensitivity. Systemic hemodynamics, renal, and neurohormonal responses to chronic changes in salt intake were examined during normal renal function, fixed low or high plasma angiotensin II (ANG II) levels, bilateral renal artery stenosis, increased renal sympathetic nerve activity (RSNA), and decreased nephron numbers. Simulations were run for 4 wk at salt intakes ranging from 30 to 1,000 mmol/day. Reducing functional kidney mass or fixing ANG II increased salt sensitivity. Salt sensitivity, associated with inability of ANG II to respond to changes in salt intake, occurred with smaller changes in renal blood flow but greater changes in glomerular filtration rate, renal sodium reabsorption, and total peripheral resistance (TPR). However, clamping TPR at normal or high levels had no major effect on salt sensitivity. There were no clear relationships between BP salt sensitivity and renal vascular resistance or extracellular fluid volume. Our robust mathematical model of cardiovascular, renal, endocrine, and sympathetic nervous system physiology supports the hypothesis that specific types of kidney dysfunction, associated with impaired regulation of ANG II or increased tubular sodium reabsorption, contribute to BP salt sensitivity. However, increased preglomerular resistance, increased RSNA, or inability to decrease TPR does not appear to influence salt sensitivity. This model provides a platform for testing competing concepts of long-term BP control during changes in salt intake.

Validation of an integrative mathematical model of dehydration and rehydration in virtual humans.

Water homeostasis is one of the body's most critical tasks. Physical challenges to the body, including exercise and surgery, almost always coordinate with some change in water handling reflecting the changing needs of the body. Vasopressin is the most important hormone that contributes to short-term water homeostasis. By manipulating vascular tone and regulating water reabsorption in the collecting duct of the kidneys, vasopressin can mediate the retention or loss of fluids quickly. In this study, we validated HumMod, an integrative mathematical model of human physiology, against six different challenges to water homeostasis with special attention to the secretion of vasopressin and maintenance of electrolyte balance. The studies chosen were performed in normal men and women, and represent a broad spectrum of perturbations. HumMod successfully replicated the experimental results, remaining within 1 standard deviation of the experimental means in 138 of 161 measurements. Only three measurements lay outside of the second standard deviation. Observations were made on serum osmolarity, serum vasopressin concentration, serum sodium concentration, urine osmolarity, serum protein concentration, hematocrit, and cumulative water intake following dehydration. This validation suggests that HumMod can be used to understand water homeostasis under a variety of conditions.

The Creation of Surrogate Models for Fast Estimation of Complex Model Outcomes.

A surrogate model is a black box model that reproduces the output of another more complex model at a single time point. This is to be distinguished from the method of surrogate data, used in time series. The purpose of a surrogate is to reduce the time necessary for a computation at the cost of rigor and generality. We describe a method of constructing surrogates in the form of support vector machine (SVM) regressions for the purpose of exploring the parameter space of physiological models. Our focus is on the methodology of surrogate creation and accuracy assessment in comparison to the original model. This is done in the context of a simulation of hemorrhage in one model, "Small", and renal denervation in another, HumMod. In both cases, the surrogate predicts the drop in mean arterial pressure following the intervention. We asked three questions concerning surrogate models: (1) how many training examples are necessary to obtain an accurate surrogate, (2) is surrogate accuracy homogeneous, and (3) how much can computation time be reduced when using a surrogate. We found the minimum training set size that would guarantee maximal accuracy was widely variable, but could be algorithmically generated. The average error for the pressure response to the protocols was -0.05±2.47 in Small, and -0.3 +/- 3.94 mmHg in HumMod. In the Small model, error grew with actual pressure drop, and in HumMod, larger pressure drops were overestimated by the surrogates. Surrogate use resulted in a 6 order of magnitude decrease in computation time. These results suggest surrogate modeling is a valuable tool for generating predictions of an integrative model's behavior on densely sampled subsets of its parameter space.

Impact of cooking, cooling, and subsequent refrigeration on the growth or survival of Clostridium perfringens in cooked meat and poultry products.

In January 1999, the Food Safety and Inspection Service (FSIS) finalized performance standards for the cooking and chilling of meat and poultry products in federally inspected establishments. More restrictive chilling (stabilization) requirements were adopted despite the lack of strong evidence of a public health risk posed by industry practices employing the original May 1988 guidelines (U.S. Department of Agriculture FSIS Directive 7110.3). Baseline data led the FSIS to estimate a "worst case" of 10(4) Clostridium perfringens cells per g in raw meat products. The rationale for the FSIS performance standards was based on this estimate and the assumption that the numbers detected in the baseline study were spores that could survive cooking. The assumptions underlying the regulation stimulated work in our laboratory to help address why there have been so few documented outbreaks of C. perfringens illness associated with the consumption of commercially processed cooked meat and poultry products. Our research took into account the numbers of C. perfringens spores in both raw and cooked products. One hundred ninety-seven raw comminuted meat samples were cooked to 73.9 degrees C and analyzed for C. perfringens levels. All but two samples had undetectable levels (<3 spores per g). Two ground pork samples contained 3.3 and 66 spores per g. Research was also conducted to determine the effect of chilling on the outgrowth of C. perfringens spores in cured and uncured turkey. Raw meat blends inoculated with C. perfringens spores, cooked to 73.9 degrees C, and chilled according to current guidelines or under abuse conditions yielded increases of 2.25 and 2.44 log10 CFU/g for uncured turkey chilled for 6 h and an increase of 3.07 log10 CFU/g for cured turkey chilled for 24 h. No growth occurred in cured turkey during a 6-h cooling period. Furthermore, the fate of C. perfringens in cooked cured and uncured turkey held at refrigeration temperatures was investigated. C. perfringens levels decreased by 2.52, 2.54, and 2.75 log10 CFU/g in cured turkey held at 0.6, 4.4, and 10 degrees C, respectively, for 7 days. Finally, 48 production lots of ready-to-eat meat products that had deviated from FSIS guidelines were analyzed for C. perfringens levels. To date, 456 samples have been tested, and all but 25 (ranging from 100 to 710 CFU/g) of the samples contained C. perfringens at levels of <100 CFU/g. These results further support historical food safety data that suggest a very low public health risk associated with C. perfringens in commercially processed ready-to-eat meat and poultry products.

Incidence of Escherichia coli O157:H7 in frozen beef patties produced over an 8-hour shiftt.

A ground beef patty processor detected Escherichia coli O157:H7 in five production lots during routine testing with polymerase chain reaction (PCR) technology. This finding stimulated research to determine the incidence and potential entry points of the pathogen during processing. One of these lots (53,960 kg) was divided into 71 pallets (760 kg each) of food service ground beef patties. Ten cartons (19 kg each) were removed from each pallet, for a total of 710 cartons. Four patties were taken from each carton and subdivided to provide comparable samples for E. coli O157:H7 analyses by three different laboratories. Two laboratories employed different immunoassay tests, and one used PCR to screen samples. One sample set was analyzed for aerobic plate, coliform, and E coli Biotype I counts to determine if any relationship existed between these microbial groups and the incidence of E. coli O157:H7. For 73 samples, presumptive positive results for E. coli O157:H7 were obtained by one or more methods. For 48 of these 73 samples, positive results for the pathogen were culture confirmed. The largest number (29) of culture-confirmed positive E. coli O157:H7 results were detected by PCR. Most positive results were obtained during a short segment of processing. All culture-confirmed E. coli O157:H7 strains were further characterized by two genetic subtyping techniques, resulting in two to four different patterns, depending on the subtyping procedure employed. For any sample tested, the aerobic plate count was < 3.0 log CFU/g, and coliform and E. coli Biotype I counts were < or = 1.00 log CFU/g. The results of this study suggest that most positive samples were associated with a contaminated batch of raw material introduced just before the 1725- to 1844-h processing segment. These results also indicate that more aggressive sampling plans and genetic screening technologies such as PCR may be used to better detect low levels of E. coli O157:H7 in ground beef products.

Standardized Microbiological Sampling and Testing Procedures for the Beef Industry †.

Standardized microbiological sampling and testing procedures were developed that can be used throughout the beef slaughter and processing industry to facilitate the collection and any desired compilation of comparative data. Twenty samples each from carcasses (brisket, flank, and rump areas combined); subprimal cuts (clods); lean trim; and cutting and/or conveyor surfaces were collected in three slaughter and processing operations, with the first operation being a preliminary trial and resulting in no reported data. Microbiological analyses for Clostridium perfringens , Escherichia coli O157:H7, Listeria monocytogenes , Salmonella spp., Staphylococcus aureus , Campylobacter jejuni/coli , total coliforms, E. coli Biotype I, and aerobic mesophilic bacteria (aerobic plate count, APC) were performed on all samples by an outside laboratory. The procedures developed were effective in allowing samples to be collected, shipped, and analyzed in the same manner for all operations. From a logistical standpoint, approximately 20 samples each of carcasses, clods, lean trim, and surfaces could be taken within 4 to 6 h by five people. Forty samples each of carcass, clod, lean trim, and conveyor surfaces from two plants tested negative for E. coli O157:H7, Salmonella spp., and Listeria spp., with the exception of L. monocytogenes being isolated from one carcass and one clod sample. APCs and total coliform counts were between 103 to 105 and 102 to 103 CFU/cm2 or CFU/g, respectively, for the 40 samples each of carcasses, clods, and lean trim. APCs for surface swab counts ranged from ≤ 10 to 103 CFU/cm2.

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.