Predicting pathogen-specific CD8 T cell immune responses from a modeling approach.

The primary CD8 T cell immune response constitutes a major mechanism to fight an infection by intra-cellular pathogens. We aim at assessing whether pathogen-specific dynamical parameters of the CD8 T cell response can be identified, based on measurements of CD8 T cell counts, using a modeling approach. We generated experimental data consisting in CD8 T cell counts kinetics during the response to three different live intra-cellular pathogens: two viruses (influenza, vaccinia) injected intranasally, and one bacteria (Listeria monocytogenes) injected intravenously. All pathogens harbor the same antigen (NP68), but differ in their interaction with the host. In parallel, we developed a mathematical model describing the evolution of CD8 T cell counts and pathogen amount during an immune response. This model is characterized by 9 parameters and includes relevant feedback controls. The model outputs were compared with the three data series and an exhaustive estimation of the parameter values was performed. By focusing on the ability of the model to fit experimental data and to produce a CD8 T cell population mainly composed of memory cells at the end of the response, critical parameters were identified. We show that a small number of parameters (2-4) define the main features of the CD8 T cell immune response and are characteristic of a given pathogen. Among these parameters, two are related to the effector CD8 T cell mediated control of cell and pathogen death. The parameter associated with memory cell death is shown to play no relevant role during the main phases of the CD8 T cell response, yet it becomes essential when looking at the predictions of the model several months after the infection.

Interleukin 2 activates extracellular signal-regulated protein kinase 2.

Interleukin 2 (IL-2) stimulated activation of the 42-kD extracellular signal-regulated kinase 2 (Erk2) in murine IL-3-dependent cells, expressing either high or intermediate affinity IL-2 receptors. Activation was both rapid, occurring within 5 min of IL-2 addition, and prolonged, remaining elevated for 30 min. Activation of Erk2 appeared to be necessary for IL-2 stimulation of proliferation, as deletion of a region of the cytoplasmic domain of the IL-2 receptor beta chain, essential for IL-2 stimulation of proliferation, abolished Erk2 activation by IL-2. Furthermore, cells that had been deprived of cytokine for 24 h were then refractory to IL-2 stimulation of both Erk2 activity and proliferation. However, elevation of Erk2 activity was not sufficient to stimulate proliferation, as protein kinase C activation stimulated Erk2 activity but not DNA synthesis. Also, cells exposed to IL-2 in the presence of rapamycin showed full Erk2 activation but not DNA synthesis. These data suggest that IL-2 must stimulate both Erk2 activity and a further pathway(s) to trigger cell proliferation.

Interleukin 3 protects murine bone marrow cells from apoptosis induced by DNA damaging agents.

Murine bone marrow-derived cells, dependent on interleukin 3 (IL-3) for their growth in culture, undergo programmed cell, or apoptosis, upon cytokine withdrawal. Here it is reported that a variety of DNA damaging agents cause a more rapid onset of apoptosis in a factor-dependent cell line, BAF3, deprived of IL-3. In contrast, when cultured in the presence of IL-3, or other growth promoting factors, BAF3 cells are highly resistant to X-irradiation and the cytotoxic drugs etoposide and cisplatin. Overexpression of the bcl2 gene product also protects BAF3 cells from DNA damage. The presence of IL-3 is not required during the initial events of DNA damage or its repair. In the absence of IL-3, cells still complete the repair of DNA breaks within 15 min, and continue to cycle for 5 h. At this time, IL-3 is necessary to prevent the accelerated onset of DNA cleavage from a G2 arrest point.

Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro.

The characteristics of CD8+ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8+ T cell populations. In most experimental systems, heterogeneous populations of primed CD8+ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8+ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8+ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8+ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8+ T cells were compared in vitro. We found that CD8+ T cells from primed mice are activated by peptide concentrations 10-50-fold lower than naive mice. In addition, the kinetics of interleukin 2R alpha chain upregulation by primed CD8+ T cells differ from naive CD8+ T cells. These primed hyperresponsive CD8+ T cells might play an important role in the memory response.

Idiotypic analysis of potential and available repertoires in the arsonate system.

We have shown that, by suitable idiotypic manipulation, BALB/c mice can express the major cross-reactive idiotype (CRI) of A/J mice in response to azophenylarsonate (Ars). In order to know if the CRIA idiotype is present in the potential repertoire of BALB/c before any intentional selection, we used polyclonal activation in vitro and limiting dilution analysis. The readout was done with two monoclonal anti-CRIA antibodies that recognize distinct idiotopes on a CRIA+ A/J germline-encoded monoclonal antibody. We studied the frequency of CRIA+ lipopolysaccharide (LPS)-reactive cells in the spleens of nonimmune and immune A/J mice and in the spleens of naive and manipulated (i.e., producing CRIA+ antibodies) BALB/c mice. A/J and BALB/c naive individuals presented very high frequencies of Ars-specific B cells while the frequency of CRIA+ B cells was only a minor subset (0.5%) of the total Ars-specific subset in the two strains. When A/J mice were immunized with Ars-keyhole limpet hemocyanin, a clear preferential expansion of the CRIA+ minor subset of A/J mice was observed (100x). No such enhancement was observed in BALB/c mice similarly treated. Manipulated BALB/c mice presented a higher frequency of CRIA+ anti-Ars B cells than naive or antigen-immunized BALB/c individuals.

Overexpression of Sp1 transcription factor induces apoptosis.

Transcription factor Sp1 has recently been shown to be overexpressed in a number of human cancers and its overexpression contributes to malignant transformation. Sp1 regulates the expression of a number of genes participating in multiple aspects of tumorigenesis such as angiogenesis, cell growth and apoptosis resistance. To better understand the role of increased Sp1 levels on apoptosis regulation we have used retroviruses to overexpress this protein in haematopoietic Baf-3 cells and in 3T3 fibroblasts. We have also used inducible expression systems to control ectopic Sp1 levels in different cell types. Surprisingly, Sp1 overexpression on its own induces apoptosis in all the cellular models tested. The apoptotic pathways induced by Sp1 overexpression are cell type specific. Finally, using a truncated form of Sp1, we show that Sp1-induced apoptosis requires its DNA-binding domain. Our results highlight that Sp1 levels in untransformed cells must be tightly regulated as Sp1 overexpression leads to the induction of apoptosis. Our results also suggest that cancer cells overexpressing Sp1 can avoid Sp1-induced apoptosis.

Bcl-X is the major pleiotropic anti-apoptotic gene activated by retroviral insertion mutagenesis in an IL-3 dependent bone marrow derived cell line.

In order to identify genes capable of inhibiting apoptosis induced by different pathways, without inducing proliferation we have performed retroviral insertion mutagenesis in the IL-3 dependent bone marrow derived Baf-3 cell line. Out of 200 mutants obtained in three separate mutagenesis experiments, four mutants were resistant to multiple apoptosis inducing pathways (including growth factor starvation, staurosporine, etoposide and cyclosporin A) and did not proliferate in the absence of IL-3. These four mutants overexpress the bcl-X gene following a retroviral insertion 5' of the translation initiation site. These results indicate that the bcl-X gene is a major pleiotropic anti-apoptotic gene in Baf-3 cells. They also suggest that the Bcl-2 family of genes might be the only one capable of inhibiting apoptosis induced by multiple pathways without inducing cell proliferation.

Growth factor starvation of bcl-2 overexpressing murine bone marrow cells induced refractoriness to IL-3 stimulation of proliferation.

Murine bone-marrow derived BAF3 cells, over-expressing the human Bcl-2 gene product, showed considerably delayed onset of apoptosis when deprived of IL-3. Such Bcl-2-BAF3 cells arrested rapidly in the G1 phase of the cell cycle upon IL-3 removal, then became refractory to IL-3 re-stimulation. The delay in IL-3 induced proliferation of Bcl-2 over-expressing cells was due to down-regulation of a specific signalling pathway. In the refractory cells, IL-3 was able to stimulate protein tyrosine phosphorylation and c-myc mRNA accumulation, but not rapid Erk2 activation or cdc2 mRNA accumulation.

The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor.

Tumorigenesis can be induced either by activating cell proliferation or by inhibiting metabolic pathways regulating programmed cell death (apoptosis). There is evidence suggesting that p60(v-src) and other tyrosine kinases protect cells against apoptosis. This effect could contribute to cell transformation by the Rous sarcoma virus. Mechanism of cell death inhibition by p60(v-src) remains largely unknown. We have recently reported that in avian cells p60(v-src) activates the expression of nr-13, a bcl-2-related gene. In this paper, we demonstrate, using the bone marrow derived cell line Baf-3 as an experimental model, that the product of this avian gene (nr-13) is a potent anti-apoptotic factor. In addition, we report that, in quail neuroretinal cells, nr-13 expression is activated upon infection by the Rous sarcoma virus (RSV) but not by other oncogenic retroviruses like FSV or MH2, suggesting that nr-13 is a specific target of v-src. Activation of nr-13 expression may be a key step in cellular transformation by v-src.

In bone marrow derived Baf-3 cells, inhibition of apoptosis by IL-3 is mediated by two independent pathways.

The inhibition of cell death by growth factors plays a key role in the maintenance of the haematopoietic system homeostasis. However the mechanisms involved in this inhibition are still poorly understood. In order to determine if inhibition of apoptosis by growth factors is dependent only on the expression of survival genes, we have studied that process in the bone marrow derived IL-3 dependent cell line Baf-3. We show that, following IL-3 starvation, mRNA and protein levels of Bcl-X but not Bcl-2 decrease rapidly preceeding the onset of death. The death of IL-3 starved cells is asynchronous, starting between 6 to 8 h with 50% death being reached after 10 to 12 h. At any time point, apoptosis can be rapidly inhibited by growth factor re-addition. This has allowed us to determine that the inhibition of apoptosis by growth factor takes place at two levels. The first one, which we have called short term inhibition, is independent of mRNA and protein synthesis i.e. it takes place in the absence of survival gene neosynthesis and can be demonstrated during the first 6 h following growth factor re-addition. The second one corresponds to long-term survival-more than 24 h survival-and is strongly correlated with the induction of Bcl-X but not Bcl-2 gene expression. This induction of Bcl-X by IL-3 is shown to be dependent on MAP-kinase activation.

Decreased glycolytic metabolism contributes to but is not the inducer of apoptosis following IL-3-starvation.

IL-3 regulates the glycolytic pathway. In Baf-3 cells IL-3 starvation leads to a decrease in glucose uptake and in lactate production. To determine if there is a link between the decreased metabolism induced by growth factor-starvation and the induction of cell death, we have compared the cell death characteristics and the metabolic modifications induced by IL-3-deprivation or glucose-deprivation in Baf-3 cells. We show that in both conditions cells die by an apoptotic process which involves the activation of similar Caspases. Different metabolic parameters (i.e. intracellular ATP levels and lactate accumulation in the culture medium) were measured. We show that IL-3 deprivation leads to a partial decrease in lactate production in contrast to glucose deprivation that completely inhibits lactate production. Similarly following IL-3-starvation a significant drop in the intracellular ATP levels in live cells is observed only after 16 h when a large fraction, more than 50 per cent of cells, is already apoptotic. On the contrary, glucose deprivation is followed by an abrupt decrease in ATP levels in the first 2 h of treatment. However, in the presence of IL-3, cells are able to survive for an extended time in these conditions since 70% of cells survived with low ATP levels for up to 16 h. This was not due to partial inhibition of the apoptotic process by the low level of ATP as glucose-deprivation in the absence of IL-3 led to faster death kinetics of Baf-3 cells compared with IL-3 starvation only. These results indicate that the drop in ATP levels and the triggering of apoptosis can be dissociated in time and that when the glycolytic pathway is strongly inhibited, cells are able to survive with relatively low ATP levels if IL-3 is present. Finally we show that induction of bcl-x by IL-3 protects cells from glucose-deprivation induced cell death.

Role of PI3-kinase in Bcl-X induction and apoptosis inhibition mediated by IL-3 or IGF-1 in Baf-3 cells.

In Baf-3 cells, IL-3 and IGF-1 both inhibit cell death. These growth factors act at least on two different pathways involved in the inhibition of apoptosis. They both upregulate Bcl-X at the mRNA and protein levels and also activate a pathway which inhibits apoptosis in the absence of protein synthesis. Recently, these two growth factors have been shown to activate the PI3-kinase-AKT pathway which leads to the phosphorylation of the pro-apoptotic Bcl-XL regulator Bad. In this study, we have investigated the role of PI3-kinase in the regulation of Bcl-X expression and in the survival of Baf-3 cells. We show that PI3-kinase activation is involved in the upregulation of Bcl-X mRNA induced by both IL-3 and IGF-1. Moreover, PI3-kinase activity is also necessary for inhibition of apoptosis and caspase regulation by IGF-1 but not IL-3.

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes.

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.

Albumin permeation of new vessels is increased in diabetic rats.

125I-bovine serum albumin (BSA) permeation of the vasculature of 3-wk-old granulation tissue (induced by subcutaneous implantation of polyester fabric) formed in the diabetic milieu was assessed in female BB/W, spontaneously diabetic rats and in male, Sprague-Dawley rats with streptozocin-induced diabetes as well as in corresponding nondiabetic controls. Albumin permeation of new granulation tissue vessels was markedly increased in both groups of diabetic animals relative to that of nondiabetic controls, while albumin permeation of vessels in most other tissues did not differ for controls and diabetics. These observations indicate that the functional integrity of new vessels formed in the diabetic milieu is impaired: (1) to a greater extent than that of older vessels formed before induction of diabetes and (2) relative to new vessels in nondiabetics. The implication of these observations is that molecular constituents of vessels synthesized in the diabetic milieu are quantitatively and/or qualitatively abnormal and/or their incorporation into vessels is defective.

Sorbinil prevents diabetes-induced increases in vascular permeability but does not alter collagen cross-linking.

In recent studies we have demonstrated a marked increase in albumin permeation of new vessels formed by angiogenesis (in subcutaneous tissue) in the diabetic milieu. Likewise, lysyl oxidase-mediated collagen cross-linking is markedly increased in the scar tissue associated with angiogenesis. The present studies were undertaken to determine whether sorbinil, a chemical inhibitor of aldose reductase that has been shown to prevent and reverse diabetic cataracts and neuropathy, also could prevent the vascular permeability and collagen cross-linking changes in this model. Vascular permeation by 125I-BSA, collagen cross-linking, and tissue levels of sorbitol, myo-inositol, and scyllo-inositol were assessed in male Sprague-Dawley rats 3 wk after injection of streptozocin and induction of angiogenesis and collagen synthesis in polyester fabric implanted subcutaneously. Sorbinil (approximately 25 mg/kg/day) added to the diet of diabetic rats reduced the diabetes-induced increases in albumin permeation by 80%, completely prevented diabetes-induced changes in tissue levels of sorbitol and myo-inositol, and markedly reduced diabetes-induced changes in tissue levels of scyllo-inositol. In contrast, sorbinil had no effect on plasma glucose levels or collagen solubility (an index of collagen cross-linking). These observations indicate that increased vascular permeability associated with diabetes is linked to imbalances in sorbitol/inositol metabolism. These findings also indicate that diabetes-induced increases in vascular permeability and in collagen cross-linking are independent phenomena and diabetes-induced increases in vascular permeability are largely preventable by treatment with an aldose reductase inhibitor in the face of high plasma glucose levels.

Sex steroid dependency of diabetes-induced changes in polyol metabolism, vascular permeability, and collagen cross-linking.

The effects of castration on diabetes-induced increases in collagen cross-linking and vascular permeability and on polyol levels in new granulation tissue formed after induction of streptozocin (STZ) diabetes were examined in male Sprague-Dawley rats. New granulation tissue formation was induced by implanting sterile polyester fabric subcutaneously (s.c.) at the time of STZ injection 3 wk before assessment of vascular permeability and collagen cross-linking. Castration was performed 10 days before implanting the fabric. The characteristic increases in collagen cross-linking (manifested by decreased solubility in 0.5 M acetic acid) and in albumin permeation of the vasculature seen in intact diabetic rats were completely prevented by castration. Net collagen accumulation was not affected by diabetes or castration. Castration also markedly diminished diabetes-induced increases in tissue levels of sorbitol and completely prevented the decreases in tissue levels of myo-inositol and scyllo-inositol observed in intact diabetic rats, but had no effect on serum glucose levels, nonenzymatic glycosylation of plasma and granulation tissue proteins, or plasma somatomedin-C levels. The demonstration that castration prevents diabetes-induced increases in vascular permeability and collagen cross-linking as well as imbalances in tissue levels of sorbitol, myo-inositol, and scyllo-inositol in this model indicates that all of these changes are sex steroid-dependent phenomena. While the pathogenesis of these vascular permeability and collagen cross-linking changes is clearly multifactorial, these new findings: indicate that the role of sex steroids in the development of late complications of diabetes may be far more important than hitherto suspected, and suggest an explanation for the clinical observation that diabetic complications are uncommon in prepubertal diabetic subjects regardless of duration of diabetes.

The influence of V kappa gene polymorphism on the induction of silent idiotypes in the arsonate system.

It has been previously shown that it is possible to modify the expressed repertoire of a given individual using idiotypic manipulation. For example, A/J mice respond to arsonate challenge by synthesizing a dominant idiotype, CRIA, whereas BALB/c mice do not. However, after treatment with rabbit polyclonal anti-CRIA antibodies (Ab2 or anti-idiotypic antibodies) and arsonate, BALB/c mice are able to synthesize a CRIA-like idiotype. To determine whether this modification of repertoire is dependent on the immunoglobulin loci (Ig-h, kappa), we have analyzed the anti-arsonate response after anti-idiotypic treatment of three strains of mice (C58, C.C58, AKR), chosen because they are among a small group of strains which express Kappa V regions not seen in other strains. There are also L chains lacking in these strains which are expressed in other mice. The C58 and C.C58 strains share the same Ig-h locus (Ig-ha) with BALB/c mice but C.C58 are congenic mice, that express the kappa loci on a BALB/c genetic background. AKR mice express the Ig-hd haplotype. AKR, C58 and C.C58 do not produce CRIA positive antibodies in response to arsonate; a defect which has been previously mapped to the kappa locus. These three strains of mice (C58, C.C58 and AKR) were treated with rabbit anti-CRIA and boosted with Ars-KLH. The results show that after such treatment, the C.C58 mice were able to express CRIA-like antibodies which are serologically identical to those of BALB/c.

System Interdependency Modeling in the Design of Prognostic and Health Management Systems in Smart Manufacturing.

The fields of risk analysis and prognostics and health management (PHM) have developed in a largely independent fashion. However, both fields share a common core goal. They aspire to manage future adverse consequences associated with prospective dysfunctions of the systems under consideration due to internal or external forces. This paper describes how two prominent risk analysis theories and methodologies - Hierarchical Holographic Modeling (HHM) and Risk Filtering, Ranking, and Management (RFRM) - can be adapted to support the design of PHM systems in the context of smart manufacturing processes. Specifically, the proposed methodologies will be used to identify targets - components, subsystems, or systems - that would most benefit from a PHM system in regards to achieving the following objectives: minimizing cost, minimizing production/maintenance time, maximizing system remaining usable life (RUL), maximizing product quality, and maximizing product output. HHM is a comprehensive modeling theory and methodology that is grounded on the premise that no system can be modeled effectively from a single perspective. It can also be used as an inductive method for scenario structuring to identify emergent forced changes (EFCs) in a system. EFCs connote trends in external or internal sources of risk to a system that may adversely affect specific states of the system. An important aspect of proactive risk management includes bolstering the resilience of the system for specific EFCs by appropriately controlling the states. Risk scenarios for specific EFCs can be the basis for the design of prognostic and diagnostic systems that provide real-time predictions and recognition of scenario changes. The HHM methodology includes visual modeling techniques that can enhance stakeholders' understanding of shared states, resources, objectives and constraints among the interdependent and interconnected subsystems of smart manufacturing systems. In risk analysis, HHM is often paired with Risk Filtering, Ranking, and Management (RFRM). The RFRM process provides the users, (e.g., technology developers, original equipment manufacturers (OEMs), technology integrators, manufacturers), with the most critical risks to the objectives, which can be used to identify the most critical components and subsystems that would most benefit from a PHM system. A case study is presented in which HHM and RFRM are adapted for PHM in the context of an active manufacturing facility located in the United States. The methodologies help to identify the critical risks to the manufacturing process, and the major components and subsystems that would most benefit from a developed PHM system.

Survival Strategies for Tsunami of ICD-10-CM for Interventionalists: Pursue or Perish!

The unfunded mandate for the implementation of International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10-CM) is scheduled October 1, 2015. The development of ICD-10-CM has been a complicated process. We have endeavored to keep Interventional Pain Management doctors apprised via a variety of related topical manuscripts. The major issues relate to the lack of formal physician participation in its preparation. While the American Health Information Management Association (AHIMA) and American Hospital Association (AHA) as active partners in its preparation. Centers for Medicare and Medicaid Services (CMS) and Centers for Disease Control and Prevention (CDC) are major players; 3M and Blue Cross Blue Shield Association are also involved. The cost of ICD-10-CM implementation is high, similar to the implementation of electronic health records (EHRs), likely consuming substantial resources. While ICD-10, utilized worldwide, includes 14,400 different codes, ICD-10-CM, specific for the United States, has expanded to 144,000 codes, which also includes procedural coding system. It is imperative for physicians to prepare for the mandatory implementation. Conversion from ICD-9-CM to ICD-10-CM coding in interventional pain management is not a conversion of one to one that can be easily obtained from software packages. It is a both a difficult and time-consuming task with each physician, early on, expected to spend on estimation at least 10 minutes per visit on extra coding for established and new patients. For interventional pain physicians, there have been a multitude of changes, including creation of new codes and confusing conversion of existing codes. This manuscript describes a variety of codes that are relevant to interventional pain physicians and often utilized in daily practices. It is our objective that this manuscript will provide coding assistance to interventional pain physicians.

Physicochemical characterization of the human nail: I. Pressure sealed apparatus for measuring nail plate permeabilities.

Diffusion characteristics of the nail plate are necessary in providing the baselines for rational topical management of nail infections. In order to develop such baselines a unique stainless steel diffusion cell has been designed. The cell permits the exposure of 0.38 cm2 of nail plate to a bathing medium which is stirred by small motors mounted above the cell. The diffusion of water, methanol and ethanol at constant temperature (37 degrees C), has been examined over periods up to 4 h. Average permeability coefficients of water, methanol and ethanol were determined as 16.5 +/- 5.9 X 10(-3) cm hr-1, 5.6 X 10(-3) cm hr-1 and 5.8 +/- 3.1 X 10(-3) cm hr-1 respectively. Moreover rates of diffusion across the nail were inversely proportional to nail thickness. Based on methanol data, nail plate barrier property appears stable for long periods of aqueous immersion.