Multi-scale models of local control of calcium induced calcium release.

Calcium-induced-calcium-release in cardiac myocytes is the release of Ca(2+) from the sarcoplasmic reticulum (SR) triggered by Ca(2+) entering the cell through L-type Ca(2+) channels. The Ca(2+) is released through ryanodine receptors which 'sense' local [Ca(2+)] in the small region (the diadic space) positioned between the t-tubules and the SR. The length-scale of a single diad is of the order of 10nm and the diffusion time-scale is of order of 1 micros with each cell containing approximately 10,000 diadic spaces which act independently. However, typically one is interested in Ca(2+) currents at the whole cell level and higher. This is a multi-scale problem and cannot be solved by direct computation. In this paper we develop a general framework for deriving approximate solutions of these models.

A simplified local control model of calcium-induced calcium release in cardiac ventricular myocytes.

Calcium (Ca2+)-induced Ca2+ release (CICR) in cardiac myocytes exhibits high gain and is graded. These properties result from local control of Ca2+ release. Existing local control models of Ca2+ release in which interactions between L-Type Ca2+ channels (LCCs) and ryanodine-sensitive Ca2+ release channels (RyRs) are simulated stochastically are able to reconstruct these properties, but only at high computational cost. Here we present a general analytical approach for deriving simplified models of local control of CICR, consisting of low-dimensional systems of coupled ordinary differential equations, from these more complex local control models in which LCC-RyR interactions are simulated stochastically. The resulting model, referred to as the coupled LCC-RyR gating model, successfully reproduces a range of experimental data, including L-Type Ca2+ current in response to voltage-clamp stimuli, inactivation of LCC current with and without Ca2+ release from the sarcoplasmic reticulum, voltage-dependence of excitation-contraction coupling gain, graded release, and the force-frequency relationship. The model does so with low computational cost.

Transgenic swine for biomedicine and agriculture.

Initial technologies for creating transgenic swine only permitted random integration of the construct. However, by combining the technology for homologous recombination in fetal somatic cells with that of nuclear transfer (NT), it is now possible to create specific modifications to the swine genome. The first such example is that of knocking out a gene that is responsible for hyperacute rejection (HAR) when organs from swine are transferred to primates. Because swine are widely used as models of human diseases, there are opportunities for genetic modification to alter these models or to create additional models of human disease. Unfortunately, some of the offspring resulting from NT have abnormal phenotypes. However, it appears that these abnormal phenotypes are a result of epigenetic modifications and, thus, are not transmitted to the offspring of the clones. Although the technique of producing animals with specific genetic modifications by NT has been achieved, improvements to the NT technique as well as improvements in the culture conditions for somatic cells and the techniques for genetic modification are still needed.

Molecular interactions between two long-QT syndrome gene products, HERG and KCNE2, rationalized by in vitro and in silico analysis.

The cardiac delayed rectifier potassium current mediates repolarization of the action potential and underlies the QT interval of the ECG. Mutations in either of the two molecular components of the rapid delayed rectifier (I(K,r)), HERG and KCNE2, have been linked to heritable or acquired long-QT syndrome. Mechanisms whereby mutations of KCNE2 produce fatal cardiac arrhythmias characteristic of long-QT syndrome remain unclear. In this study, we characterize functional interactions between HERG and KCNE2 with a view to defining underlying mechanisms for action potential prolongation and long-QT syndrome. Whereas coexpression of hKCNE2 with HERG alters both kinetics and density of ionic current, incorporation of these effects into a quantitative model of the action potential predicts that only changes in current density significantly affect repolarization. Thus, the primary functional consequence of hKCNE2 on action potential morphology is through modulation of I(K,r) density, as predicted by the model. Mutations associated with long-QT syndrome that result only in modest changes of gating kinetics may be epiphenomena or may modulate action potential repolarization via interaction with alternative pore-forming potassium channel alpha subunits.

Role of the calcium-independent transient outward current I(to1) in shaping action potential morphology and duration.

The Kv4.3-encoded current (I:(Kv4.3)) has been identified as the major component of the voltage-dependent Ca(2+)-independent transient outward current (I:(to1)) in human and canine ventricular cells. Experimental evidence supports a correlation between I:(to1) density and prominence of the phase 1 notch; however, the role of I:(to1) in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35 degrees C are developed on the basis of experimental measurements. A model of canine I:(to1) is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca(2+) current and I:(Kv4.3) and predict a bimodal relationship between I:(Kv4.3) density and APD whereby perturbations in I:(Kv4.3) density may produce either prolongation or shortening of APD, depending on baseline I:(to1) current level.

Production of transgenic porcine blastocysts by nuclear transfer.

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.

Identification of a full-length cDNA for an endogenous retrovirus of miniature swine.

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attachment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.

Current and future prospects for xenotransplantation.

The transplantation of organs and tissues between animal species, or xenotransplantation, is the focus of a growing field of research, owing primarily to the increasing shortage of allogeneic donor organs. The pig stands out as the most suitable donor animal for humans; however, xenografts (e.g. pig organs) used for human transplantation are normally destroyed by the host within minutes by hyperacute xenograft rejection. An improved understanding of the immune recognition and rejection of xenografts has resulted in new therapies that can partially overcome hyperacute rejection (HAR), delayed xenograft rejection (DXR) or acute vascular xenograft rejection. Strategies to diminish immunogenicity following xenotransplantation can be divided into two approaches: those directed at the recipient (e.g. antibodies or complement depletion or inhibition and tolerance induction) and those directed at the donor (e.g. transgenic modifications to express human complement-regulatory proteins or removal or displacement of alphaGal epitopes). DXR is likely to be controlled by transgenic inhibition of endothelial cell activation (e.g. inhibition of NF-kappaB). Transgenic pigs required for xenotransplantation will soon be generated at a greater efficiency and precision using nuclear transfer and cloning when compared to pronuclear injection. Of greater significance is that nuclear transfer offers the ability to target gene insertion selectively to specific gene loci and to delete specific genes in the pig. Experimental pig-to-primate organ xenotransplantation is currently under way, and results show increased transplant function from minutes to days and weeks. The final therapeutic regimen that allows survival of a discordant xenograft is likely to involve a combination of 'modified' functional genes in the donor organ, the development of immunological tolerance to pig antigens and administration of novel therapeutic agents, including immunosuppressants, that can control natural killer (NK) cell and monocyte mediated responses.

The use of tolerance for transplantation across xenogeneic barriers.

The success of human organ transplantation as a clinical treatment has created a conundrum for the transplant community. It has caused a shortage of human donor organs and uncovered problems of chronic immunosuppression in those lucky enough to receive organ transplants due to their use of chronic immunosuppressive drugs. Our aim is to attempt to approach both issues by establishing specific transplantation tolerance to pig organ grafts.

Definition of the human T-cell epitopes of Fel d 1, the major allergen of the domestic cat.

BACKGROUND: A heterodimeric acidic glycoprotein (Fel d 1) has been defined as the major allergen of the domestic cat. Because T-cell help is required for the initiation and maintenance of allergic responses, it is of importance to determine the T-cell-reactive regions of the Fel d 1 molecule. METHODS: Overlapping peptides corresponding to the two chains of Fel d 1 were tested in proliferation assays on polyclonal T-cell lines and for the ability to bind Fel d 1-specific IgE in ELISA and histamine release assays. RESULTS: Assay of T-cell lines derived from 53 subjects allergic to cats demonstrated that the majority of T-cell reactivity is found in chain 1 of Fel d 1. Two peptides (Fel-1 and Fel-2) containing major epitopes, alone or as a mixture, efficiently activated T cells and exhibited minimal detectable reactivity with IgE by ELISA or histamine release assay. CONCLUSIONS: Two Fel d 1 peptides containing major T-cell epitopes have been identified, have been shown to bind minimal Fel d 1-specific IgE, and are now being tested for the ability to decrease T-cell responses in patients with cat allergy as a new form of immunotherapy.

Efficacy of a therapeutic cocaine vaccine in rodent models.

Cocaine abuse is a major medical and public health concern in the United States, with approximately 2.1 million people dependent on cocaine. Pharmacological approaches to the treatment of cocaine addiction have thus far been disappointing, and new therapies are urgently needed. This paper describes an immunological approach to cocaine addiction. Antibody therapy for neutralization of abused drugs has been described previously, including a recent paper demonstrating the induction of anti-cocaine antibodies. However, both the rapidity of entry of cocaine into the brain and the high doses of cocaine frequently encountered have created challenges for an antibody-based therapy. Here we demonstrate that antibodies are efficacious in an animal model of addiction. Intravenous cocaine self-administration in rats was inhibited by passive transfer of an anti-cocaine monoclonal antibody. To actively induce anti-cocaine antibodies, a cocaine vaccine was developed that generated a high-titer, long-lasting antibody response in mice. Immunized mice displayed a significant change in cocaine pharmacokinetics, with decreased levels of cocaine measured in the brain of immunized mice only 30 seconds after intravenous (i.v.) administration of cocaine. These data establish the feasibility of a therapeutic cocaine vaccine for the treatment of cocaine addiction.

How inhomogeneities and airway walls affect frequency dependence and separation of airway and tissue properties.

It has been proposed that during mild-to-moderate bronchoconstriction one can partition airway and tissue properties on the basis of input impedance (Zin) acquired from 0.1 to 5 Hz (K.R. Lutchen, B. Suki, Q. Zhang, F. Peták, B. Daróczy, and Z. Hantos. J. Appl. Physiol. 77: 373-385, 1994). The approach is to apply a homogeneous lung model that contains airway resistance and viscoelastic tissue damping and elastance parameters. The tissue parameters account for the frequency dependence in lung resistance (RL) and elastance (EL). We present an anatomically consistent asymmetrically branching airway model to address two key questions: 1) How will lung inhomogeneities, airway wall shunting, and tissue viscoelasticity contribute to increased frequency dependence and levels of RL and EL during lung constriction? and 2) How much can lung inhomogeneities and airway wall shunting contribute to our assessment of airway, tissue, and overall lung properties derived from Zin? The model incorporates nonrigid airway walls and allows for explicit control over the type and degree of inhomogeneous airway constriction or tissue changes. Our results indicate that, from 0.1 to 5 Hz, airway wall shunting does not become important unless the entire lung periphery experiences significant constriction. Mild-to-moderate inhomogeneous peripheral airway constriction produces a relatively minor additional frequency dependence in RL and EL beyond that due to the tissues alone. With more extreme constriction, however, there is a marked frequency-dependent increase in EL. This phenomenon may render it impossible to distinguish from a single frequency measurement whether an increase in EL during bronchoconstriction is a consequence of a true increase in tissue stiffening or simply a consequence of airway phenomena. Finally, Zin from 0.1 to 5 Hz can be used to provide a reasonable separation of airway and tissue properties for mild-to-moderate homogeneous or inhomogeneous lung constriction. However, during more severe disease, inhomogeneities and/or wall shunting will produce substantial overestimation of tissue damping and hysteretic properties. In fact, the only reliable indicator of a real change in the tissues may be a change in the estimate of tissue elastance that is based on data extending to a sufficiently low frequency.

T cell epitope mapping of ragweed pollen allergen Ambrosia artemisiifolia (Amb a 5) and Ambrosia trifida (Amb t 5) and the role of free sulfhydryl groups in T cell recognition.

Ambrosia artemisiifolia (Amb a 5; Ra5S) and Ambrosia trifida (Amb t 5; Ra5G) are homologous allergens purified from short and giant ragweed pollen, respectively. Allergic human sera and hyperimmunized animal antisera directed against Amb a 5 or Amb t 5 show a high degree of species specificity, with little or no cross-reactivity between these two allergens, suggesting that the major Ab binding epitopes of Amb a 5 and Amb t 5 are distinct. Overlapping synthetic peptides derived from the allergen sequences were used to investigate the specificity of T cell responses in four strains of mice, BALB/c (H-2d), CBA (H-2k), C57BL/6 (H-2b), and A/J (H-2a). All four strains of mice responded to purified Amb a 5 and Amb t 5. Cross-reactivity was found at the T cell level between Amb a 5 and Amb t 5 in T cells from BALB/c, A/J, and CBA mice, but not in T cells from C57BL/6 mice. A T cell epitope from Amb a 5, residues 27-36 (PWQVVCYESS), was mapped using T cell hybridomas from BALB/c mice. A T cell epitope in Amb t 5 was mapped in the same strain to residues 24-34 (KYCVCYDSKAI). Disulfide bonds in Amb a 5 and Amb t 5 were found to be involved in T cell reactivity. Conversion of disulfide bridges into free sulfhydryl (SH) forms was required for the response of T cell hybridomas to peptide t5 (residues 27-40) from Amb t 5. Reduction of peptide a4 (residues 21-37) from Amb a 5 was essential for inducing the cross-reactivity observed with Amb t 5-specific T cell hybridomas. It is concluded that free sulfhydryl groups play a major role in the T cell recognition of cross-reactivity T cell epitopes within these related allergens.

Peripheral T-cell tolerance induced in naive and primed mice by subcutaneous injection of peptides from the major cat allergen Fel d I.

T cells control the majority of antigen-specific immune responses. Therefore, influencing the activation of the T-cell response in order to modify immune responsiveness is an obvious therapeutic goal. We have used a mouse model of response to Fel d I, the major cat protein allergen in humans, to explore the ability of peptides derived from Fel d I to inhibit T-cell-dependent immune responses to the peptides themselves and to larger polypeptides. T cells from B6CBAF1 mice respond to the Fel d I peptide IPC-2 after challenge with IPC-2. However, subcutaneous tolerization with IPC-2 prevents this response as measured by production of interleukins 2 and 4 and interferon gamma. Fel d I immunization of B6D2F1 mice results in T-cell responses primarily to one peptide derived from Fel d I. Injecting this peptide in soluble form inhibits T-cell activation (as measured by interleukin 2 production) and antibody production in Fel d I-primed animals when they are subsequently challenged with peptide in adjuvant. Most of the cat-allergic human T-cell response to Fel d I is specific for two peptides on one of its two chains. Immunization of B6CBAF1 mice with recombinant Fel d I chain 1 results in T-cell responses to the same peptides. Subcutaneous administration of these two peptides, which contain some, but not all, of the T-cell epitopes from Fel d I chain I, decreases the T-cell response to the entire recombinant Fel d I chain 1. The ability to tolerize T-cell responses with subcutaneous injections suggests a practical approach to treating human diseases with peptides containing T-cell epitopes.

Specific V beta T cell subsets mediate the immediate hypersensitivity response to ragweed allergen.

T and B cell responses after sensitization to ragweed (RW) were examined in a mouse model in which BALB/c mice were exposed to the allergen by ultrasonic nebulization. Sensitization resulted in the stimulation of an IgE anti-RW response and was paralleled by a rise in IgG1 anti-RW titers. Skin testing for immediate cutaneous hypersensitivity revealed the presence of allergic type I reactions to RW. Sensitization to RW in this way was also associated with the development of increased airways responsiveness as determined by electrical field stimulation of preparations of tracheal smooth muscle. Histologic examination of the airways and the lung indicated the presence of a mononuclear cell infiltrate in the mucosa and submucosa of the airways that was accompanied by an enlargement of local draining lymph nodes of the airways and the lung. T cell populations were analyzed for the frequency of V beta-expressing T cells. Such analysis indicated that RW sensitization stimulated the expression of V beta 8.1+, V beta 8.2+, and V beta 13+ T cells in the local lymphoid tissue and of V beta 8.1+, V beta 8.2+, V beta 8.3+, V beta 9+ and V beta 14+ T cells in the spleen. Co-culture of these T cell populations with RW-primed B cells indicated that in the presence of RW, V beta 8.2 T cells stimulated IgE and IgG1 production, whereas the other T cell populations showed a different stimulation profile for Ig isotypes and IgG subclasses. The transfer of V beta 8.2 T cells from sensitized but not from nonsensitized control mice stimulated an allergen-specific IgE and IgG1 response and increased airways responsiveness in naive recipients. These data provide additional support for the pivotal role of specific V beta-expressing T cell subpopulations in the stimulation of IgE/IgG1 production and increased airways responsiveness.

An ancient revisits cosmology.

In this after-dinner speech, a somewhat light-hearted attempt is made to view the observational side of physical cosmology as a subdiscipline of astrophysics, still in an early stage of sophistication and in need of more theoretical understanding. The theoretical side of cosmology, in contrast, has its deep base in general relativity. A major result of observational cosmology is that an expansion of the Universe arose from a singularity some 15 billion years ago. This has had an enormous impact on the public's view of both astronomy and theology. It places on cosmologists an extra responsibility for clear thinking and interpretation. Recently, gravitational physics caused another crisis from an unexpected observational result that nonbaryonic matter appears to dominate. Will obtaining information about this massive nonbaryonic component require that astronomers cease to rely on measurement of photons? But 40 years ago after radio astronomical techniques uncovered the high-energy universe, we happily introduced new subfields, with techniques from physics and engineering still tied to photon detection. Another historical example shows how a subfield of cosmology, big bang nucleosynthesis, grew in complexity from its spectroscopic astrophysics beginning 40 years ago. Determination of primordial abundances of lighter nuclei does illuminate conditions in the Big Bang, but the observational results faced and overcame many hurdles on the way.

Development and transfer of immediate cutaneous hypersensitivity in mice exposed to aerosolized antigen.

We previously showed that BALB/c mice sensitized to ovalbumin (OVA) by brief daily inhalations of antigen over 10 consecutive days exhibit elevated antigen-specific serum IgE antibody levels and increased airways responsiveness. For the first time, we now show that animals sensitized in this fashion to either OVA or ragweed (RGW) develop immediate hypersensitivity skin test reactions when challenged 2 d after completion of the sensitization protocol. Skin testing, performed by direct assessment of wheal formation after intradermal injection of allergen, was sensitive and specific, since animals exposed to RGW by inhalation only responded to RGW, and OVA-sensitized animals responded only to OVA. Positive reactions were associated with mast cell degranulation, whereas control injections were not. Since only sensitized IgE high responder BALB/c mice but neither nonsensitized BALB/c mice nor OVA-sensitized IgE low responder SJL/J mice exhibited wheal responses, induction of OVA-specific IgE appeared to be essential for the mediation of OVA-specific immediate hypersensitivity reactions of the skin in this model. Passive cutaneous anaphylaxis (PCA) testing confirmed the presence of antigen-specific IgE in the serum. Mice that developed IgG (predominantly IgG2b) anti-OVA antibodies did not respond to OVA injection, indicating that OVA-specific IgG was not involved in this system. Further support for the role of IgE in the immediate hypersensitivity response included the wheal response to intradermal injection of anti-IgE antibody that occurred in OVA- and RGW-sensitized mice at 10-fold lower concentrations than in nonsensitized BALB/c mice and not in sensitized SJL/J mice. After transfer of mononuclear cells from peribronchial lymph nodes of OVA- or RGW-sensitized BALB/c mice, naive, syngeneic recipients developed antigen-specific IgE and specific immediate hypersensitivity responses, indicating that the local lymphoid tissue at the site of sensitization can transfer responsiveness to these allergens. These results demonstrate for the first time the ability to elicit and study IgE-mediated immediate skin hypersensitivity responses in the mouse and illustrate the association of increased antigen-specific and total serum IgE levels, airways hyperresponsiveness, and antigen-specific immediate cutaneous reactivity after sensitization to allergen via the airways.