Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-ß1.

Cardiac fibroblasts are exposed to both cyclic strain and interstitial fluid flow in the myocardium. The balance of these stimuli is affected by fibrotic scarring, during which the fibroblasts transition to a myofibroblast phenotype. The present study investigates the mechanisms by which cardiac fibroblasts seeded in three-dimensional (3D) collagen gels differentiate between strain and fluid flow. Neonatal cardiac fibroblast-seeded 3D collagen gels were exposed to interstitial flow and/or cyclic strain and message levels of collagens type I and III, transforming growth factor ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA) were assessed. Flow was found to significantly increase and strain to decrease expression of myofibroblast markers. Corresponding immunofluorescence indicated that flow and strain differentially regulated α-SMA protein expression. The effect of flow was inhibited by exposure to losartan, an angiotensin II type 1 receptor (AT1R) blocker, and by introduction of shRNA constructs limiting AT1R expression. Blocking of TGF-ß also inhibited the myofibroblast transition, suggesting that flow-mediated cell signaling involved both AT1R and TGF-ß1. Reduced smad2 phosphorylation in response to cyclic strain suggested that TGF-ß is part of the mechanism by which cardiac fibroblasts differentiate between strain-induced and flow-induced mechanical stress. Our experiments show that fluid flow and mechanical deformation have distinct effects on cardiac fibroblast phenotype. Our data suggest a mechanism in which fluid flow directly acts on AT1R and causes increased TGF-ß1 expression, whereas cyclic strain reduces activation of smad proteins. These results have relevance to the pathogenesis and treatment of heart failure.

The development of community health indicators: a district-wide approach.

INTRODUCTION: In response to high rates of chronic disease, the Capital District Health Authority in Nova Scotia recognized a need to move from a focus on acute care in decision making to one that also values a population health approach guided by community health indicators. METHODS: Stakeholders were surveyed on the choice, knowledge and utility of selected indicators. RESULTS: Respondents reported high scores for changes in their knowledge and attitude regarding community health indicators, and identified priority indicators for action.Decision makers' use of community health indicators was increased by stakeholder involvement, supporting evidence in plain language, and wide dissemination.

Role of serum IgA. Hepatobiliary transport of circulating antigen.

The IgA mediated hepatobiliary excretion of antigen from the circulation was studied using a radiolabeled haptenated protein (dinitrophenyl-human serum albumin) injected intravenously in mice together with monoclonal anti-dinitrophenyl antibodies of different immunoglobulin classes. Antibodies were obtained from ascitic fluids of mice bearing the MOPC315 myeloma (IgA), or immune spleen cell hybridomas (IgG and IgM). IgA antibody brought about the transport of large amounts of antigen from the circulation to the bile during 1-3h. Analysis of bile by gel filtration showed that a large part of the transported antigen remained intact and complexed with IgA. Neither IgA of different specificity nor anti-dinitrophenyl IgM medicated biliary transport of antigen. With anti-dinitrophenyl IgG, only small amounts of low molecular weight fragments of labeled antigen were found in he bile. Preformed immune complex of radiolabeled antigen and IgA antibody were rapidly transported from the circulation to the bile, resulting in threefold-higher levels of radioactivity in bile than in serum. It is proposed that an important function of serum IgA is to mediate the hepatobiliary excretion of corresponding circulating antigens.

Matrix-Mediated Synthesis of Nanocrystalline ggr-Fe2O3: A New Optically Transparent Magnetic Material.

A new magnetic material with appreciable optical transmission in the visible region at room temperature has been isolated as a gamma-Fe(2)O(3)/polymer nanocomposite. The synthesis is carried out in an ion-exchange resin at 60 degrees C. Magnetization and susceptibility data demonstrate loading-dependent saturation moments as high as 46 electromagnetic units per gram and superparamagnetism for lower loadings where particle sizes are less than 100 angstroms. Optical absorption studies show that the small-particle form of gamma-Fe(2)O(3) is considerably more transparent to visible light than the single-crystal form. The difference in absorption ranges from nearly an order of magnitude in the "red" spectral region to a factor of 3 at 5400 angstroms. The magnetization of the nanocomposite is greater by more than an order of magnitude than those of the strongest room-temperature transparent magnets, FeBO(3) and FeF(3).

Experimental vaccine induces Th1-driven immune responses and resistance to Neisseria gonorrhoeae infection in a murine model.

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

A method for quantification of absolute amounts of nucleic acids by (RT)-PCR and a new mathematical model for data analysis.

Accurate quantification of nucleic acids by competitive (RT)-PCR requires a valid internal standard, a reference for data normalization and an adequate mathematical model for data analysis. We report here an effective procedure for the generation of homologous RNA internal standards and a strategy for synthesizing and using a reference target RNA in quantification of absolute amounts of nucleic acids. Further, a new mathematical model describing the general kinetic features of competitive PCR was developed. The model extends the validity of quantitative competitive (RT)-PCR beyond the exponential phase. The new method eliminates the errors arising from different amplification efficiencies of the co-amplified sequences and from heteroduplex formation in the system. The high accuracy (relative error <2%) is comparable to the recently developed real time detection 5'-nuclease PCR. Also, corresponding computer software has been devised for practical data analysis.

Molecular cloning of the human HAND2 gene.

We have cloned and characterized the coding sequence of the human HAND2 basic helix-loop-helix transcription factor. The amino acid sequence includes an amino-terminal polyalanine repeat which is precisely conserved in the rat HAND2 gene. Northern analysis indicates that the HAND2 transcript is 2.3 kb in length and strongly expressed in the human heart.

Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections.

The heat-labile enterotoxins, such as cholera toxin (CT), and the labile toxins types I and II (LT-I and LT-II) of Escherichia coli have been extensively studied for their immunomodulatory properties, which result in the enhancement of immune responses. Despite superficial similarity in structure, in which a toxic A subunit is coupled to a pentameric binding B subunit, different toxins have different immunological properties. Administration of appropriate antigens admixed with or coupled to these toxins by oral, intranasal, or other routes in experimental animals induces mucosal IgA and circulating IgG antibodies that have protective potential against a variety of enteric, respiratory, or genital infections. These include the generation of salivary antibodies that may protect against colonization with mutans streptococci and the development of dental caries. However, exploitation of these adjuvants for human use requires an understanding of their mode of action and the separation of their desirable immunomodulatory properties from their toxicity. Recent findings have revealed that adjuvant action is not critically dependent upon the enzymic activity of the A subunits, and that the isolated B subunits may exert different effects on cells of the immune system than do the intact toxins. Interaction of the toxins with immunocompetent cells is not exclusively dependent upon their conventional ganglioside receptors. Immunomodulatory effects have been observed on dendritic cells, macrophages, CD4(+) and CD8(+) T-cells, and B-cells. Numerous factors-including the precise form of the toxin adjuvant, properties of the antigen, whether and how they are coupled, route of administration, and species of animal model-affect the outcome, whether this is enhanced humoral and cellular immunity, or specific induced tolerance toward the antigen.

Dual function of human IgA antibodies: inhibition of phagocytosis in circulating neutrophils and enhancement of responses in IL-8-stimulated cells.

We have sought to elucidate the responses of human peripheral blood neutrophils to antigenic surfaces complexed with human specific IgA antibodies obtained either as myeloma proteins that recognize staphylococcal alpha-toxin, or from the serum of patients with subacute bacterial endocarditis due to Streptococcus mutans, or from colostrum. In contrast to IgG, IgA antibodies bound to antigen-coated fluorescent microspheres, and subsequently exposed to complement (or not), did not promote phagocytosis, as measured by flow cytometric enumeration of cell-associated microspheres. Instead, IgA antibodies interfered with complement-dependent phagocytosis mediated by IgG antibodies. These properties were shown by different forms of IgA antibodies, including serum and secretory IgA, as well as by monoclonal or polyclonal antibodies. Neutrophils did not respond to the production of superoxide to IgA antibodies complexed with antigen-coated microspheres or with antigen deposited on a solid surface and IgA antibodies suppressed IgG antibody- and complement-mediated superoxide release. However, neutrophils pretreated with interleukin-8 ingested IgA-opsonized microspheres and released superoxide when exposed to IgA antibody-antigen complexes. IgG antibody-antigen complexes did not stimulate increased superoxide release in interleukin-8-treated neutrophils. These findings were consistent with a selective increase in the surface expression of Fc alpha R by interleukin-8-treated neutrophils. We conclude that IgA antibodies interfere with the phagocytic activities of normal circulating human neutrophils and may promote these activities in inflammatory neutrophils activated by interleukin-8 in which Fc alpha R is up-regulated.

Systemic treatment for moderate to severe psoriasis: estimates of failure rates and direct medical costs in a north-eastern US managed care plan.

BACKGROUND: Estimates of US medical costs related to psoriasis treatment are limited and tend to understate the economic burden of moderate to severe psoriasis, which often requires the use of systemic agents, phototherapy or both. OBJECTIVE: To estimate treatment failure rates and direct medical costs associated with the use of systemic agents and phototherapy in US patients with psoriasis. METHODS: Claims records from a large New England-based health insurer were used to obtain patient-level data. Eligible patients with at least one claim listing an ICD-9-CM code for psoriasis (696.0; 696.1) were identified. Patients not receiving systemic treatments (methotrexate, cyclosporine, acitretin) or phototherapy (ultraviolet B with or without tar or petrolatum, psoralen and ultraviolet A [PUVA]) were excluded. Treatment failure was defined as a switch in therapy, augmentation with non-topical therapies, discontinuation following uptitration of dose or discontinuation following hospitalization. Medical costs included those related to pharmacy (over-the-counter medication excluded), institutional services (inpatient and outpatient) and professional services. RESULTS: A total of 2068 patients with moderate to severe psoriasis were included in the analysis. Over a 1-year period, approximately 20% of patients experienced treatment failure. The mean time to failure among patients who switched therapy ranged from 3 to 6 months. Mean annual pharmacy costs in the various treatment groups (categorized according to initial therapy received) ranged from 257 dollars to 1992 dollars per patient. Mean annual costs for institutional and professional services ranged from 156 dollars to 799 dollars and 183 dollars to 481 dollars per patient, respectively. The 99th percentile annual pharmacy and institutional costs exceeded 10,000 dollars and 18,000 dollars, respectively. CONCLUSION: Treatment of moderate to severe psoriasis with traditional systemic agents or phototherapy is associated with a high likelihood of treatment failure and a considerable economic burden.

Preferential transport of IgA and IgA-immune complexes to bile compared with other external secretions.

When radiolabeled dinitrophenyl-human serum albumin was injected intravenously in mice together with M315 IgA, labeled antigen was specifically transported into bile, but not into saliva, urine, milk or bronchial and intestinal secretions. Ultracentrifugal analysis showed that the antigen transported into bile was intact and partly complexed with IgA. The radioactivity that was present in other secretions regardless of M315 IgA, represented free and degraded fragments of antigen. M315 IgA alone was readily transported into bile, where it was detected at high titer by hemagglutination, but not into other secretions, apart from milk which contained only very low titers. The liver therefore appears to be singularly capable of transporting both free and complexed IgA into its secretion, the bile.

Selective hepatobiliary transport of human polymeric IgA in mice.

Both subclasses of human polymeric IgA (pIgA) were selectively transported from the serum into the bile of mice relative to human IgG or IgM. Removal of human pIgA from serum corresponded to the clearance kinetics shown for murine pIgA. The biliary pIgA was intact as determined by sucrose density gradient ultracentrifugation. This hepatic uptake was specific for the IgA isotype and occurred independently of receptors in the liver specific for glycoproteins that terminate with galactose or mannose moieties. Desialylation of human pIgA resulted in its rapid clearance from serum and subsequent deposition in the liver in a manner similar to most other desialylated serum glycoproteins. The desialylated pIgA present in bile was also an intact molecule; thus the asialoglycoprotein receptor may represent an additional mechanism for the transport of serum pIgA into bile.

Role of hepatocytes in the uptake of IgA and IgA-containing immune complexes in mice.

The role of parenchymal and nonparenchymal mouse liver cells in the uptake of polymeric IgA (pIgA) and pIgA-containing immune complexes (IC) of low mol. wt (less than 1 x 10(6)) was studied. As detected by immunofluorescence and immunoelectron microscopy, pIgA were bound on the surface of isolated hepatocytes. Following the injection of radiolabeled pIgA or pIgA-IC into mice, the total radioactivity recovered from isolated liver cells was preferentially associated with parenchymal cells. The ability to inhibit the transport of pIgA and pIgA-IC by pIgA of an irrelevant specificity suggests that pIgA-IC and pIgA are bound and transported by the same mechanism. These results indicate that mouse hepatocytes are involved in the uptake and hepatobiliary transport of pIgA and pIgA-IC of low mol. wt.

Analysis of the hepatobiliary transport of IgA with monoclonal anti-idiotype and anti-allotype antibodies.

The processing and fate of mixed immune complexes is influenced by the antibody isotypes present. The hepatobiliary transport of mixed immune complexes containing the mouse IgA myeloma protein J558 and corresponding monoclonal IgG or IgM anti-J558 idiotype or monoclonal IgG anti-mouse IgA allotype antibodies has been studied. The anti-idiotype or anti-allotype antibodies were radiolabeled and injected into mice with or without mouse polymeric IgA (J558). IgG anti-idiotype antibodies to J558 IgA were selectively transported into bile by J558 IgA. This process occurred with a radiolabeled Fab preparation of the IgG anti-idiotype and was inhibitable with IgA of an irrelevant antigenic specificity. Thus, polymeric IgA influenced the fate of IgA-IgG idiotype-anti-idiotype serum immune complexes. A monoclonal anti-idiotype antibody of the IgM isotype (D8-3) was not selectively transported into bile by itself or as an IgA-IgM complex. A monoclonal IgG antibody (CB5-6) to a mouse allotype determinant in the Fc portion of IgA was not selectively transported into bile. This anti-allotype monoclonal antibody inhibited the hepatobiliary transport of 125I-polymeric J558 IgA and therefore appeared to directly or indirectly block the site in the Fc region of IgA recognized by the hepatic receptor.

Nasal lymphoid tissue, intranasal immunization, and compartmentalization of the common mucosal immune system.

Mucosal application of vaccines with an appropriate adjuvant can induce immune responses at both systemic and mucosal sites, and therefore may prevent not only infectious disease, but also colonization of mucosal surfaces. Intranasal is more effective than intragastric immunization at generating earlier and stronger mucosal immune response. Nasal lymphoid tissue (NALT) and its local draining lymph nodes may retain long-term immune memory. IgA isotype switching, and the differentiation and maturation of IgA antibody-secreting cells (ASC) may occur before these cells migrate out of NALT, whereas IgG ASC responses require passage of the cells through draining lymph nodes of the NALT. Knowledge of whether immune memory cells can recirculate to and reside in the inductive sites other than their origin after encountering antigen will be helpful for understanding the compartmentalization of the common mucosal immune system as well as for determining the best route for delivering a mucosal vaccine against a particular pathogen.

Assay of human IgA subclass antibodies in serum and secretions by means of monoclonal antibodies.

Solid-phase radioimmunoassays have been developed for the detection and quantification of human serum and secretory IgA antibodies to a variety of food, bacterial and viral antigens. Monoclonal antibodies specific for IgA1 and IgA2 and capable of binding to serum and secretory IgA were used. The assays were calibrated by reference to standard serum or purified myeloma proteins bound to solid-phase anti-immunoglobulin reagents, and sigmoid calibration curves were constructed by means of computer programs using 4-parameter logistic or weighted logit-log principles. Polymeric and monomeric forms of IgA antibodies were assayed in fractions separated by high performance size exclusion chromatography. These techniques have demonstrated the expected predominance of IgA1 antibodies in serum, and these included polymeric forms. Saliva contained both IgA1 and IgA2 antibodies, and increased proportions of IgA2 antibodies to lipopolysaccharides and lipoteichoic acid were observed.

Direct medical costs of coronary artery disease in the United States.

To generate current incidence-based estimates of the direct medical costs of coronary artery disease (CAD) in the United States, a Markov model of the economic costs of CAD-related medical care was developed. Risks of initial and subsequent CAD events (sudden CAD death, fatal/nonfatal acute myocardial infarction [AMI], unstable angina, and stable angina) were estimated using new Framingham Heart Study risk equations and population risk profiles derived from national survey data. Costs were assumed to be those related to treatment of initial and subsequent CAD events ("event-related") and follow-up care ("nonevent-related"), respectively. Cost estimates were derived primarily from national public-use databases. First-year direct medical costs of treating CAD events are estimated to be $17,532 for fatal AMI, $15,540 for nonfatal AMI, $2,569 for stable angina, $12,058 for unstable angina, and $713 for sudden CAD death. Nonevent-related direct costs of CAD treatment are estimated to be $1,051 annually. The annual incidence of CAD in the United States is estimated at 616,900 cases, with first-year costs of treatment totaling $5.54 billion. Five- and 10-year cumulative costs in 1995 dollars for patients who are initially free of CAD are estimated at $9.2 billion and $16.5 billion, respectively; for all patients with CAD, these costs are estimated to be $71.5 billion and $126.6 billion, respectively. The direct medical costs of CAD create a large economic burden for the United States health-care system.

Cost-effectiveness of statins.

Currently, 6 hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are marketed in the United States (US). Given the wide variation in the prices and efficacy of statins, formal cost-effectiveness analysis may improve drug selection decisions. To assess the cost-effectiveness of statin therapy in primary and secondary prevention of coronary heart disease, we developed a model of the costs and consequences of lipid-regulating therapy and estimated the incremental cost-effectiveness of 5 statins (atorvastatin, fluvastatin, lovastatin, pravastatin, simvastatin) at usual starting doses versus no therapy. Drug effects on serum lipids were assessed using data approved by the US Food and Drug Administration for product labeling. Annual risks of coronary event occurrence were estimated using Framingham Heart Study coronary risk equations developed for use in this model. Current estimates of direct medical costs of coronary heart disease were used to assign costs to health states and acute coronary events. Main outcome measurements were net cost (statin therapy minus savings in coronary heart disease treatment), gain in life expectancy, and cost per life-year saved. The maximum gain in life expectancy was achieved with atorvastatin, which also had a lower net cost than lovastatin, pravastatin, and simvastatin. Compared with fluvastatin, atorvastatin's greater effectiveness is attained at a lower cost per life-year saved. The cost-effectiveness of HMG-CoA reductase inhibition in primary and secondary prevention of coronary heart disease has been improved with the introduction of atorvastatin.

Accident- and injury-related health-care utilization among benzodiazepine users and nonusers.

Benzodiazepine tranquilizers have been found to cause psychomotor and cognitive impairment, and there is evidence of an increased rate of automobile accidents among users of these drugs. To determine whether benzodiazepine users are more likely than nonusers to experience accidental injury requiring medical attention, we examined health-care utilization among 7,271 such users and an age- and sex-matched sample of 65,439 nonusers, all of whom were enrolled in an "HMO-like" health insurance plan. Benzodiazepine users and nonusers were identified through a review of 4 months' prescription drug claims. Six months' health-care claims for each user and nonuser subsequent to the first observed claim for a benzodiazepine or nonbenzodiazepine agent, respectively, were compiled. Claims related to accidents were identified on the basis of physician-recorded diagnoses. Our results indicate that benzodiazepine users were significantly (p less than .01) more likely than nonusers to experience (1) at least one accident-related episode of care; (2) a greater number of accident-related hospital admissions; and (3) a greater number of accident-related inpatient days. Accident-related utilization was also significantly higher when users had recently filled a prescription for a benzodiazepine agent. Benzodiazepine users, however, also utilized significantly more non-accident-related health-care services than nonusers. The nature of the association between benzodiazepine use and a higher accident-related utilization is thus unclear. A pretest-posttest study is now being undertaken to ascertain the significance of these findings.

Systemic and mucosal protective immunity to pneumococcal surface protein A.

To date our studies demonstrate that PspA is a highly immunogenic molecule in mice and that it can elicit immunity to otherwise fatal infections following iv, ip, in, and it challenge. Although the molecule is serologically variable, it is sufficiently cross-reactive so that immunization with a single PspA can protect against strains of highly diverse serotypes. It is anticipated that a vaccine composed of a mixture of carefully chosen PspA molecules will be able to elicit protective immunity to virtually all pneumococci. If this vaccine proved efficacious in man, it would provide a more simple and less costly means of immunizing against pneumococcal infection than using recombinant vaccines. This could be especially important in the developing world where the cost of successful vaccines must be no more than pennies per dose. If PspA is found to be less efficacious than capsular polysaccharides, it may be valuable as a protein component of a PS-protein conjugate vaccine. In this capacity, PspA might expand the breath of protection elicited by a vaccine composed of only a few polysaccharide-protein conjugates representing capsule types most commonly associated with infectious pneumococci.