Cytokine kinetic profiles in children with acute lower respiratory tract infection: a post hoc descriptive analysis from a randomized control trial.

OBJECTIVES: Standard inflammatory markers and chest radiography lack the ability to discriminate bacterial from non-bacterial lower respiratory tract infection (LRTI). Cytokine profiles may serve as biomarkers for LRTI, but their applicability to identify aetiology, severity of disease and need for antibiotic prescription in children remains poorly defined. Objectives were to determine the cytokine kinetic profiles over 5 days in paediatric patients with LRTI, to investigate the relationship between cytokine patterns, and clinical and laboratory variables. METHODS: We included patients aged 1 month to 18 years, with febrile LRTI and three consecutive cytokines measurements on days 1, 3 and 5 of a randomized controlled trial (ProPAED study). We evaluated differences in cytokine concentrations between days and associations with clinical and laboratory variables. RESULTS: A total of 181 patients (median age 4.1 years) were included; 72/181 (40%) received antibiotics. Serum concentrations of interferon (IFN)-γ, interleukin (IL)-1ra, IL-6, IL-10, IFN-γ-inducible protein (IP)-10 and tumor necrosis factor-α were elevated on day 1 and decreased subsequently, with the greatest decline between day 1 and 3 (by -8 to >-94%). Procalcitonin (PCT) and C-reactive protein (CRP) values showed a protracted decrease with the most prominent reduction in concentrations between days 3 and 5. Significantly elevated IL-6 concentrations were associated with hospital admission, antibiotic treatment, and prolonged antibiotic treatment. Bacteraemic LRTI patients had higher concentrations of IL-1ra (p <0.0055) and IL-6 (p <0.0055) on day 1. CONCLUSIONS: We observed an earlier decrease of elevated cytokines compared to PCT or CRP. Both pro- and anti-inflammatory cytokines may serve as markers for severity of LRTI.

Myofibrillar disruption in hypocontractile myocardium showing perfusion-contraction matches and mismatches.

Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization.

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture.

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists. Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading.

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading.

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes.

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non-beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.

Catecholamines modulate protein turnover in cultured, quiescent rabbit cardiac myocytes.

When rabbit ventricular myocytes were cultured for 1 wk and then exposed to alpha- and/or beta-adrenergic agonists, such nonbeating heart cell preparations disclosed increased protein-to-DNA ratios and elevated RNA content, indicative of cellular hypertrophy. Norepinephrine, isoproterenol, and phenylephrine provoked hypertrophy with norepinephrine eliciting a greater response than isoproterenol or phenylephrine. Specific alpha- and beta-antagonists blocked growth by inhibiting catecholamine-induced changes in protein turnover. Each catecholamine enhanced the fractional rate of protein synthesis within 48 h; however, changes in growth rates appeared to be modulated, in part, by alterations in protein degradation. Even though rates of total protein and actin synthesis resembled values measured in vivo, myosin heavy chain fractional rate of synthesis was only 22% of in vivo levels. Double label immunofluorescence microscopy further illustrated that catecholamine treatment accelerated myofibrillar disruption in these quiescent heart cells. These observations suggested that in the absence of beating, neurohumoral modulation of contractile protein turnover was not associated with the maintenance of myofibrillar integrity even though catecholamines induced cellular hypertrophy.

Morphometric evaluation of the contractile apparatus in primary cultures of rabbit cardiac myocytes.

Rabbit cardiac myocytes remain quiescent for more than 1 month when cultured at low density. During this period, myofibrillar volume density declines sixfold as myofibrils are disassembled or degraded and are replaced by actin and alpha-actinin-positive, myosin-negative structures that resemble myofibrils but lack thick filaments. Such structures are termed minute myofibrils. The length of the sarcomeres in these altered myofibrils is significantly less than length values obtained from freshly isolated heart cells or from contracting myocytes. A number of high density cultures develop spontaneous, synchronous contraction during the second week of culture. Myofibrillar volume density is stabilized when beating begins, and no further decline is observed in the succeeding weeks of culture. Such contracting myocytes display myofibrils typical of normal heart with no visible evidence of minute myofibrils. The volume density of the transverse tubular system also declines significantly in both beating and nonbeating myocytes, and its reduction appears more closely correlated with cell spreading than with beating per se. No quantitative changes in volume density of mitochondria or sarcoplasmic reticulum could be documented, but the structural organization of the sarcoplasmic reticulum seems to be greatly influenced by the physiological state of the heart cell. The present observations document the importance of mechanical factors in regulating the integrity of the contractile apparatus in cardiac myocytes and emphasize the utility of the cultured heart cell to directly investigate structure-function relations in individual myocytes.

Cell shape and organization of the contractile apparatus in cultured adult cardiac myocytes.

The isolation and culture of adult cardiac myocytes has proved to be an ideal model system to explore myocardial biology at the cellular level. A major criticism of this model, however, has been that organ-specific characteristics such as cell shape and subcellular structural organization cannot be retained in vitro for prolonged periods of time. Encasing freshly isolated myocytes in a matrix of calcium alginate enables one to maintain the rod-like, three-dimensional (3D) shape of the cultured myocyte. Such preparations more closely resemble their in vivo counterparts with respect to the organization of the contractile apparatus, the transverse tubular system and the sarcoplasmic reticulum than do heart cells cultured on a two-dimensional (2D) plastic surface. Stereologic measurements reveal that myofibrillar volume density (VvMYF) decreases in both non-beating preparations over a 2-week interval, but VvMYF is conserved in cells cultured in an alginate matrix when compared to those myocytes maintained on a laminin-coated substratum. The present observations suggest that in the absence of contractile function myofibrillar atrophy appears responsible for the decline in VvMYF in alginate (3D) preparations, whereas atrophy and subcellular remodelling probably mediate the myofibrillar loss and reorganization that develops when adult heart cells are cultured on a 2D surface.

Morphological analysis of contracting and quiescent adult rabbit cardiac myocytes in long-term culture.

Isolated rabbit ventricular cardiac myocytes adapt readily to primary culture. As the myocytes spread and flatten over the culture substratum, the myofibrillar apparatus retains a "rod-like" orientation. Development of contractile activity is crucial in the maintenance of the integrity of the myofibrillar apparatus during prolonged culture. Myocytes that fail to beat display morphological indications of atrophy; conversely, myocytes that commence beating show no such morphological signs of myofibrillar disorganization. The subcellular organization of other elements of the contractile apparatus, including the transverse tubular system and the sarcoplasmic reticulum, retain their structural relationship with the myofibrils in beating myocytes but not in quiescent cells. Cultured adult myocytes represent an important model to investigate the influence of mechanical factors on the organization and maintenance of the adult cardiac phenotype.

Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture.

The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.

Lysosomal changes during thyroxine-induced left ventricular hypertrophy in rabbits.

By use of a combined morphologic, immunocytochemical, and biochemical approach, this study demonstrates the changes in the lysosomal vacuolar apparatus that accompany thyroxine-induced cardiac hypertrophy. During the 1st wk of thyroxine administration, immunocytochemical studies revealed a decline in cathepsin D within many myocytes, but an increase within interstitial cells. These events transpired with only a modest rise in cathepsin D activity. During the 2nd wk, cathepsin D reappeared within most myocytes and continued to increase within the interstitial population and was associated with a general rise in lysosomal enzyme activity. These data demonstrate the importance of employing both immunocytochemical and biochemical approaches to evaluate the status of the lysosomal vacuolar apparatus, and suggests that the lysosomal vacuolar apparatus may be involved in the apparent remodeling that attends rapid cardiac growth, which may represent one component of the enhanced rates of proteolysis documented in this model of cardiac hypertrophy.

Responses of cultured cardiac myocytes to lysosomotropic compounds and methylated amino acids.

Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to 'lysosomotropic' weak bases (chloroquine, methylamine, and NH4Cl) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4Cl, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.

The distribution of lysosomal cathepsin D in cardiac myocytes.

Lysosomal cathepsin D has been localized with the electron microscope employing an indirect immunohistochemical method using peroxidase labeled, monospecific antibody Fab' subunits. The acid proteinase has been demonstrated within secondary lysosomes of cardiac myocytes and interstitial cells, but not in components of the Golgi apparatus or endoplasmic reticulum. Incubations with a variety of peroxidatic inhibitors suggests that the staining that is observed in secondary lysosomes is attributable to the peroxidase-labeled antibody and not to endogenous oxidation of DAB. The protocol outlined here provides a reproducible method to localize the major lysosomal acid proteinase of the heart at the subcellular level.