CSF from Parkinson disease patients differentially affects cultured microglia and astrocytes.

BACKGROUND: Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging. RESULTS: After PD-CSF treatment, microglia growth was reduced extensively, and a non-confluent pattern with morphological changes developed, that was not evident in disease control CSF and no-CSF treated cultures. Astrocyte growth rates were similarly reduced by exposure to PD-CSF, but morphological changes were not consistently noted. PD-CSF treated microglia showed a significant increase in α-synuclein content by day 4 compared to other treatments (p ≤ 0.02). In microglia only, α-synuclein aggregated and redistributed to peri-nuclear locations. CONCLUSIONS: Cultured microglia and astrocytes are differentially affected by PD-CSF exposure compared to non-PD-CSF controls. PD-CSF dramatically impacts microglia cell growth, morphology, and α-synuclein deposition compared to astrocytes, supporting the hypothesis of cell specific susceptibility to PD-CSF toxicity.

An inducible cartilage oligomeric matrix protein mouse model recapitulates human pseudoachondroplasia phenotype.

Cartilage oligomeric matrix protein (COMP) is a pentameric extracellular protein expressed in cartilage and other musculoskeletal tissues. Mutations in the COMP gene cause pseudoachondroplasia (PSACH), a severe dwarfing condition that has a growth plate chondrocyte pathology. PSACH is characterized by intracellular retention of COMP and other extracellular matrix (ECM) proteins, which form an ordered matrix within large rough endoplasmic reticulum cisternae. This accumulation is cytotoxic and causes premature chondrocyte cell death, thereby depleting chondrocytes needed for normal long bone growth. Research to define the underlying molecular mechanisms of PSACH has been hampered by the lack of a suitable model system. In this study, we achieved robust expression of human mutant (MT) or wild-type (WT) COMP in mice by using a tetracycline-inducible promoter. Normal growth plate distribution of ECM proteins was observed in 1-month-old WT-COMP and C57BL\6 control mice. In contrast, the structure of the MT-COMP growth plate recapitulated the findings of human PSACH growth plate morphology, including (1) retention of ECM proteins, (2) intracellular matrix formation in the rER cisternae, and (3) increased chondrocyte apoptosis. Therefore, we have generated the first mouse model to show extensive intracellular retention of ECM proteins recapitulating the human PSACH disease process at the cellular level.

Fluorescence microscopy and 3D image reconstruction of cytokine initiated disruption of the Parkinson disease associated proteins alpha-synuclein, tau and ubiquitin in cultured glial cells.

Human derived glioblastoma cells were cultured and treated with cytokines interleukin-6 (IL6), tumor necrosis factor alpha (TNF) and interferon-gamma (IFN) and imaged by fluorescence deconvolution microscopy to localize alpha-synuclein, tau and ubiquitin. Exposures were for short (2 h) and prolonged times (up to 96 h), with doses at both low (10 ng/ml), and high (100 ng/ml) concentrations. Further experiments used additive doses up to 200 ng/ml (2 x 100 ng), mimicking a super-infection state. Single, low doses of the cytokines initiated changes in levels of intracellular proteins, but these changes, be they increases or decreases, were not sustained, so we added higher doses of cytokine to the culture medium or fresh aliquots of cytokines over time. Finally, we treated cells with high, single doses of cytokine (200 ng/ml), to try to sustain perturbations of the proteins with cytokines. IFN caused a disruption and reduction of peripheral synuclein, TNF treatment resulted in increased levels of ubiquitin and IL6 disrupted and appeared to fragment tau. Of note, each of the proteins was found in a specific locale, tau being perinuclear, ubiquitin residing in the cytoplasm, and alpha-synuclein occupying the tips of cellular processes, exhibiting the characteristics of an adhesion protein/molecule [Word count=198].

Cytokines disrupt intracellular patterns of Parkinson's disease-associated proteins alpha-synuclein, tau and ubiquitin in cultured glial cells.

The purpose of this study was to determine the effects of specific proinflammatory cytokines interleukin-6 (Il-6), interleukin-1beta (Il-1beta), interferon-gamma (IFN), and tumor necrosis factor-alpha (TNFalpha), on content and distribution of alpha-synuclein (alpha-synuclein), tau and ubiquitin in human derived cultured glial cells. Exposure paradigms mimicked acute (2 h), intermediate (18 h) and prolonged time frames (96 h); consisting of single or repeated low doses (10 ng/ml) or high doses (50 ng/ml), consistent with either mild or serious systemic infectious/inflammatory responses. Images of intracellular protein content and distribution were reconstructed from emission patterns generated by fluorescence deconvolution microscopy. Minor alterations were seen in protein content with IFN; Il-1beta decreased alpha-synuclein and tau at 18 and 96 h; TNFalpha inversely reduced alpha-synuclein and increased ubiquitin content. Combinations of Il-1beta and IFN produced a robust increase of alpha-synuclein and tau at 2 h. Consecutive low doses of Il-6 produced only minor increases in alpha-synuclein and ubiquitin after 4 h, whereas a single high dose resulted in major increases for all three proteins over the first 18 h. Protein localization patterns were distinctly different and were altered dependent upon cytokine treatment. A high dose exposure (2 x 50 ng/ml) with Il-6 and IFN demonstrated that protein increases and dispersals could be sustained and that the normal perinuclear tau and peripheral alpha-synuclein patterns were disrupted. These results support the postulate that specific cytokines affect temporal protein changes with concomitant pattern disruptions, possibly reflecting a mechanism of cell dysfunction in Parkinson's degeneration.

Cardiovascular distribution of the calcium sensing receptor before and after burns.

Due to up-regulation of the parathyroid gland calcium-sensing receptor (CaR), burned children have hypocalcemic hypoparathyroidism, and decreased myocardial contractility. Our aim was to localize the CaR in the heart and measure receptor density changes due to burns. Heart and aorta samples from sheep subjected to 40% burn or sham conditions were probed for CaR via fluorescence microscopy. CaR was localized to endocardial endothelium, myocardial microvasculature, and fibroblasts and vessels of the aortic adventitia. CaR was not found in cardiomyocytes or smooth muscle cells. No differences in density of CaR or beta-adrenergic receptors were noted. No differences in CaR distribution were seen in the myocardium or aorta, in contrast to the parathyroid where burn injury up-regulates CaR. We suggest that CaR has a local, tissue-specific role, and functions in vascular calcium sensing for intravascular calcium deposition or regulation of other calcium channels after trauma or burn.

Oleandrin produces changes in intracellular calcium levels in isolated cardiomyocytes: a real-time fluorescence imaging study comparing adult to neonatal cardiomyocytes.

Oleanders are common, hardy shrubs that grow throughout the southern United States. They contain cardiotonic steroids formed from cardenolides and bufadienolides, making the plant poisonous to both animals and humans. Aliquots of both commercially available oleander and fresh oleander extracts were prepared. Fresh, rod-like, calcium-tolerant adult rat cardiomyocytes and cultured neonatal cardiomyocytes were isolated and treated with 0-4 ng/ml of both preparations, challenged with verapamil and ouabain, and real-time spectrophotometric calcium transients and images were acquired. A number of effects were observed with the adult cells: (1) intracellular calcium levels were increased in a concentration-dependent manner: (2) reduced calcium transient heights and eventual cessation of beating resulted; and (3) increased sparking intensity led to subsequent beating and eventual calcium overload. In the spontaneously beating cultured neonatal myocytes increased intramyocytic calcium levels were also seen, with retention of this calcium rise leading to overload and, as in the adult myocytes, cessation of beating. These observations demonstrate that oleander extract is markedly potent with respect to the elevation of calcium concentrations in cardiomyocytes, and that the inability of the cardiomyocytes to release the accumulated calcium possibly indicates a role for oleandrin in inhibition of ryanodine receptor calcium release channels, calcium uptake via Na+,K+-ATPase inhibition [EC 3.6.1.3], and/or dysfunction of sarcolemmal calcium release channels.

Unlocking Aspirin's Chemopreventive Activity: Role of Irreversibly Inhibiting Platelet Cyclooxygenase-1.

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirin's ability to irreversibly inhibit COX-1-mediated platelet activation, thereby blocking platelet-cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial-mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, ß-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirin's chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142-52. ©2016 AACR.

Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes.

Ginsengs are widely used to improve cardiac health and circulation. Loosely termed as ginsengs, Asian (Panax), Siberian and Ashwagandha (Indian Ginseng) Indian ginsengs are prepared from different plants. We tested the popular belief of cardiotonic effects of ginsengs using both neonatal and adult rat cardiomyocytes, comparing extracts from the three ginsengs. Addition of 10% v/v of extract (100 microl of extract/ml of culture medium) of each of the ginsengs resulted in a rapid (<10 s) cessation of beating in neonatal cardiomyocytes due to calcium overload, while sequential dilutions revealed that treatment with a low dose (0.01% v/v, 0.1 microl/ml of the medium) resulted in constant, regular beats (transients), and a slight elevation of diastolic calcium without overload. Addition of extracts to sparking, calcium-tolerant adult cardiomyocytes resulted in initiation of calcium transients, and adult cells were able to tolerate exposure to high concentrations of extract. Cardiotonic effects in adult cells (cardiotoxicity in neonatal cells) were most profound with Asian ginseng (2.6 times that of Siberian ginseng, 1.6 times that of Indian ginseng) probably due to the active ingredients (ginsenosides in Asian, eleutherosides in Siberian and withanolides in Indian) being structurally different. We conclude that fully developed cardiomyocytes are able to accommodate higher doses of ginseng than neonatal cells, and that the effects of ginseng on newly formed, developing myocytes, could be extremely deleterious to the fetus. However, for adults, ginseng might well be a 'tonic' in its ability to increase beating and intramyocytic calcium levels.

Localization of antimicrobial peptides in normal and burned skin.

Severe burn causes immunosuppression, and the eschar remains a perfect culture medium for microbial growth. The resulting sepsis is a common complication of burns with a high mortality. The skin produces a number of molecules including antimicrobial peptides (AMPs) that act in the first line of host defense. Previous studies from our laboratory suggested decreased expression of human beta-defensin-2 (HBD-2) in burned wounds. Here, we have expanded our work by identifying HBD-1, HBD-2, HBD-3 and human neutrophil peptide (HNP) in normal and burn skin samples using fluorescence deconvolution microscopy. In normal skin, HBD-1 was localized to the perinuclear region of keratinocytes, while HBD-2 was seen primarily in the stratum basale of the epidermis. HBD-3 was found in dendritic cells of the stratum spinosum. HNP was distributed in a somewhat random pattern in the papillary dermis. In burned skin, in which the epidermis had been destroyed or disrupted, the presence of HBD-1 was localized to dermal glandular structures and hair shafts. HBD-2 was found in both the upper portions of the remaining keratin layers, and localized to lower, f-actin containing, acini-like structures, a pattern also evident with HBD-3. We conclude that although the upper layers of skin are destroyed and disrupted by burn, cells in the lower portions of the skin demonstrate an ability to synthesize most of the AMPs, thereby maintaining some barrier against infection. The results of these studies further contribute to an understanding of the role of AMPs in the pathophysiology of cutaneous burn and the possibility of using these sites for upregulation of AMP synthesis in the prevention of burn sepsis.

Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Are Temporal Differences in GDNF and NOS Isoform Induction Contributors to Neurodegeneration? A Fluorescence Microscopy-Based Study.

BACKGROUND: Specific factors in Parkinson's disease have become targets as to their protective and degenerative effects. We have demonstrated that cytokines and PD-CSF detrimentally affect microglia and astrocyte growth. While glial cell-derived neurotrophic factor (GDNF) has been recognized as a possible neuron-rescue agent, nitric oxide synthase (NOS) has been implicated in neurodegenerative processes. OBJECTIVE: To demonstrate that glial cell activation, cytokine production, and NOS induction, play an intimate role in the loss of dopaminergic signaling, via mechanisms that are a result of inflammation and inflammatory stimuli. METHODS: Study animals were sacrificed following endotoxin treatment and tissue sections were harvested and probed for GDNF and NOS isomers by fluorescence deconvolution microscopy. Fluorescence was mapped and quantified for each probe. RESULTS: An immune cell influx into 'vulnerable' areas of the brain was seen, and three NOS isomers, inducible (iNOS), neuronal (nNOS) and endothelial (eNOS), were synthesized in the brains, a finding which suggests that each isomer has a role in neurodegeneration. eNOS was found associated with blood vessels, while iNOS was associated with glial and matrix cells and nNOS was located with both glia and neurons. Following endotoxin treatment, serum levels of nitric oxide were higher at 6-8 hours, while tissue levels of NOS were elevated for much longer. Thus, induction of NOS occurred earlier than the induction of GDNF. CONCLUSION: Our findings suggest that the protective abilities of GDNF to combat neural destruction are not available rapidly enough, and do not remain at sufficiently high levels long enough to assert its protective effects. (250).

Fluorescence imaging microscopy of cellular markers in ischemic vs non-ischemic cardiomyopathy after left ventricular unloading.

BACKGROUND: The heart undergoes repair and initiates protective mechanisms via ventricular unloading. We examined the presence of 2 markers in pre-unloaded and post-unloaded human cardiac tissue that are important indicators of cardiac failure, tumor necrosis factor-alpha and inducible nitric oxide synthase. We also measured 2 nuclear transcription factors, NFkappaB50 and NFkappaB65, comparing quantities and localizations to determine if mechanical unloading reduced their presence, as these markers are also thought to be indicators of impending heart failure. Amounts and localizations in patients that had been diagnosed with either ischemic or non-ischemic cardiomyopathy were compared after mechanical unloading with a left ventricular assist device. To establish that unloading had been achieved, levels of atrial natriuretic protein were determined. METHODS: Core biopsies were harvested at assist device implantation and removal. Fluorescence deconvolution microscopy image reconstructions of fluorescence probes were correlated with data obtained by western Blot and electrobility shift assays. RESULTS: Statistically significant differences in localization and amounts of tumor necrosis factor and nitric oxide synthase were seen between pre- and post-assist device samples. Amounts of tumor necrosis factor and nitric oxide synthase in ischemic tissue were increased at the time of assist device removal, but decreased in dilated or idiomyopathic samples. Ventricular unloading resulted in reduced levels of natriuretic protein, with the greatest reduction being seen in ischemic tissue. Both NFkappaB50 and NFkappaB65 increased in ischemic tissue, but only NFkappaB50 in non-ischemic samples. CONCLUSIONS: Changes in localization of the factors and altered levels of cytokine and nitric oxide synthase indicate that the heart switches to a "protective and repair" mode, and mechanical unloading allows this transition to occur. Observed changes were dependent on the etiology of the disease.

Localization of calcitonin gene-related peptide in cardiomyocytes: comparison of neonatal and dedifferentiating cells to adult myocytes.

The purpose of this study was to localize sites of calcitonin gene-related peptide binding in neonatal, freshly isolated and dedifferentiated adult cardiac myocytes in order to help us elucidate the mechanisms of action of this neuropeptides. Previous work has shown that treatment with calcitonin gene-related peptide results in dramatic changes in calcium transients, so we carried out multi-channel acquisitions of fluorescently labeled images to reveal where calcitonin gene-related protein and the L-type calcium channel were localized. Calcitonin gene-related protein was sparse and randomly distributed in rod-like adult cardiomyocytes, found in abundance in areas of the cell where striations were apparent and not where adhesion proteins predominated in dedifferentiating adult myocytes, and in a large perinuclear concentration, with some spreading into the cytoplasm in neonatal cells. Subsequent modeling demonstrated that calcitonin gene-related peptide and the L-type calcium channel protein were closely associated in each of the three myocyte types, suggesting that while the peptide has dramatic and different effects on intracellular calcium levels of the various cardiomyocytes, the action is probably via diverse mechanisms as a result of effects on different channels or pump proteins due to alterations in intracellular calcium concentrations.

Determination of the site of action of calcitonin gene-related peptide in the alteration of intracellular calcium levels in adult and neonatal rodent myocytes.

The purpose of this study is to elucidate the mechanism of action and site of action of calcitonin gene-related peptide (CGRP) and its effects on calcium concentrations in two types of cardiomyocytes, neonatal and adult, by employing real-time fluorescence imaging. CGRP caused an increase in intramyocytic calcium with adult cells, but a decrease with neonates. Treatment of adult myocytes with ouabain and ryanodine yielded results suggesting that CGRP action is not at the ryanodine receptor (RyR) and does not involve Na+ +K+ ATPase. Furthermore, in neonatal cardiomyocytes CGRP caused a reduction in intramyocytic calcium levels, and challenges with ryanodine and ouabain gave results supporting the hypothesis that CGRP acts at the sarcolemmal L-type calcium channel. Employing real-time fluorescence measurements in cultured, dedifferentiated adult cardiomyocytes, which are known to express a fetal phenotype and exhibit neonatal-like calcium transients, our acquisitions demonstrated a major reduction in intracellular calcium levels. Finally, our collaborative studies in human myocardium using fluorescence deconvolution microscopy revealed that CGRP localization was found in a pattern similar to that of the sarcolemmal L-type calcium channel.

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function.

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.

Enteral glutamine but not alanine maintains small bowel barrier function after ischemia/reperfusion injury in rats.

We previously demonstrated that glucose and glutamine, solutes metabolized by the gut, replenish ATP and enhance gut function compared with alanine, a solute not metabolized by the gut, following mesenteric ischemia/reperfusion (I/R). The purpose of the present study was to determine if the nonmetabolizable solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R. At laparotomy, rats had jejunal sacs filled with 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mm, osmotic control) followed by superior mesenteric artery clamping for 60 min and 30 min of reperfusion or sham laparotomy. Jejunum was harvested for evaluation by deconvolution microscopy, fluorescent measurement of F:G actin ratio, or mounted in an Ussing chamber for determination of intestinal permeability. Deconvolution microscopy revealed that the actin cytoskeleton was preserved by enteral glutamine, comparable to shams, but disrupted by enteral alanine. Glucose and controls resulted in comparable disruption, which was less than that with alanine. The F:G actin ratio was highest for glutamine and lowest for alanine; glucose was comparable to controls. Intestinal permeability was highest for alanine and lowest for glutamine, which was comparable to shams. Permeability following glucose and controls was higher than that following glutamine but lower than that following alanine. The nonmetabolizable solute alanine resulted in disruption of the actin cytoskeleton and enhanced intestinal permeability under conditions of mesenteric I/R. The metabolizable solute glutamine was protective under these conditions, whereas glucose exerted minimal effect on the integrity of the cytoskeleton and intestinal permeability. The individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications when administered to critically injured patients.

Effects of Chan Su, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: cardiotoxicity due to more than Na, K-ATPase blocking.

Extracts of Chan Su, a traditional Chinese medication used as a topical anesthetic and cardiac medication, were incubated with cardiomyocytes that had been loaded with a calcium specific fluorescent probe. Calcium transients were measured by real-time fluorescence spectrophotometry following treatment. The transients were rapidly abolished following addition of a moderate concentration of the extract (400 ng/ml), resulting in high levels of intracellular calcium, while the lower amount (40 ng/ml) blocked the sodium-potassium adenosine triphosphatase. Treatments with ouabain and nifedipine were also made, either prior to, or after the addition of the Chan Su, in an attempt to better delineate the site(s) of action. The moderate concentration of Chan Su (400 ng/ml) extract caused the myocytes to cease beating within seconds of addition, even in experiments when saturating concentrations of nifedipine or ouabain had been previously added to the cells. As expected bufalin, the active component of Chan Su has similar effects. Our findings indicate that this compound is extremely cardiotoxic, even in small dose and acts rapidly to alter intracellular calcium stores in cardiomyocytes and possibly acts at sites other than the Na(+)+K(+) ATPase, either directly, or indirectly via changes in calcium concentrations.

Effects of IgM from rheumatic fever patients on intracellular calcium levels of neonatal rat cardiac myocytes.

Rheumatic fever (RF), a potential sequela of Streptococcus pyogenes pharyngitis, sometimes results in myocarditis and heart failure. Antibodies have been implicated in the pathogenesis of RF and anti-cardiac myosin antibody levels are elevated in RF patients. Since myocarditis is associated with altered cardiomyocyte calcium transients it was of interest to determine the direct effects of RF patient antibodies on calcium transients in cultured myocytes. RF patient polyclonal IgM treatment caused increased calcium retention by neonatal rat heart cells in vitro as determined with isotopically labeled calcium. Therefore, to further characterize this finding, calcium transients were evaluated by real time fluorescence spectroscopy and deconvolution imaging. RF patient polyclonal IgM produced increased calcium retention during the relaxation stage of the contraction cycle leading to a slowing of contraction rate, disorganized calcium transients, and eventual tetany. In contrast, calcium transient studies of cardiomyocytes following treatment with monoclonal anti-myosin antibodies revealed declining intracellular calcium levels, accompanied by disorganized transients and tetany. Treatment with both antibodies led to myocyte dysfunction and these novel findings suggest a role for antibodies in the pathogenesis of the myocarditis associated with rheumatic carditis.

Expression of human beta defensin 2 in thermal injury.

Sepsis is a common and serious complication of major burn injury and accounts for over 54% of deaths in burn patients. Burns are associated with high levels of circulating pro-inflammatory cytokines and immunosuppression, promoting systemic inflammatory response syndrome (SIRS) and sepsis, for which no effective treatment is currently available. Defensins, a family of cationic, naturally occurring, antimicrobial peptides are important components of the innate immune system, playing a major role in the body's defence by inhibiting activities of bacteria, fungi and enveloped viruses. These natural antimicrobials also chemoattract immature dendritic cells, some types of T and B-lymphocytes, neutrophils and macrophages, and act as an adjuvant, enhancing adaptive immunity. Our prior studies suggested a decreased expression of human beta defensin 2 (HBD2) in burn wounds. Here we have identified HBD2 protein in skin samples of partial and full thickness burns and in normal skin using fluorescence deconvolution microscopy. Images showed that in normal skin the majority of HBD2 is located in the Malpighian layer and, in smaller amounts, in the more superficial layers, a pattern that is absent in burned skin in which the epidermis is destroyed or damaged. However, surviving dermal and subcutaneous layers revealed the presence of HBD2 in a number of other cell types and structures, such as hair follicles and sweat gland acini, but not in vascular endothelium and fat cells. The results of these studies further contribute to an understanding of the role of antimicrobial peptides in the pathophysiology of burn injury, associated immunosuppression and sepsis and the possibility of using these other sites of HBD2 deposition for upregulation of antimicrobial synthesis in the treatment of burns.

Effects of cytokines and heat shock on defensin levels of cultured keratinocytes.

Burns have been associated with high levels of circulating pro-inflammatory cytokines which promote systemic inflammatory response syndrome (SIRS), immunosuppression and sepsis for which no effective treatment is currently available. Defensins, a family of cationic naturally occurring antimicrobial peptides, are considered important components of the innate immune system and enhance adaptive immunity. This study examines the effects of pro-inflammatory cytokines, interleukin-1beta (IL-1beta), gamma-interferon (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on human beta-defensin-2 (HBD-2) levels in cultured keratinocytes. We also examined the effects of heat shock at 42 degrees C. The results demonstrate that only TNFalpha shows significant induction of HBD-2 but this induction was not sustained in the long-term. In addition, endogenous levels of defensin were significantly reduced by exposure to heat shock. The keratinocytes also responded to IL-1beta by becoming hypertrophic. These results indicate that stress-related, pro-inflammatory cytokines can induce keratinocytes to synthesize HBD-2, while heat shock appears to reduce its production. These experiments give us further insight into the role of natural antimicrobial peptides under conditions of stress.