Traumatic right atrial rupture bridge to definitive repair with extra-corporeal life support.

Cardiac injury secondary to non-penetrating trauma is more common than thought, albeit, the injury is usually minor and goes undiagnosed without significant sequelae in most cases. Blunt cardiac rupture is much rarer accounting for <0.05% of all trauma cases but lethal in most circumstances. We present a case report of a young trauma victim who presented with both right atrial rupture and traumatic atrial septal disruption (ASD) requiring extra-corporeal life support (ECLS) and surgical repair. Blunt cardiac trauma with chamber rupture and septal disruption is a devastating injury. Stopping the hemorrhage and using ECLS gave our patient time to stabilize before definitive management of her traumatic ASD.

An investigation of the impact of the location and timing of antigen-specific T cell division on airways inflammation.

It is widely accepted that allergic asthma is orchestrated by T helper type 2 lymphocytes specific for inhaled allergen. However, it remains unclear where and when T cell activation and division occurs after allergen challenge, and whether these factors have a significant impact on airways inflammation. We therefore employed a CD4-T cell receptor transgenic adoptive transfer model in conjunction with laser scanning cytometry to characterize the location and timing of T cell division in asthma in vivo. Thus, for the first time we have directly assessed the division of antigen-specific T cells in situ. We found that accumulation of divided antigen-specific T cells in the lungs appeared to occur in two waves. The first very early wave was apparent before dividing T cells could be detected in the lymph node (LN) and coincided with neutrophil influx. The second wave of divided T cells accumulating in lung followed the appearance of these cells in LN and coincided with peak eosinophilia. Furthermore, accumulation of antigen-specific T cells in the draining LN and lung tissue, together with accompanying pathology, was reduced by intervention with the sphingosine 1-phosphate receptor agonist FTY720 2 days after challenge. These findings provide greater insight into the timing and location of antigen-specific T cell division in airways inflammation, indicate that distinct phases and locations of antigen presentation may be associated with different aspects of pathology and that therapeutics targeted against leukocyte migration may be useful in these conditions.

Dissecting the contribution of innate and antigen-specific pathways to the breach of self-tolerance observed in a murine model of arthritis.

BACKGROUND: The relative roles of innate immunity and antigen-specific T cells in rheumatoid arthritis remain controversial. Previous studies demonstrated that T-helper type 1 cells of irrelevant antigen specificity (ovalbumin) induced a transient arthritis in BALB/c mice, which recapitulates many of the pre-articular and articular features of human disease and is associated with the emergence of autoreactive T and B-cell responses to joint-specific antigens. However, the mechanisms underlying this phenomenon were unclear. OBJECTIVES: The aim of this study was to dissect the relative contribution of innate and heterologous antigen-specific pathways to the breach of self-tolerance and pathology observed in this model and how this may result from modified T and B-cell interactions. METHODS: To address this issue, experimental arthritis was elicited either by a non-specific inflammatory stimulus alone, by activation of T cells of an irrelevant specificity or a combination of both. RESULTS: The non-specific inflammatory response generated by lipopolysaccharide led to articular inflammation and cartilage erosion, but did not break tolerance to joint-specific antigens. In contrast, local activation of T cells of an irrelevant specificity produced a similar pathological picture but, in addition, induced T-cell responses to unrelated joint-specific antigens with associated activation of autoreactive B cells. These effects could be further potentiated by the addition of lipopolysaccharide. CONCLUSION: These data demonstrate that non-specific inflammation alone is insufficient to breach self-tolerance. In contrast, T cells of an irrelevant specificity, when triggered locally in an antigen-specific manner, can breach self-tolerance leading to arthritis and autoantibody production, which can then be amplified in a non-specific manner.

Artifact produced in disc electrophoresis by ammonium persulfate.

Ammonium persulfate, a common polymerizing agent for acrylamide gels, can inactivate yeast enolase and produce increased electrophoretic heterogeneity during disc electrophoresis in gels containing 8M urea. The use of riboflavin and light for polymerization or thioglycolate for removal of the persulfate are feasible alternatives.

Imaging T-cell movement in the brain during experimental cerebral malaria.

T-cells are known to play a role in the pathology associated with experimental cerebral malaria, although it has not previously been possible to examine their behaviour in brain. Using multiphoton laser scanning microscopy, we have examined the migration and movement of these cells in brain tissue. We believe that this approach will help define host-parasite interactions and examine how intervening in these relationships affects the development of cerebral pathology.

Tumour necrosis factor-alpha blockade suppresses murine allergic airways inflammation.

Asthma is a heterogeneous disease that has been increasing in incidence throughout western societies and cytokines, including proinflammatory tumour necrosis factor alpha (TNF-alpha), have been implicated in the pathogenesis of asthma. Anti-TNF-alpha therapies have been established successfully in the clinic for diseases such as rheumatoid arthritis and Crohn's disease. TNF-alpha-blocking strategies are now being trialled in asthma; however, their mode of action is poorly understood. Based on the observation that TNF-alpha induces lymph node hypertrophy we have attempted to investigate this as a mechanism of action of TNF-alpha in airway inflammation by employing two models of murine airway inflammation, that we have termed short and long models, representing severe and mild/moderate asthma, respectively. The models differ by their immunization schedules. In the short model, characterized by eosinophilic and neutrophilic airway inflammation the effect of TNF-alpha blockade was a reduction in draining lymph node (DLN) hypertrophy, eosinophilia, interleukin (IL)-5 production and immunoglobulin E (IgE) production. In the long model, characterized by eosinophilic inflammation, TNF-alpha blockade produced a reduction in DLN hypertrophy and IL-5 production but had limited effects on eosinophilia and IgE production. These results indicate that anti-TNF-alpha can suppress DLN hypertrophy and decrease airway inflammation. Further investigations showed that anti-TNF-alpha-induced inhibition of DLN hypertrophy cannot be explained by preventing l-selectin-dependent capture of lymphocytes into the DLN. Given that overall TNF blockade was able to suppress the short model (severe) more effectively than the long model (mild/moderate), the results suggest that TNF-alpha blocking therapies may be more effective in the treatment of severe asthma.

What can transgenic parasites tell us about the development of Plasmodium-specific immune responses?

Malaria infects 500 million people and kills an estimated 2.7 million annually, representing one of the most significant diseases in the world. However, efforts to develop effective vaccines have met with limited success. One reason is our lack of basic knowledge of how and where the immune system responds to parasite antigens. This is important as the early events during induction of an immune response influence the acquisition of effector function and development of memory responses. Our knowledge of the interactions of Plasmodia with the host immune system has largely been derived through in vitro study. This is a significant issue as the component parts of the immune system do not work in isolation and their interactions occur in distinct and specialized micro- and macro-anatomical locations that can only be assessed in the physiological context, in vivo. In this context, the availability of transgenic malaria parasites over the last 10 years has greatly enhanced our ability to understand and evaluate factors involved in host-parasite interactions in vivo. In this article, we review the current status of this area and speculate on what parasite transgenesis approaches will tell us about the development of Plasmodium-specific immune responses in the future.

Neutrophil swarming and extracellular trap formation play a significant role in Alum adjuvant activity.

There are over 6 billion vaccine doses administered each year, most containing aluminium-based adjuvants, yet we still do not have a complete understanding of their mechanisms of action. Recent evidence has identified host DNA and downstream sensing as playing a significant role in aluminium adjuvant (aluminium hydroxide) activity. However, the cellular source of this DNA, how it is sensed by the immune system and the consequences of this for vaccination remains unclear. Here we show that the very early injection site reaction is characterised by inflammatory chemokine production and neutrophil recruitment. Intravital imaging demonstrates that the Alum injection site is a focus of neutrophil swarms and extracellular DNA strands. These strands were confirmed as neutrophil extracellular traps due to their sensitivity to DNAse and absence in mice deficient in peptidylarginine deiminase 4. Further studies in PAD4-/- mice confirmed a significant role for neutrophil extracellular trap formation in the adjuvant activity of Alum. By revealing neutrophils recruited to the site of Alum injection as a source of the DNA that is detected by the immune system this study provides the missing link between Alum injection and the activation of DNA sensors that enhance adjuvant activity, elucidating a key mechanism of action for this important vaccine component.

Characterization of the interaction of yeast enolase with polynucleotides.

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties.

The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211. The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822). Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca. 10% and 0.0003% of the native enzyme activity, respectively. H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase. H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP. H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase. Substrate and Mg2+ increase subunit association in both mutants. Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373. We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.

Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism.

The S39A mutant of yeast enolase (isozyme 1), prepared by site-directed mutagenesis, has a relative Vmax of 0.01% and an activation constant for Mg2+ ca. 10-fold higher, compared with native enzyme. It is correctly folded. There is little effect of solvent viscosity on activity. We think that the loop Ser36-His43 fails to move to the 'closed' position upon catalytic Mg2+ binding, weakening several electrostatic interactions involved in the mechanism.

Preparation by site-directed mutagenesis and characterization of the E211Q mutant of yeast enolase 1.

The published 'charge shuttle' mechanism of enolase (Lebioda, L. and Stec, B. (1991) Biochemistry 30, 2817-2822) assigns Glu-211 the task of orienting a water molecule that serves as the catalytic base which removes the proton from carbon-2 of the substrate. We prepared the E211Q mutant of yeast enolase 1 by site-directed mutagenesis. It appears to be folded correctly and to respond similarly to many of the normal ligands of enolase: it is stabilized against thermal denaturation by conformational Mg2+ and by Mg2+ and substrate and binds the chromophoric substrate analogue D-tartronate semialdehyde-2-phosphate (TSP) with affinity comparable to that of the native enzyme. However, it has only 0.01% (10(-4)) of the activity of native enolase under standard assay conditions and does not exhibit significantly more activity at various pH values or higher concentrations of substrate and Mg2+. Its ability to produce the form of enzyme-bound and reacted TSP that absorbs at shorter wavelengths is greatly slowed, while the longer wavelength absorbing form is produced rapidly. Overall, these observations are consistent with the hypothetical mechanism.

Crystallization and preliminary crystallographic data for a tetragonal form of yeast enolase.

Yeast enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) has been crystallized by vapor diffusion and equilibrium dialysis of solutions of ammonium sulfate. The crystals with the dimer have 2-fold axial symmetry and appear to be suitable for high-resolution X-ray diffraction analysis. Our potential heavy-atom derivative of the native crystals has been prepared.

The amino acid sequence of avian thymic hormone, a parvalbumin.

The complete amino acid sequence of 'avian thymic hormone' (ATH), a protein from thymus tissue that appears to promote immune maturation in chicken bone marrow cells in culture, is presented. The sequence was obtained from sequences of ATH peptides isolated by HPLC after tryptic, chymotryptic, peptic or S aureus V8 protease digestions. The protein is a parvalbumin consisting of 108 residues with a blocked amino terminus, a single cysteine, tyrosine, proline and arginine and no histidine, methionine or tryptophan. This is the first amino acid sequence of a parvalbumin which is not derived from muscle tissue.

Homodimer formation by the individual subunits of bovine lutropin as determined by sedimentation equilibrium.

Although differing in their amino acid sequences, the folding patterns of the alpha and beta subunits of human choriogonadotropin are similar in the crystal structure of the HF-treated glycoprotein hormone. Each subunit forms a cystine-knot motif like that found in several growth factors that form homodimers and heterodimers. In order to ascertain if the alpha and beta subunits can self-associate, e.g. to form homodimers, sedimentation equilibrium at various glycoprotein concentrations and temperatures was used to study the subunits of bovine lutropin, which are expected to exhibit conformations like those of the choriogonadotropin subunits. Each subunit was found to form homodimers with Kd values of 0.3 and 0.1 mM for alpha and beta respectively at 37 degrees C. Self-association was weakly exothermic for alpha and endothermic for beta; entropic factors made a major contribution for each. It is unlikely that homodimer formation of either subunit would be physiologically important, although homodimers may form to some extent intracellularly because of the relatively high concentrations during biosynthesis.

Percutaneous transluminal coronary angioplasty failures in patients with multivessel disease. Is there an increased risk?

In recent years, there has been a nationwide trend toward performing percutaneous transluminal coronary angioplasty in patients with multivessel coronary artery disease. The clinical course of 57 consecutive patients who required emergency first-time coronary artery bypass grafting operations were reviewed to assess for difference in outcome between the 28 patients (49%) with single-vessel disease and the 29 patients (51%) with multivessel disease. The two groups were similar in preoperative characteristics except for a higher proportion of chronic obstructive pulmonary disease in the patients with multivessel disease (p = 0.03). Twice as many patients with multivessel disease were in shock (single-vessel disease = 4 [14%], multivessel disease = 8 [28%], p = not significant) en route to the operating room and significantly more patients with multivessel disease required on-going cardiopulmonary resuscitation (single-vessel disease = 0 [0%], multivessel disease = 5 [17%], p = 0.03). Significantly more coronary artery bypass grafts were placed in the patients with multivessel disease (single-vessel disease = 1.5 +/- 0.6, multivessel disease = 2.9 +/- 0.7, p < 0.01), which required longer aortic clamping time (p = 0.02) and cardiopulmonary bypass time (p < 0.01). There were seven postoperative deaths; all but one occurred in patients with multivessel disease (single-vessel disease = 1 [4%], multivessel disease = 6 [21%], p = 0.05). According to multivariate analysis, incremental risk factors of mortality were preoperative shock (p < 0.01), urgent or emergency percutaneous transluminal coronary angioplasty (p = 0.06), and multivessel disease (p = 0.12). Despite a similar incidence of myocardial infarction (single-vessel disease = 8 [29%], multivessel disease = 12 [41%], p = not significant), patients with multivessel disease had a higher incidence of cardiac morbidity (single-vessel disease = 4 [14%], multivessel disease = 11 [38%], p = 0.04) and noncardiac morbidity (single-vessel disease = 4 [14%], multivessel disease = 12 [41%], p = 0.02). By multivariate analysis, incremental risk factors of morbidity were preoperative shock (p < 0.01), multivessel disease (p = 0.02), and ejection fraction < 50% (p = 0.07). In the subset of patients with multivessel disease, preoperative shock, ejection fraction < 50, and an age of 60 years or greater were associated with higher morbidity and mortality. In conclusion, the risk of percutaneous transluminal coronary angioplasty failure is considerably higher in patients with multivessel disease. In certain subsets of patients with multivessel disease, coronary artery bypass grafting would be a safer procedure when compared with percutaneous transluminal coronary angioplasty for initial myocardial revascularization.

Neuropeptide Y rapidly reduces Period 1 and Period 2 mRNA levels in the hamster suprachiasmatic nucleus.

The mammalian suprachiasmatic nucleus (SCN) contains the main circadian clock. Neuropeptide Y (NPY) that is released from the intergeniculate leaflet of the lateral geniculate body to the SCN, acts in the SCN to advance circadian phase in the subjective day via the NPY Y2 receptor. We used semi-quantitative in situ hybridization to determine the effect of NPY on circadian clock genes, Period 1 (Per1) and Period 2 (Per2), expression in SCN slices. Addition of NPY to the brain slices in the subjective day resulted in reduction of Per1 and Per2 mRNA levels 0.5 and 2 h after treatment. NPY Y1/Y5 and Y2 agonists decreased Per1 within 0.5 h. These results suggest that NPY may induce phase shifts by mechanisms involving or resulting in reduction of Per1 and Per2 mRNA levels.

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases.

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.

Fluorescence lifetime and spectral study of the acid expansion of bovine serum albumin.

The fluorescence lifetimes of the tryptophan residues of bovine serum albumin were measured in the native and acid-expanded conformation. A three-exponential process is required to fit the fluorescence decay data. The results are interpreted empirically in terms of two emitting species. The emission at longer wavelength (360 nm) has slower rates of decay than that at shorter wavelength (325 nm). For both emitting species the average lifetime decreases when the N-F transition occurs and shortens further when the protein expands. Rotational correlation times, derived from the decay of the fluorescence anisotropy of the tryptophan residues, suggest that longer emission wavelengths are associated with somewhat shorter correlation times. There is no certain indication of any independent motion of the tryptophans in any conformation, although some very fast process, perhaps Raman scattering, appears to occur. On acid expansion the long correlation times decrease to around 10 ns in the fully expanded form. Static quenching experiments using I- or acrylamide suggest a greater average exposure of the tryptophans when the protein is most greatly expanded. This is despite the fact that the fluorescence emission maximum shifts to shorter wavelength under these conditions. Also, there is no difference in accessibility to quenching between the longer and shorter wavelength emissions.