ABSTRACT
A more complete understanding of immune-mediated damage to the coronary arteries in children with Kawasaki disease (KD) is required for improvements in patient treatment and outcomes. We recently reported the transcriptional profile of KD coronary arteritis, and in this study sought to determine protein expression of transcriptionally up-regulated immune genes in KD coronary arteries from the first 2 months after disease onset. We examined the coronary arteries of 12 fatal KD cases and 13 childhood controls for expression of a set of proteins whose genes were highly up-regulated in the KD coronary artery transcriptome: allograft inflammatory factor 1 (AIF1), interleukin 18 (IL-18), CD74, CD1c, CD20 (MS4A1), Toll-like receptor 7 (TLR-7) and Z-DNA binding protein 1 (ZBP1). Immunohistochemistry and immunofluorescence studies were performed to evaluate protein expression and co-localization, respectively. AIF1 was expressed transmurally in KD arteritis and localized to macrophages and myeloid dendritic cells. CD74, which interacts with major histocompatibility complex (MHC) class II on antigen-presenting cells, localized to the intima-media. CD1c, a marker of myeloid dendritic cells, was expressed in a transmural pattern, as were IL-18 and CD20. ZBP1 and TLR-7 were up-regulated compared to controls, but less highly compared to the other proteins. These findings provide evidence of antigen presentation and interferon response in KD arteritis. In combination with prior studies demonstrating T lymphocyte activation, these results demonstrate the complexity of the KD arterial immune response.
Subject(s)
Arteritis/immunology , Coronary Vessels/immunology , Gene Expression , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/metabolism , Antigen Presentation , Antigens, CD/genetics , Antigens, CD1/genetics , Antigens, CD20/genetics , Arteritis/physiopathology , Calcium-Binding Proteins , Coronary Aneurysm/immunology , Coronary Vessels/physiopathology , DNA-Binding Proteins/genetics , Female , Fluorescent Antibody Technique , Gene Expression Profiling , Glycoproteins/genetics , Humans , Immunohistochemistry , Infant , Interleukin-18/genetics , Male , Microfilament Proteins , Mucocutaneous Lymph Node Syndrome/complications , Mucocutaneous Lymph Node Syndrome/mortality , RNA-Binding Proteins , Sialyltransferases/genetics , Toll-Like Receptor 7/geneticsABSTRACT
Introduction Heavy alcohol and illicit drug use has been documented amongst medical and dental professionals and educational programs have been developed to attempt to reduce such behaviour in clinical undergraduates. This pilot study aims to investigate the legal and moral perceptions of substance use in clinical and non-clinical undergraduates.Method A cross-sectional self-report questionnaire was administered to 107 clinical and non-clinical undergraduates to investigate their moral and legal perceptions of alcohol and illicit substance use.Results More clinical (72.5%) than non-clinical students (66%) drink alcohol regularly. Both groups perceive ecstasy, cocaine and ketamine as 'high risk' drugs. A third of both clinical (34%) and non-clinical (36%) students support the legalisation of illicit drugs. Forty-seven percent of non-clinical students would consider changing their behaviour if illicit substances were legalised compared to 32% of clinical students. Clinical students believe the legal punishment for Class A drugs is appropriate, but disagree with that for Class C drug use. Personal values of clinical students differ regarding some immoral activities. Social perceptions of illicit substance users are similar for both clinical and non-clinical students with those who use heroin perceived most negatively by 86.5% of all undergraduates.Conclusion Individual substance use behaviours may be influenced by legal perceptions of illicit substance use. Personal values and social norms are also likely to be important. Further research is required to investigate how these perceptions affect a clinical student's decision to participate in excessive alcohol and illicit substance use behaviours.
Subject(s)
Attitude to Health , Illicit Drugs/legislation & jurisprudence , Morals , Students , Substance-Related Disorders , Alcoholism , Cross-Sectional Studies , Female , Humans , Male , Pilot Projects , Self Report , United Kingdom , UniversitiesABSTRACT
The major goals of Kawasaki disease (KD) therapy are to reduce inflammation and prevent thrombosis in the coronary arteries (CA), but some children do not respond to currently available non-specific therapies. New treatments have been difficult to develop because the molecular pathogenesis is unknown. In order to identify dysregulated gene expression in KD CA, we performed high-throughput RNA sequencing on KD and control CA, validated potentially dysregulated genes by real-time reverse transcription-polymerase chain reaction (RT-PCR) and localized protein expression by immunohistochemistry. Signalling lymphocyte activation molecule CD84 was up-regulated 16-fold (P < 0·01) in acute KD CA (within 2 months of onset) and 32-fold (P < 0·01) in chronic CA (5 months to years after onset). CD84 was localized to inflammatory cells in KD tissues. Genes associated with cellular proliferation, motility and survival were also up-regulated in KD CA, and immune activation molecules MX2 and SP140 were up-regulated in chronic KD. CD84, which facilitates immune responses and stabilizes platelet aggregates, is markedly up-regulated in KD CA in patients with acute and chronic arterial disease. We provide the first molecular evidence of dysregulated inflammatory responses persisting for months to years in CA significantly damaged by KD.
Subject(s)
Antigens, CD/metabolism , Antigens, Nuclear/metabolism , Blood Platelets/immunology , Mucocutaneous Lymph Node Syndrome/immunology , Myxovirus Resistance Proteins/metabolism , Transcription Factors/metabolism , Vascular Calcification/immunology , Acute Disease , Antigens, CD/genetics , Antigens, Nuclear/genetics , Cell Growth Processes/genetics , Cell Movement/genetics , Cell Survival/genetics , Chronic Disease , Coronary Vessels/pathology , Female , High-Throughput Screening Assays , Humans , Infant , Male , Mucocutaneous Lymph Node Syndrome/blood , Mucocutaneous Lymph Node Syndrome/genetics , Myxovirus Resistance Proteins/genetics , Platelet Aggregation/genetics , RNA, Messenger/analysis , Signaling Lymphocytic Activation Molecule Family , Transcription Factors/genetics , Up-Regulation , Vascular Calcification/blood , Vascular Calcification/geneticsABSTRACT
Both wild and cultured mussels (Mytilus edulis, Mytilus galloprovincialis and hybrids), are found along most of the Irish coastline. M. edulis is widespread along all Irish coasts and is the only mussel species present on both the east coast of Ireland and the Welsh coast in the Irish Sea. M. galloprovincialis and hybrids are found along the Irish coastline except for the east coast. Samples of Mytilus spp. were collected from twenty-four sites, encompassing all coasts of Ireland and the Welsh coast, at different times of the year and over several years (2008-2011). In total, 841 mussels were examined histologically to assess their health status and the presence of any parasites or commensals. Mussels from 14 of the 24 sites were screened using polymerase chain reaction (PCR) to determine which mytilid species were present. A range of parasites were observed, generally at low levels. The most diverse community of parasites was observed at a sheltered site with poor water quality. Of significance, a previously undescribed haplosporidian was detected in a single mussel sample in the Menai Strait, Wales, by PCR and was confirmed by direct sequencing and is most closely related to Minchina chitonis and a haplosporidian of the Florida marsh clam Cyrenoida floridana. While M. edulis were infected by a variety of micro- and macro-parasites, only trematodes were observed in M. galloprovincialis and hybrids. Habitat description and the environmental factors influencing the study sites, including water quality and exposure, were recorded.
Subject(s)
Haplosporida/genetics , Mytilus edulis/parasitology , Animals , Ireland , Polymerase Chain Reaction , Wales , Water QualityABSTRACT
Juvenile edible crabs, Cancer pagurus L., were surveyed from Mumbles Head and Oxwich Bay in South Wales, UK, and the number of heterotrophic bacteria and vibrios in the hemolymph was determined. The percentage of crabs with hemolymph containing bacteria was variable over the survey with higher numbers of animals affected in summer than in winter. Post-moult crabs contained significantly higher numbers of heterotrophic bacteria in the hemolymph than pre- and intermoult animals. Crabs with cuticular damage to the gills also had significantly higher numbers of bacteria in the hemolymph. Crabs were found to have a high prevalence of infection by the dinoflagellate, Hematodinium. Such animals had significantly fewer bacteria in the blood in comparison with Hematodinium-free animals. Of the 463 crabs surveyed, only 3 individuals had hemolymph containing 2000 + CFU mL(-1). Based on 16S rRNA gene sequences, two of these crabs contained a Vibrio pectenicida-like isolate, while the other had a mixed assemblage of vibrios. Although 59% of the crabs surveyed had culturable bacteria in the hemolymph, the majority only had small numbers (<2000 CFU mL(-1) ), suggesting that such infections may be of limited importance to the sustainability of the crab fishery in this region.
Subject(s)
Brachyura/microbiology , Hemolymph/microbiology , Vibrio/isolation & purification , Animals , Dinoflagellida/isolation & purification , Female , Gills/microbiology , Host-Pathogen Interactions , Male , Time FactorsABSTRACT
BACKGROUND AND PURPOSE: γ-Secretase modulators represent a promising therapeutic approach for Alzheimer's disease (AD) because they selectively decrease amyloid ß 42 (Aß42), a particularly neurotoxic Aß species that accumulates in plaques in the brains of patients with AD. In the present study, we describe the in vitro and in vivo pharmacological properties of a potent novel γ-secretase modulator, 2-(S)-(3,5-bis(4-(trifluoromethyl)phenyl)phenyl)-4-methylpentanoic acid (JNJ-40418677). EXPERIMENTAL APPROACH: The potency and selectivity of JNJ-40418677 for Aß reduction was investigated in human neuroblastoma cells, rat primary neurones and after treatment with single oral doses in non-transgenic mouse brains. To evaluate the effect of JNJ-40418677 on plaque formation, Tg2576 mice were treated from 6 until 13 months of age via the diet. KEY RESULTS: JNJ-40418677 selectively reduced Aß42 secretion in human neuroblastoma cells and rat primary neurones, but it did not inhibit Notch processing or formation of other amyloid precursor protein cleavage products. Oral treatment of non-transgenic mice with JNJ-40418677 resulted in an excellent brain penetration of the compound and a dose- and time-dependent decrease of brain Aß42 levels. Chronic treatment of Tg2576 mice with JNJ-40418677 reduced brain Aß levels, the area occupied by plaques and plaque number in a dose-dependent manner compared with transgenic vehicle-treated mice. CONCLUSIONS AND IMPLICATIONS: JNJ-40418677 selectively decreased Aß42 production, showed an excellent brain penetration after oral administration in mice and lowered brain Aß burden in Tg2576 mice after chronic treatment. JNJ-40418677 therefore warrants further investigation as a potentially effective disease-modifying therapy for AD.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases/metabolism , Pentanoic Acids/therapeutic use , Plaque, Amyloid/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Cell Line, Tumor , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Female , Humans , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Neurons/drug effects , Neurons/metabolism , Pentanoic Acids/pharmacokinetics , Pentanoic Acids/pharmacology , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Rats , Receptors, Notch/metabolismABSTRACT
AIMS: The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei. METHODS AND RESULTS: Of the ten species tested, only Vibrio alginolyticus (NCIMB 1339) and Vibrio gazogenes (NCIMB 2250) showed antagonistic activity towards a panel of shrimp pathogenic vibrios. In the case of V. alginolyticus, this activity depended on the presence of live bacteria while in V. gazogenes both live and dead bacteria showed anti-Vibrio activity. Injection of shrimp with either V. alginolyticus or V. gazogenes at 3 × 10(7) or 3 × 10(5) total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10(7) bacteria). Juvenile shrimp were fed commercial diets top-coated with either chitin (an immune stimulant) or chitin + V. gazogenes. Both chitin and V. gazogenes caused a significant decline in the number of Vibrio-like bacteria in the fore and hind gut, and changes were also seen in the hepatosomatic index (a measure of digestive health) and the total number of blood cells in circulation. Analysis of mid/hindgut and faecal samples obtained using terminal restriction fragment length polymorphism showed that the gut microbiota of shrimp has limited bacterial diversity and that after 8 weeks exposure to the experimental diets there were significant changes in the microbial flora of the GI tract of shrimp as a result of the presence of V. gazogenes. CONCLUSIONS: Of the vibrios tested, V. gazogenes has potential as a probiotic for the control of bacterial diseases in shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, this study shows the promise of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions.
Subject(s)
Penaeidae/microbiology , Probiotics , Vibrio/physiology , Animals , Aquaculture , Gastrointestinal Tract/microbiology , Microbial Interactions , Penaeidae/immunology , Vibrio alginolyticus/physiologyABSTRACT
Epiretinal prostheses used to treat degenerative retina diseases apply stimulus via an electrode array fixed to the ganglion cell side of the retina. Mechanical pressure applied by these arrays to the retina, both during initial insertion and throughout chronic use, could cause sufficient retinal damage to reduce the device's effectiveness. In order to understand and minimize potential mechanical damage, we have used finite element analysis to model mechanical interactions between an electrode array and the retina in both acute and chronic loading configurations. Modeling indicates that an acute tacking force distributes stress primarily underneath the tack site and heel edge of the array, while more moderate chronic stresses are distributed more evenly underneath the array. Retinal damage in a canine model chronically implanted with a similar array occurred in correlating locations, and model predictions correlate well with benchtop eyewall compression tests. This model provides retinal prosthesis researchers with a tool to optimize the mechanical electrode array design, but the techniques used here represent a unique effort to combine a modifiable device and soft biological tissues in the same model and those techniques could be extended to other devices that come into mechanical contact with soft neural tissues.
Subject(s)
Electrodes, Implanted , Models, Biological , Prostheses and Implants , Retina/physiology , Animals , Compressive Strength , Computer Simulation , Dogs , Elastic Modulus , Equipment Failure Analysis , Finite Element Analysis , Hardness/physiology , Prosthesis DesignABSTRACT
Wavelet cross-correlation (WCC) is used to analyse the relationship between low-frequency oscillations in near-infrared spectroscopy (NIRS) measured cerebral oxyhaemoglobin (O(2)Hb) and mean arterial blood pressure (MAP) in patients suffering from autonomic failure and age-matched controls. Statistically significant differences are found in the wavelet scale of maximum cross-correlation upon posture change in patients, but not in controls. We propose that WCC analysis of the relationship between O(2)Hb and MAP provides a useful method of investigating the dynamics of cerebral autoregulation using the spontaneous low-frequency oscillations that are typically observed in both variables without having to make the assumption of stationarity of the time series. It is suggested that for a short-duration clinical test previous transfer-function-based approaches to analyse this relationship may suffer due to the inherent nonstationarity of low-frequency oscillations that are observed in the resting brain.
Subject(s)
Blood Pressure/physiology , Brain Chemistry/physiology , Oxyhemoglobins/analysis , Adult , Aged , Algorithms , Autonomic Nervous System Diseases/metabolism , Autonomic Nervous System Diseases/physiopathology , Data Interpretation, Statistical , Female , Homeostasis/physiology , Humans , Male , Middle Aged , Plethysmography , Spectroscopy, Near-Infrared , Supine Position/physiology , Tilt-Table TestSubject(s)
Coronavirus/metabolism , Mucocutaneous Lymph Node Syndrome/virology , Acute Disease , Child , Child, Preschool , Coronavirus Infections/virology , DNA Primers/chemistry , Genome, Viral , Humans , Infant , Models, Genetic , Mucocutaneous Lymph Node Syndrome/etiology , RNA, Viral/metabolism , Respiratory Tract Infections/virology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Angiogenesis has been shown to be dysregulated in coronary artery (CA) aneurysms in the chronic phase of Kawasaki disease (KD). Neovascularization may occur in inflammatory-related vascular diseases because many angiogenesis mediators are secreted by inflammatory cells. We hypothesized that inflammation of the acute KD CA aneurysm could lead to dysregulation of angiogenesis mediators and subsequent neovascularization. To investigate this hypothesis, acute fatal KD cardiac tissues were immunostained for angiogenic inducers and inhibitors. Microvessel density was determined and the degree of inflammation assessed. Marked inflammation and angiogenesis were found in acute KD CA aneurysms and myocardium, with the highest microvessel density seen in patients who died 2-3 weeks after onset of the disease. Expression of proangiogenic proteins was higher than expression of inhibitors in KD CA aneurysms and myocardium. Angiogenesis mediators were localized to inflammatory cells in the myointima, adventitia, and myocardium. We conclude that significant neovascularization occurs in acute KD CA aneurysms and myocardium much sooner after onset of the disease than has been previously reported, that multiple angiogenesis factors are involved, and that dysregulation of angiogenesis likely contributes to KD vasculopathy.
Subject(s)
Coronary Aneurysm/mortality , Coronary Vessels/pathology , Mucocutaneous Lymph Node Syndrome/mortality , Mucocutaneous Lymph Node Syndrome/pathology , Myocardium/pathology , Neovascularization, Pathologic/mortality , Acute Disease , Aneurysm, Ruptured/mortality , Angiostatins/metabolism , Case-Control Studies , Child , Coronary Aneurysm/metabolism , Coronary Aneurysm/pathology , Coronary Vessels/metabolism , Female , Fibroblast Growth Factor 2/metabolism , Humans , Immunohistochemistry , Infant , Infant, Newborn , Male , Mast Cells/metabolism , Microcirculation , Mucocutaneous Lymph Node Syndrome/metabolism , Myocarditis/metabolism , Myocarditis/mortality , Myocarditis/pathology , Myocardium/metabolism , Neovascularization, Pathologic/metabolism , Nerve Growth Factor/metabolism , Platelet-Derived Growth Factor/metabolism , Thrombospondins/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Kawasaki disease (KD) is an acute vasculitis of young childhood predominantly affecting the coronary arteries. IgA plasma cells have been found to infiltrate vascular and nonvascular tissues in fatal acute KD. To determine whether IgA B-lymphocytes were increased in the peripheral blood of patients with KD, we performed three-color flow cytometry to detect surface and cytoplasmic immunoglobulin expression (IgA, IgM, IgD, and IgG) of peripheral B-lymphocytes in KD patients during the acute, subacute, and convalescent stages of illness and in age-matched febrile and afebrile pediatric controls. Surprisingly, absolute numbers of B-lymphocytes expressing IgA were found to be significantly lower in peripheral blood of acute KD patients compared with febrile and afebrile pediatric controls. These findings indicate that IgA plasma cells are not present in KD tissue as a result of excess numbers of these IgA B-lymphocytes in peripheral blood. We speculate that IgA B-lymphocytes are selectively withdrawn from the peripheral circulation into KD target tissues as part of a specific IgA immune response.
Subject(s)
B-Lymphocytes/immunology , Cytoplasm/immunology , Immunoglobulins/blood , Mucocutaneous Lymph Node Syndrome/immunology , Acute Disease , Flow Cytometry , Humans , Immunophenotyping , Mucocutaneous Lymph Node Syndrome/bloodABSTRACT
The pathogenesis of coronary arterial inflammation in acute Kawasaki disease (KD) is unclear. To test the hypothesis that the KD vascular lesion is an activated T lymphocyte-dependent process, immunohistochemical studies were done on coronary artery aneurysms from 8 fatal acute KD cases by using antibodies to CD45RO (activated or memory T lymphocyte), CD8 (cytotoxic T lymphocyte), CD4 (helper T lymphocyte), HAM56 (macrophage), and CD20 (B lymphocyte). Acute KD coronary arteritis was characterized by transmural infiltration of CD45RO T lymphocytes with CD8 T lymphocytes predominating over CD4 T lymphocytes. Macrophages were present primarily in the adventitial layer; B lymphocytes were notably absent. These data lend support to the hypotheses that KD results from infection with an intracellular pathogen, such as a virus, whose antigens are presented by major histocompatibility complex class I molecules, and that CD8 T lymphocytes and macrophages are important in the pathogenesis of KD coronary aneurysms.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Coronary Aneurysm/immunology , Macrophages/immunology , Mucocutaneous Lymph Node Syndrome/immunology , Antigens, CD20/analysis , CD8 Antigens/analysis , Child , Child, Preschool , Coronary Aneurysm/complications , Coronary Vessels/immunology , Endothelium, Vascular/immunology , Female , Humans , Immunohistochemistry , Infant , Leukocyte Common Antigens/analysis , Male , Mucocutaneous Lymph Node Syndrome/complicationsABSTRACT
Chlamydia pneumoniae has been associated with atherosclerosis and myocardial infarction in adults. Coronary artery tissues of five fatal cases of Kawasaki disease were examined by PCR; none was positive.
Subject(s)
Chlamydophila pneumoniae/isolation & purification , Coronary Vessels/microbiology , Mucocutaneous Lymph Node Syndrome/microbiology , Aneurysm, Ruptured/etiology , Child , Chlamydophila pneumoniae/genetics , DNA, Viral/analysis , Female , Humans , Infant , Male , Mucocutaneous Lymph Node Syndrome/etiology , Mucocutaneous Lymph Node Syndrome/pathology , Polymerase Chain ReactionABSTRACT
Kawasaki Disease (KD) is a potentially fatal acute vasculitis of childhood. Although KD is the leading cause of acquired heart disease in children in developed nations, its pathogenesis remains unknown. We previously reported the novel observation that IgA plasma cells infiltrate the vascular wall in acute KD. We have now examined the clonality of this IgA response in vascular tissue from three fatal cases of KD to determine whether it is oligoclonal, suggesting an Ag-driven process, or polyclonal, suggesting nonspecific B cell activation or a response to a superantigen. We first sequenced VDJ junctions of 44 alpha genes isolated from a primary, unamplified KD vascular cDNA library. Five sets of clonally related alpha sequences were identified, comprising 34% (15 of 44) of the isolated alpha sequences. Furthermore, point mutations consistent with somatic mutation were detected in the related sequences. Next, using formalin-fixed coronary arteries from two additional fatal KD cases, we sequenced VDJ junctions of alpha genes isolated by RT-PCR, and a restricted pattern of CDR3 usage was observed in both. We conclude that the vascular IgA response in acute KD is oligoclonal. The identification of an oligoclonal IgA response in KD strongly suggests that the immune response to this important childhood illness is Ag-driven.
Subject(s)
Coronary Vessels/immunology , Coronary Vessels/metabolism , Immunoglobulin A/genetics , Mucocutaneous Lymph Node Syndrome/genetics , Mucocutaneous Lymph Node Syndrome/immunology , Acute Disease , Amino Acid Sequence , Base Sequence , Child , Clone Cells , Cloning, Molecular , Female , Gene Amplification , Gene Library , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Genes, Immunoglobulin , Humans , Immunoglobulin A/biosynthesis , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin alpha-Chains/biosynthesis , Immunoglobulin alpha-Chains/genetics , Infant , Male , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Shell disease syndrome is characterised by the external manifestation of black spot lesions in the exoskeletons of crustaceans. In the present study, gills, hepatopancreas and hearts from healthy (<0.05% black spot coverage) and diseased (5 to 15% coverage) edible crabs, Cancer pagurus, were examined histologically to determine whether this disease can cause internal damage to such crabs. There was clear evidence of cuticular damage in the gills of diseased crabs leading to the formation of haemocyte plugs termed nodules. Nephrocytes found within the branchial septa of the gills showed an increase in the accumulation of dark material in their vacuoles in response to disease. In the hepatopancreas, various stages of tubular degradation were apparent that correlated with the severity of external disease. Similarly, there was a positive correlation between the number of viable bacteria in the haemolymph and the degree of shell disease severity. Approximately 21% of the haemolymph-isolated bacteria displayed chitinolytic activity. Overall, these findings suggest that shell disease syndrome should not be considered as a disease of the cuticle alone. Furthermore, it shows that in wild populations of crabs shell perforations may lead to limited septicaemia potentially resulting in damage of internal tissues. Whether such natural infections lead to significant fatalities in crabs is still uncertain.
Subject(s)
Brachyura/microbiology , Animals , Bacteria/isolation & purification , Digestive System/pathology , Female , Gills/pathology , Hemolymph/microbiology , Male , Myocardium/pathology , Severity of Illness Index , SyndromeABSTRACT
A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.
Subject(s)
Golgi Apparatus/chemistry , Membrane Proteins/analysis , Membrane Proteins/chemistry , Proteome/analysis , Amino Acid Sequence , Animals , Cells, Cultured , Golgi Apparatus/ultrastructure , Molecular Sequence Data , Neurons/chemistry , Octoxynol , Polyethylene Glycols/chemistry , Rats , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , rab GTP-Binding Proteins/analysis , rab GTP-Binding Proteins/chemistryABSTRACT
The etiology and pathogenesis of Kawasaki disease (KD) remain unknown. As previously reported, in US patients with acute KD, IgA plasma cells (PCs) infiltrate the vascular wall. To determine whether IgA PCs are increased at mucosal sites in KD and to determine whether other nonvascular KD tissues are infiltrated by IgA PCs, the cells were immunolocalized and quantitated in tissue sections taken from 18 US and Japanese patients who died of acute KD and from 10 age-matched controls. IgA PCs were significantly increased in the trachea of patients who died of acute KD, compared with controls (P<.01), a finding that was similar to findings in children with fatal respiratory viral infection. IgA PCs also infiltrated coronary artery, pancreas, and kidney in all KD patients. These findings strongly support entry of the KD etiologic agent through the upper respiratory tract, resulting in an IgA immune response, with systemic spread to vascular tissue, pancreas, and kidney.
Subject(s)
Immunoglobulin A/analysis , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/pathology , Plasma Cells/pathology , Acute Disease , Cause of Death , Child , Child, Preschool , Coronary Vessels/immunology , Coronary Vessels/pathology , Ethnicity , Female , Humans , Infant , Japan , Kidney/immunology , Kidney/pathology , Male , Pancreas/immunology , Pancreas/pathology , Plasma Cells/immunology , Respiratory System/immunology , Respiratory System/pathology , United StatesABSTRACT
Advances in mass spectrometry combined with accelerated progress in genome sequencing projects have facilitated the rapid identification of proteins by enzymatic digestion, mass analysis, and sequence database searching. Applications for this technology range from the surveillance of protein expression in cells, tissues, and whole organisms, to the identification of proteins and posttranslational modifications. Here we consider practical aspects of the application of mass spectrometry in cell biology and illustrate these with examples from our own laboratories.