Protein array technology to investigate cytokine production by monocytes from patients with advanced alcoholic cirrhosis: An ex vivo pilot study

Aim:  In patients with advanced cirrhosis, little is known about the ability of peripheral blood monocytes to spontaneously produce signaling proteins such as cytokines. The aim of this ex vivo study was to evaluate cytokine production under baseline conditions and after stimulation by lipopolysaccharide (LPS), a toll-like receptor (TLR) agonist.
Methods:  Peripheral blood monocytes were isolated from patients with advanced alcoholic cirrhosis (without ongoing bacterial infections) and normal subjects. Cells were left unstimulated or were stimulated with LPS. The abundance of 24 cytokines was measured using a filter-based, arrayed sandwich enzyme-linked immunosorbent assay (ELISA) in the supernatant of cultured monocytes.
Results:  Cirrhotic monocytes spontaneously produced six proteins (TNF-α, IL-6, IL-8, MCP-1, RANTES and Gro), whereas normal monocytes produced only small amounts of IL-8 and RANTES. Analyses with the online gene set analysis toolkit WebGestalt ( http://bioinfo.vanderbilt.edu/webgestalt ) found enrichment for the six proteins in the human gene ontology subcategory ( http://www.geneontology.org ), Kyoto Encyclopedia of Genes and Genome pathways ( http://www.genome.ad.jp/kegg/ ) and BioCarta pathways ( http://www.biocarta.com/genes/index.asp ) consistent with a proinflammatory phenotype of cirrhotic monocytes resulting from activated TLR signaling. Interestingly, LPS-elicited TLR engagement further increased the production of the six proteins and did not induce the secretion of any others, in particular the anti-inflammatory cytokine IL-10. LPS-stimulated normal monocytes produced TNF-α, IL-6, IL-8, MCP-1, RANTES, Gro and IL-10.
Conclusion:  In patients with advanced cirrhosis, peripheral blood monocytes spontaneously produce proinflammatory cytokines, presumably in response to unrestricted TLR signaling.     

Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review

Sepsis and non-infectious systemic inflammatory response syndrome (SIRS) are paradoxically associated with an exacerbated production of cytokines, as assessed by their presence in biological fluids, and a diminished ability of circulating leukocytes to produce cytokine upon in vitro activation. In this review, we depict that the observed cellular hyporeactivity is not a global phenomenon and that some signalling pathways are unaltered and allow the cells to respond normally to certain stimuli. Furthermore, we illustrate that during sepsis and SIRS, cells derived from tissues are either fully responsive to ex vivo stimuli or even primed, in contrast to cells derived from hematopoietic compartments (blood, spleen, etc.) which are hyporeactive. In addition to cytokine production, nuclear factor-kappa B (NF-kappa B) status within leukocytes can be used as a useful marker of hypo- or hyper-reactivity. We illustrate that the immune-depression reported in sepsis and SIRS patients, often revealed by a diminished capacity of leukocytes to respond to lipopolysaccharide, is not a generalized phenomenon and that SIRS is associated with a compartmentalized responsiveness which involves either anergic or primed cells.     

Deletion of mitochondrial DNA in the endomyocardial biopsy sample from a patient with Kearns-Sayre syndrome.

A 15-year-old boy with Kearns-Sayre syndrome is reported. The deletion of mitochondrial DNA in the endomyocardial biopsy sample from the patient was confirmed by the polymerase chain reaction method, and was identified to that in the skeletal muscle.     

Manometric characteristics of cervical dysphagia in a patient with the Kearns-Sayre syndrome.

The manometric findings of deglutitive pharyngoesophageal function in a patient with the Kearns-Sayre syndrome and cervical dysphagia are described. These findings indicate that striated muscles of the pharynx, upper esophageal sphincter (UES), and proximal esophagus are involved. Near absence of pharyngeal peristalsis, abnormally low UES resting pressure, and absence of proximal esophageal peristalsis characterize the manometric findings in this patient. It is conceivable that in mild cases, a combination of various degrees of severity of the above findings may exist.     

Ultrastructural findings in endomyocardial biopsy of patients with Kearns-Sayre syndrome

Kearns-Sayre syndrome is clinically defined by progressive external ophthalmoplegia, atypical retinitis pigmentosa and the potential occurrence of complete atrioventricular (AV) block. Right septal endomyocardial biopsy specimens from nine patients (four men and five women with a mean [+/- SD] [corrected] age of 36.3 +/- 14.4 years) with chronic progressive external ophthalmoplegia and mitochondrial skeletal myopathy were studied. Three patients had atypical retinal pigmentation. An atrioventricular or intraventricular conduction defect was observed in five patients. A pacemaker was prophylactically implanted in one patient because of abnormal conduction distal to the His bundle. Ultrastructural investigations revealed mitochondriosis in many heart muscle cells and an increased variability of mitochondrial form and size in all patients. In seven patients, 0.4 to 2.1% of all examined myocytes contained exclusively abnormal mitochondria. Three main types were observed: huge, mainly round mitochondria with concentric cristae; large, round or oval mitochondria with transverse or curved cristae; and small, vacuolated mitochondria. The volume density of myofibrils was reduced (41.9 +/- 11.1 compared with the normal value of 56.5 +/- 2.5 volume density [in percent], p less than 0.01) in these myocytes. Increasing numbers of vacuolated mitochondria correlated significantly with a reduction of myofibrils (r = -0.64, p less than 0.01). The data suggest that the ventricular myocardium of most patients with complete and even incomplete Kearns-Sayre syndrome is affected by disseminated mitochondrial cytopathy.     

Cord blood transplantation supported with ex vivo expanded fraction for a patient with myelodysplastic syndrome and metastatic breast cancer

Despite the promising outcomes of unrelated cord blood transplantations (UCBT) in pediatric recipients, the major limitation in the widespread use of cord blood (CB) as a source of hematopoietic stem cells (HSC), particularly in adults, is the physiological small number of cells. To overcome this limitation, we developed an ex vivo expansion system for HSC, in which CB CD34+ cells are cultured on feeder cells (HESS-5 cells) in the presence of cytokines (TPO, SCF and Flt3 ligand). A phase I/II clinical trial, approved by our institutional review board, has been started to assess the safety and effectiveness of this system. A 52-year-old woman with metastatic breast cancer and myelodysplastic syndrome (MDS) received a non-myeloablative preparative regimen followed by UCBT. On day 0, 75% of the whole CB and a fraction of CD34 negative cells were transplanted. The remaining 25% of the CB was CD34-selected and expanded on HESS-5 in a non-serum media in the presence of TPO, SCF, and Flt3-L. On day 5, the ex vivo-expanded, CD34+ cells were transplanted. The patient received 1.83 x 10(7)/kg of total nucleated cells and 7.7 X 10(4)/kg of CD34+ cells (expanded and unexpanded). No acute adverse effects were observed after the infusion of the cultured cells. She suffered from pneumonia on day 37, a cerebral hemorrhage on day 48, and died on day 50. Further studies are required to assess the effectiveness of this protocol.     

Raised plasma concentration and ex vivo production of inflammatory chemokines in patients with systemic lupus erythematosus

BACKGROUND: Chemokines are involved in leucocyte chemotaxis. Infiltrating leucocytes play an important role of tissue injury in systemic lupus erythematosus (SLE). OBJECTIVE: To investigate the role of inflammatory chemokines and their association with interleukin 18 (IL18) in SLE pathogenesis and disease activity. METHODS: Plasma concentrations and ex vivo peripheral blood mononuclear cell production of inflammatory chemokines IP-10, RANTES, MIG, MCP-1, TARC, IL8, and GROalpha, and proinflammatory cytokines IL18, IFNgamma, IL2, IL4, and IL10 were assayed in 80 SLE patients with or without renal disease and 40 healthy controls by immunofluorescence flow cytometry and enzyme linked immunosorbent assay. RESULTS: Plasma IP10, RANTES, MIG, MCP-1, GROalpha, and IL18 concentrations in all SLE patients were higher than in controls, and correlated significantly with SLEDAI score (all p<0.05). In SLE patients without renal disease, IP10, RANTES, MIG, MCP-1, IL8, and IL18 correlated positively with SLEDAI score, while in those with renal derangement, IP10, IL8, IL10, and IL18 correlated with disease activity (all p<0.05). Plasma IL18 concentration correlated positively with IP10, MIG, GROalpha, and IL8 in all SLE patients (all p<0.005). Mitogen induced increases in ex vivo production of IP10, MCP-1, TARC, IFNgamma, IL4, and IL10 were higher in all SLE patients regardless of their difference in disease activity (all p<0.05). Patients with renal disease had an augmented ex vivo release of RANTES. CONCLUSIONS: The correlation of raised plasma concentration and ex vivo production of inflammatory chemokines with disease activity, and their association with IL18, supports the view that chemotaxis of Th1/Th2 lymphocytes and neutrophils is important in SLE pathogenesis.     

Mitochondrial DNA deletion diagnosed by analysis of an endomyocardial biopsy specimen from a patient with Kearns-Sayre syndrome and complete heart block

Defects of mitochondrial DNA have been found at necropsy in the myocardium of patients with Kearns-Sayre syndrome. A patient with characteristics typical of Kearns-Sayre syndrome and a complete heart block is described. Southern blot analysis showed a deletion of 3·3 kb in the mitochondrial DNA in an endomyocardial biopsy specimen and in skeletal muscle. The deletion led to the disappearance of the genes for four transfer RNAs and four subunits of complex I (NADH:ubiquinone oxidoreductase) in the mitochondrial respiratory chain. The defect could not be demonstrated in whole blood despite amplification of the mitochondrial DNA region of interest by the polymerase chain reaction technique. There can be heteroplasmy—that is, normal and abnormal mitochondrial DNA populations in one cell—in different tissues, and the degree of heteroplasmy may be crucial in the development of organ-specific symptoms. This patient raises the possibility that some tissues can be specifically enriched with mitochondria with DNA defects and emphasises the need for elective sampling of the target tissue and polymerase chain reaction technique to exclude these defects. The role of mitochondrial DNA defects in idiopathic cardiomyopathies could perhaps be studied by analysis of mitochondrial DNA from endomyocardial biopsy specimens.     

Mitochondrial DNA deletion diagnosed by analysis of an endomyocardial biopsy specimen from a patient with Kearns-Sayre syndrome and complete heart block

Defects of mitochondrial DNA have been found at necropsy in the myocardium of patients with Kearns-Sayre syndrome. A patient with characteristics typical of Kearns-Sayre syndrome and a complete heart block is described. Southern blot analysis showed a deletion of 3·3 kb in the mitochondrial DNA in an endomyocardial biopsy specimen and in skeletal muscle. The deletion led to the disappearance of the genes for four transfer RNAs and four subunits of complex I (NADH:ubiquinone oxidoreductase) in the mitochondrial respiratory chain. The defect could not be demonstrated in whole blood despite amplification of the mitochondrial DNA region of interest by the polymerase chain reaction technique. There can be heteroplasmy—that is, normal and abnormal mitochondrial DNA populations in one cell—in different tissues, and the degree of heteroplasmy may be crucial in the development of organ-specific symptoms. This patient raises the possibility that some tissues can be specifically enriched with mitochondria with DNA defects and emphasises the need for elective sampling of the target tissue and polymerase chain reaction technique to exclude these defects. The role of mitochondrial DNA defects in idiopathic cardiomyopathies could perhaps be studied by analysis of mitochondrial DNA from endomyocardial biopsy specimens.     

Detection of "deleted" mitochondrial genomes in cytochrome-c oxidase-deficient muscle fibers of a patient with Kearns-Sayre syndrome

Using in situ hybridization and immunocytochemistry, we studied a muscle biopsy sample from a patient with Kearns-Sayre syndrome (KSS) who had a deletion of mitochondrial DNA (mtDNA) and partial deficiency of cytochrome-c oxidase (COX; EC 1.9.3.1). We sought a relationship between COX deficiency and abnormalities of mtDNA at the single-fiber level. COX deficiency clearly correlated with a decrease of normal mtDNA and, conversely, deleted mtDNA was more abundant in COX-deficient fibers, especially ragged-red fibers. The distribution of mtRNA had a similar pattern, suggesting that deleted mtDNA is transcribed. Immunocytochemistry showed that the nuclear DNA-encoded subunit IV of COX was present but that the mtDNA-encoded subunit II was markedly diminished in COX-deficient ragged-red fibers. Because the mtDNA deletion in this patient did not comprise the gene encoding COX subunit II, COX deficiency may have resulted from lack of translation of mtRNA encoding all three mtDNA-encoded subunits of COX.     

Mitochondrial DNA deletion diagnosed by analysis of an endomyocardial biopsy specimen from a patient with Kearns-Sayre syndrome and complete heart block.

Defects of mitochondrial DNA have been found at necropsy in the myocardium of patients with Kearns-Sayre syndrome. A patient with characteristics typical of Kearns-Sayre syndrome and a complete heart block is described. Southern blot analysis showed a deletion of 3.3 kb in the mitochondrial DNA in an endomyocardial biopsy specimen and in skeletal muscle. The deletion led to the disappearance of the genes for four transfer RNAs and four subunits of complex I (NADH:ubiquinone oxidoreductase) in the mitochondrial respiratory chain. The defect could not be demonstrated in whole blood despite amplification of the mitochondrial DNA region of interest by the polymerase chain reaction technique. There can be heteroplasmy--that is, normal and abnormal mitochondrial DNA populations in one cell--in different tissues, and the degree of heteroplasmy may be crucial in the development of organ-specific symptoms. This patient raises the possibility that some tissues can be specifically enriched with mitochondria with DNA defects and emphasises the need for elective sampling of the target tissue and polymerase chain reaction technique to exclude these defects. The role of mitochondrial DNA defects in idiopathic cardiomyopathies could perhaps be studied by analysis of mitochondrial DNA from endomyocardial biopsy specimens.     

Comorbidities and factors influencing frequent gout attacks in patients with gout: a cross-sectional study

Clinical Rheumatology - The objective was to identify the comorbidities of gout, to compare gender difference and independent factors of frequent gout attacks (&gt; 20 times). Demographic,...     

Restless legs syndrome: a common disorder in patients with rheumatologic conditions

OBJECTIVES: To review the symptoms, differential diagnosis, and treatment of the restless legs syndrome (RLS), and its relevance within rheumatologic practice. METHODS: Review of the scientific literature on RLS to summarize symptom presentation, burden, diagnosis, treatment, and association with rheumatologic conditions. RESULTS: RLS is a sensorimotor neurological disorder characterized by an irresistible urge to move the legs, usually accompanied or caused by unpleasant sensations within the legs. These sensations are sometimes described as achy or painful. They may cause sleep disruption and impair quality of life. RLS may be primary, of unknown etiology, with a likely genetic basis, or secondary, provoked by other conditions. Secondary RLS often improves when the underlying condition is treated or resolves. Since RLS is common in rheumatologic disorders such as rheumatoid arthritis or Sj?gren's syndrome, rheumatologists need to be familiar with the condition. Primary care physicians may misattribute RLS symptoms to other conditions and refer patients to specialists for treatment. Since RLS symptoms can be similar to, and mistaken for, symptoms in rheumatologic diseases, patients may be referred to rheumatologists. Therefore, it is important that rheumatologists be able to recognize, differentiate, diagnose, and treat RLS. CONCLUSIONS: The clinical diagnosis of RLS is based on 4 essential diagnostic criteria related to the urge to move that characterizes this disorder. Beyond good sleep hygiene and behavioral measures, dopaminergic agents are first-line treatments for primary RLS. Anticonvulsants, opioids, and sedative/hypnotics may also have a role in management.     

Left common carotid artery arising from the pulmonary artery in a patient with DiGeorge syndrome.

A female infant, born at 33 weeks' gestation with tetralogy of Fallot, died of severe perinatal asphyxia 6 hours after birth. Necropsy disclosed two associated vascular anomalies: a right aortic arch with a left common carotid artery arising from the pulmonary artery (isolated left common carotid artery) and an aberrant left subclavian artery arising from the descending aorta. Agenesis of the thymus and parathyroid gland was also found, suggesting that the child also had DiGeorge syndrome. Origin of the left common carotid artery from the pulmonary artery is exceedingly rare. When planning surgical treatment it is important to be aware of the possibility of this anomaly occurring in association with congenital heart disease, particularly in the presence of tetralogy of Fallot, right sided aortic arch, or DiGeorge syndrome.