A longitudinal study of the relationship between galactosylation degree of IgG and rheumatoid factor titer and avidity during long-term immunization of rabbits with BSA

Although IgG with reduced content of galactose has been implicated as important in the autoimmune rheumatoid factor (RF) response in rheumatoid arthritis (RA), relatively little is known about the temporal relationship between RF and the degree of galactosylation of IgG in vivo. We established an experimental model for studying the dynamic association between changes in the relative extent of galactosylation of IgG antigen(s) and the main parameters of RF activity, such as the titer, specificity and functional affinity/avidity. Rabbits hyperimmunized with BSA were used for examining the influence of long-term antigenic stimulation on the galactosylation status of IgG and rheumatoid factor production. The results showed that the galactosylation profile of IgG varied during the humoral anti-BSA response in rabbits and that the accompanying RF response fluctuated in titer and binding avidity for differently galactosylated IgG. The immune complexes (IC) were found to be composed of differently galactosylated IgG differing in capacity to inhibit the agglutination activity of RF. Moreover, the ability of circulating RF to react avidly with rather small IC was associated with a lower content of galactose in complexed IgG. The results suggest that a certain dynamic relationship exists between the oligosaccharide moiety of IgG and the titer and avidity of RF during the normal anti-BSA response of rabbits.     

Adhesion capacity and integrin expression by dendritic-like cells generated from acute myeloid leukemia blasts by calcium ionophore treatment

OBJECTIVE: Dendritic cells (DC) play a key role in initiation of immune responses. In vitro modified acute myeloid leukemia (AML) blasts acquire certain specific features of DC and are suggested as a potential source of anti-leukemia vaccines. AML-DC have been characterized in terms of costimulatory molecule expression and cytokine production. In contrast, migratory capacity of AML-DC, which is a major attribute of DC required for their in vivo function, remains unknown. Here we present data on adhesion properties and profile of integrin expression of AML-DC. MATERIALS AND METHODS: Blasts from nine patients were used to generate AML-DC by calcium ionophore treatment. Adhesion of AML-DC to the major components of the extracellular matrix and the profile of integrin expression was studied using flow cytometry. RESULTS: Similar to their normal counterparts, calcium ionophore-induced AML-DC acquired the ability to bind to fibronectin and in 4 of 7 studied cases to bind to denatured collagen. Adhesion to native collagen remained unchanged during DC-type differentiation of AML blasts. AML-DC and DC obtained from monocytes of healthy donors expressed CD49d, CD49e, alphavbeta3, and alphavbeta5. However, AML-DC from 3 of 8 patients down-regulated CD49d, which plays an important role in cell-to-cell and cell-to-matrix interactions and normally is coexpressed with CD83. CONCLUSION: The results provide further evidence that AML blasts can be induced to display functional properties characteristic for DC and may prove useful for in vivo delivery and presentation of tumor antigens to the immune system. Abnormal CD49d expression and variability in AML-DC adhesion to denatured collagen indicate that motility of AML-DC from individual patients may vary, and a customized approach is essential for evaluating leukemic cell feasibility for vaccine design.     

Distinct cytotoxic mechanisms of pristine versus hydroxylated fullerene.

The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C60) and water-soluble polyhydroxylated fullerene [C60(OH)n] were investigated. Crystal violet assay for cell viability demonstrated that nano-C60 was at least three orders of magnitude more toxic than C60(OH)n to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines. Flow cytometry analysis of cells stained with propidium iodide (PI), PI/annexin V-fluorescein isothiocyanate, or the redox-sensitive dye dihydrorhodamine revealed that nano-C60 caused rapid (observable after few hours), reactive oxygen species (ROS)-associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C60(OH)n caused delayed, ROS-independent cell death with characteristics of apoptosis, including DNA fragmentation and loss of cell membrane asymmetry in the absence of increased permeability. Accordingly, the antioxidant N-acetylcysteine protected the cell lines from nano-C60 toxicity, but not C60(OH)n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C60(OH)n-induced apoptosis, but not nano-C60-mediated necrosis. Finally, C60(OH)n antagonized, while nano-C60 synergized with, the cytotoxic action of oxidative stress-inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C60 that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C60 seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death.     

Thermodynamic aspects of solubility process of some sulfonamides

The thermodynamic aspects of solubility process of sulfonamides with the general structures C₆H₅–SO₂NH–C₆H₄–R (R = 4-NO₂; 4-Cl) and 4-NH₂–C₆H₄–SO₂NH–C₆H₄–R (R = 4-NO₂; 4-CN; 4-Cl; 4-OMe; 4-C₂H₅) in water, phosphate buffer with pH 7.4 and n-octanol (as phases modeling various drug delivery pathways) were studied using the isothermal saturated method.     

Markers of lipid oxidative damage in the exhaled breath condensate of nano TiO2 production workers

Nanoscale titanium dioxide (nanoTiO₂) is a commercially important nanomaterial. Animal studies have documented lung injury and inflammation, oxidative stress, cytotoxicity and genotoxicity. Yet, human health data are scarce and quantitative risk assessments and biomonitoring of exposure are lacking. NanoTiO₂ is classified by IARC as a group 2B, possible human carcinogen. In our earlier studies we documented an increase in markers of inflammation, as well as DNA and protein oxidative damage, in exhaled breath condensate (EBC) of workers exposed nanoTiO₂. This study focuses on biomarkers of lipid oxidation. Several established lipid oxidative markers (malondialdehyde, 4-hydroxy-trans-hexenal, 4-hydroxy-trans-nonenal, 8-isoProstaglandin F2α and aldehydes C₆–C₁₂) were studied in EBC and urine of 34 workers and 45 comparable controls. The median particle number concentration in the production line ranged from 1.98?×?10⁴ to 2.32?×?10⁴ particles/cm³ with ～80% of the particles?<100?nm in diameter. Mass concentration varied between 0.40 and 0.65?mg/m³. All 11 markers of lipid oxidation were elevated in production workers relative to the controls (p?2 and markers of lipid oxidation in the EBC. These markers were not elevated in the urine samples. Lipid oxidation in the EBC of workers exposed to (nano)TiO₂ complements our earlier findings on DNA and protein damage. These results are consistent with the oxidative stress hypothesis and suggest lung injury at the molecular level. Further studies should focus on clinical markers of potential disease progression. EBC has reemerged as a sensitive technique for noninvasive monitoring of workers exposed to engineered nanoparticles.     

Urinary Plasmin Inhibits TRPV5 in Nephrotic-Range Proteinuria

Urinary proteins that leak through the abnormal glomerulus in nephrotic syndrome may affect tubular transport by interacting with membrane transporters on the luminal side of tubular epithelial cells. Patients with nephrotic syndrome can develop nephrocalcinosis, which animal models suggest may develop from impaired transcellular Ca²⁺ reabsorption via TRPV5 in the distal convoluted tubule (DCT). In nephrotic-range proteinuria, filtered plasminogen reaches the luminal side of DCT, where it is cleaved into active plasmin by urokinase. In this study, we found that plasmin purified from the urine of patients with nephrotic-range proteinuria inhibits Ca²⁺ uptake in TRPV5-expressing human embryonic kidney 293 cells through the activation of protease-activated receptor-1 (PAR-1). Preincubation with a plasmin inhibitor, a PAR-1 antagonist, or a protein kinase C (PKC) inhibitor abolished the effect of plasmin on TRPV5. In addition, ablation of the PKC phosphorylation site S144 rendered TRPV5 resistant to the action of plasmin. Patch-clamp experiments showed that a decreased TRPV5 pore size and a reduced open probability accompany the plasmin-mediated reduction in Ca²⁺ uptake. Furthermore, high-resolution nuclear magnetic resonance spectroscopy demonstrated specific interactions between calmodulin and residues 133–154 of the N-terminus of TRPV5 for both wild-type and phosphorylated (S144pS) peptides. In summary, PAR-1 activation by plasmin induces PKC-mediated phosphorylation of TRPV5, thereby altering calmodulin-TRPV5 binding, resulting in decreased channel activity. These results indicate that urinary plasmin could contribute to the downstream effects of proteinuria on the tubulointerstitium by negatively modulating TRPV5.In the kidney, the fine regulation of Ca²⁺ balance occurs through the activity of the epithelial Ca²⁺ channel TRPV5.¹ TRPV5 is mostly expressed in the distal convoluted tubule (DCT) and connecting tubule of the nephron, where it constitutes the apical entry mechanism for transcellular Ca²⁺ reabsorption. TRPV5 is a constitutively active ion channel that bears unique electrophysiologic characteristics, including calmodulin (CaM) and Ca²⁺-dependent inactivation and high selectivity for Ca²⁺.^2,3 The activity of TRPV5 is tightly controlled at multiple levels by an array of different factors, including parathyroid hormone and the serine protease tissue kallikrein. Both parathyroid hormone and tissue kallikrein initiate the phosphorylation of TRPV5 through the cAMP/protein kinase A (PKA) and phospholipase C (PLC)/ diacylglycerol (DAG)/protein kinase C (PKC) signaling cascades, respectively.^4,5Proteinuria is a hallmark of nephrotic syndrome, in which large plasma proteins pass through the disrupted glomerular basement membrane (GBM). Pathologic leakage of glomerular proteins causes multiple tubulointerstitial abnormalities, such as interstitial inflammation and eventually fibrosis, but does not affect tubular structure.⁶ However, recent data showed that tubular transport processes could be affected by direct effect of urinary protein on membrane transporters at the luminal side of the DCT, such as epithelial sodium channel.⁷ The observation of nephrocalcinosis in patients with nephrotic-range proteinuria,^8,9 associated with impaired transcellular Ca²⁺ reabsorption via TRPV5 in a mouse model,¹⁰ suggests that proteinuria might affect tubular Ca²⁺ handling.Patients with nephrotic syndrome (NS) have elevated serum plasminogen levels.¹¹ After leakage into the urine, plasminogen is converted into active plasmin by tubular urokinase-type plasminogen activator and has recently been reported to regulate renal ion transport in nephrotic syndrome by effects on the epithelial sodium channel.^7,12 Whether urinary plasmin also can affect tubular Ca²⁺ handling and, if so, by which mechanisms, has not been established so far. This study aimed to investigate the effects and mechanism of urinary plasmin on TRPV5-mediated Ca²⁺ reabsorption.     

High salt intake causes adverse fetal programming--vascular effects beyond blood pressure

Background High salt intake causes hypertension, adverse cardiovascular outcomes and potentially also blood pressure (BP)-independent target organ damage. Excess salt intake in pregnancy is known to affect BP in the offspring. The present study was designed to assess whether high salt intake in pregnancy affects BP and vascular morphology in the offspring. Methods Sprague-Dawley rats were fed a standard rodent diet with low-normal (0.15%) or high (8.0%) salt content during pregnancy and lactation. After weaning at 4 weeks of age, offspring were maintained on the same diet or switched to a high- or low-salt diet, respectively. Vascular geometry was assessed in male offspring at 7 and 12 weeks postnatally. Results Up to 12 weeks of age, there was no significant difference in telemetrically measured BP between the groups of offspring. At 12 weeks of age, wall thickness of central (aorta, carotid), muscular (mesenteric) and intrapulmonary arteries was significantly higher in offspring of mothers on a high-salt diet irrespective of the post-weaning diet. This correlated with increased fibrosis of the aortic wall, more intense nitrotyrosine staining as well as elevated levels of marinobufagenin (MBG) and asymmetric dimethyl arginine (ADMA). Conclusions High salt intake in pregnant rats has long-lasting effects on the modeling of central and muscular arteries in the offspring independent of postnatal salt intake and BP. Circulating MBG and ADMA and local oxidative stress correlate with the adverse vascular modeling.     

Adult-onset autosomal dominant leukodystrophy without early autonomic dysfunctions linked to lamin B1 duplication: a phenotypic variant

The early presentation of autonomic dysfunctions at the disease onset has been considered the mandatory clinical feature in adult-onset autosomal dominant leukodystrophy, which is a rarely recognised leukodystrophy caused by duplication of the lamin B1 gene. We report the first family with adult-onset autosomal dominant leukodystrophy and lamin B1 duplication, without the distinguishing early-appearing autonomic dysfunctions. Subjects from three consecutive generations of a multi-generational Serbian family affected by adult-onset autosomal dominant leukodystrophy underwent clinical, biochemical, neurophysiological, neuroradiological, and genetic studies. The patients atypically exhibited late autonomic dysfunctions commencing at the disease end-stages in some. Genetic findings of lamin B1 duplication verified adult-onset autosomal dominant leukodystrophy, which was supported also by neuroimaging studies. Exclusively, proton magnetic spectroscopy of the brain revealed a possibility of neuro-axonal damage in the white matter lesions, while magnetic resonance imaging of the spinal cord excluded spinal myelin affection as a required finding in this leukodystrophy. The detection of lamin B1 duplication, even when autonomic dysfunctions do not precede the other symptoms of the disease, proves for the first time that lamin B1-duplicated adult-onset autosomal dominant leukodystrophy may have a phenotypic variant with delayed autonomic dysfunctions. Prior to this report, such a phenotype had been speculated to represent an entity different from lamin B1-duplicated leukodystrophy. Hereby we confirm the underlying role of lamin B1 duplication, regardless of the autonomic malfunction onset in this disorder. It is the only report on adult-onset autosomal dominant leukodystrophy from Southeastern Europe.     

Ribavirin shows immunomodulatory effects on activated microglia

Ribavirin (RBV) is synthetic purine nucleoside analogue, licensed as anti-viral drug that displays immunomodulatory actions on various immune cells. Our previous ex vivo studies have demonstrated immunosuppressive effects of RBV on reactive T-lymphocytes in experimental autoimmune encephalomyelitis. Here, we examined the effects of RBV on inflammatory response of microglia. RBV potency to down-regulate microglia inflammatory response was assessed by measuring microglia cell body size, and the production of nitric oxide (NO) and pro- and anti-inflammatory cytokines. RBV exerted cytotoxic effects on LPS-stimulated microglia, leaving non-stimulated microglia unaffected. The exposure of activated microglia to RBV led to: decrease in the level of NO as a result of decreased cell number, lower average cell surface, the reduction of membrane ruffling, the suppression of interleukin-6 release and promoted interleukin-10 production. On the other hand, RBV promoted LPS-induced interleukin-1 beta release. Our results imply that RBV is a complex immunomodulator showing both anti- and pro-inflammatory effects on activated microglia.