HIV-induced IL-6/IL-10 dysregulation of CD4 cells is associated with defective B cell help and autoantibody formation against CD4 cells

To analyse CD4 cell cytokine secretion and helper/suppressor function at a clonal level we established 446 CD4⁺ T cell clones (TCC) in four healthy controls, three HIV^? haemophilia patients, four CDC II,III and four CDC IV patients. Spontaneous TCC secretion of Th1 cytokines (IL-2, interferon-gamma (IFN-γ)) and Th2 cytokines (IL-4, IL-6, IL-10) was determined by ELISA. TCC helper and suppressor functions were tested in a pokeweed mitogen (PWM)-stimulated allogeneic co-culture system using a reverse haemolytic plaque assay for assessment of B cell responses. There was a significant association of TCC surface marker expression (Leu-8, CD45RA) with TCC IL-6 secretion in healthy controls (P < 0.01), HIV^? patients (P 0.001) and CDC II,III patients (P 0.01) but not in CDC IV patients. Likewise, TCC expression of Leu-8 and CD45RA was significantly associated with TCC suppressor function in healthy controls (P 0.0005) but not in HIV-infected patients. A reduced TCC helper frequency (10% of TCC) and an enhanced TCC suppressor frequency (> 80% of TCC) were detected only in those HIV-infected patients who showed an excessively increased TCC IL-6 secretion (> 70% of TCC) together with a significantly diminished TCC IL-10 secretion (10% of TCC). CD4 cell autoantibodies also were found only in patients with this type of cytokine dysregulation. These data indicate that CD4 cell surface markers lose their functional relevance in HIV-infected patients. HIV-induced IL-6/IL-10 dysregulation of CD4⁺ T cells, i.e. the up-regulation of spontaneous IL-6 and down-regulation of spontaneous IL-10 secretion, appears to be involved in inducing CD4 helper defects and may promote autoantibody formation against CD4 cells.     

A prospective observational study of the rapid detection of clinically-relevant plasma direct oral anticoagulant levels following acute traumatic injury

In urgent clinical situations, such as trauma, urgent surgery or before thrombolysis, rapid quantification of direct oral anticoagulant plasma drug levels is warranted. Using the ClotPro® analyser, we assessed two novel viscoelastic tests for detection of clinically-relevant plasma drug levels in trauma patients. The ecarin clotting time was used to assess the plasma concentration of dabigatran and Russell´s viper venom clotting time to determine the plasma concentration of direct factor Xa inhibitors. In parallel, plasma concentrations were analysed using plasma-based chromogenic assays. A total of 203 simultaneous measurements were performed. Strong to very strong linear correlations were detected between ecarin clotting time and plasma concentration of dabigatran (r = 0.9693), and between Russell´s viper venom clotting time and plasma concentrations of apixaban (r = 0.7391), edoxaban (r = 0.9251) and rivaroxaban (r = 0.8792), all p < 0.001. An ecarin clotting time ≥ 189 seconds provided 100% sensitivity and 90% specificity for detecting plasma dabigatran concentrations ≥ 50 ng.ml^-1. Corresponding Russell´s viper venom clotting time cut-off values were ≥ 136 seconds for apixaban (80% sensitivity, 88% specificity), ≥ 168 seconds for edoxaban (100% sensitivity, 100% specificity) and ≥ 177 seconds for rivaroxaban (90% sensitivity, 100% specificity). Detection of drug levels ≥ 100 ng.ml^-1 was also investigated: for dabigatran, an ecarin clotting time ≥ 315 seconds yielded 92% sensitivity and 100% specificity; while Russell´s viper venom clotting time cut-offs of 191, 188 and 196 seconds were calculated for apixaban (67% sensitivity, 88% specificity), edoxaban (100% sensitivity, 75% specificity) and rivaroxaban (100% sensitivity, 91% specificity), respectively. We have demonstrated strong positive correlations between plasma drug levels and clotting time values in the specific ClotPro assays. Cut-off values for detecting clinically-relevant drug levels showed high levels of sensitivity and specificity.     

Haemostatic profile of reconstituted blood in a proposed 1:1:1 ratio of packed red blood cells,platelet concentrate and four different plasma preparations

The concept of haemostatic resuscitation implies early and high‐volume plasma transfusion. We investigated the haemostatic profile of reconstituted whole blood prepared in a 1:1:1 ratio of blood, platelets and plasma. This consisted of packed red blood cells, platelet concentrate and four different plasma variants: fresh frozen; solvent‐detergent; lyophilised quarantine; and lyophilised methylene blue‐inactivated plasma. Haematocrit, platelet count, endogenous thrombin potential and coagulation factor activity were significantly lower in reconstituted blood compared with citrated whole blood (p < 0.01). Except for lyophilised methylene blue‐inactivated plasma, no substantial differences between plasma variants in coagulation factor activity, endogenous thrombin potential and standard coagulation tests were observed. After reconstitution, haematocrit and platelet counts were slightly above recommended transfusion triggers, most thromboelastometry (ROTEM^®) parameters were within the normal range and fibrinogen concentrations were between 1.57 g.l^?1 and 1.91 g.l^?1. Reconstitution of whole blood in a 1:1:1 ratio resulted in significant dilution of haematocrit and platelet count, but values remained above limits recommended by transfusion guidelines. Fibrinogen concentrations of reconstituted whole blood were also significantly reduced, and these were below the threshold value for supplementation recommended by recent guidelines.     

High-calcium intake abolishes hyperoxaluria and reduces urinary crystallization during a 20-fold normal oxalate load in humans

Background: The aim of the study was to test whether increasing dietary calcium intake lowers intestinal oxalate absorption and thereby prevents hyperoxaluria and urinary crystallization during a 20-fold normal oxalate load in healthy subjects. Methods: Fourteen healthy male volunteers (age 23-44 years, BMI 21.5-27.7 kg/m²) collected 24-h urines while on free choice diet as well as on two standardized diets. The latter contained 2545 kcal, 2500 ml of mineral water, 102 g of protein, 13.6 g of sodium chloride and 2220 mg of oxalate ( 20-fold content of an average diet). Subjects were studied twice while on the standardized diet, once while eating a normal amount of calcium (1211 mg/day, oxalate-rich, diet), and once while eating 3858 mg of calcium /day (calcium and oxalate-rich diet). Results: Compared with the free-choice diet (322±36 &mgr;mol/d), UOx X V increased to 780±722 &mgr;mol/d on the oxalate-rich diet (P=0.001) and fell again to 326±31 &mgr;mol/d on calcium and oxalate-rich diet (P=0.001 vs oxalate-rich diet). Urinary glycolate (a metabolic precursor of Ox) always remained below the upper limit of the normal range and did not change between different diets, indicating that changes in UOx x V reflect respective variations in intestinal absorption of Ox. UCa x V was 4.60±0.45 mmol/d on the free choice diet and 3.20±0.32 mmol/d on the oxalate-rich diet (P=0.011 vs free-choice diet); it increased to 7.28±0.74 mmol/d on the calcium- and oxalate-rich diet (P=0.001 vs free-choice and oxalate-rich diets). As indicated by the AP (CaOx) index (Tiselius), urinary supersaturation did not vary significantly between the three diets. In freshly voided morning urines (studied in 8/14 subjects) on the oxalate-rich diet, CaOx crystals or crystal aggregates of up to 80 &mgr;m diameter were found in 5/8 urines, whereas this never occurred on the free-choice diet and only once on the calcium- and oxalate-rich diet. Conclusion: Increasing calcium intake while eating Ox-rich food prevents dietary hyperoxaluria and reduces CaOx crystallization in healthy subjects. This further illustrates that dietary counseling to idiopathic calcium-stone formers should ensure sufficient calcium intake, especially during oxalate-rich meals.     

Tamm-Horsfall protein in recurrent calcium kidney stone formers with positive family history: abnormalities in urinary excretion,molecular structure and function

Tamm-Horsfall protein (THP) powerfully inhibits calcium oxalate crystal aggregation, but structurally abnormal THPs from recurrent calcium stone formers may promote crystal aggregation. Therefore, increased urinary excretion of abnormal THP might be of relevance in nephrolithiasis. We studied 44 recurrent idiopathic calcium stone formers with a positive family history of stone disease (RCSF_fam) and 34 age- and sex-matched healthy controls (C). Twenty-four-hour urinary THP excretion was measured by enzyme linked immunosorbent assay. Structural properties of individually purified THPs were obtained from analysis of elution patterns from a Sepharose 4B column. Sialic acid (SA) contents of native whole 24-h urines, crude salt precipitates of native urines and individually purified THPs were measured. THP function was studied by measuring inhibition of CaOx crystal aggregation in vitro (pH 5.7, 200 mM sodium chloride). Twenty-four-hour urine excretion of THP was higher in RCSF_fam (44.0 ± 4.0 mg/day) than in C (30.9 ± 2.2 mg/day, P = 0.015). Upon salt precipitation and lyophilization, elution from a Sepharose 4B column revealed one major peak (peak A, cross-reacting with polyclonal anti-THP antibody) and a second minor peak (peak B, not cross-reacting). THPs from RCSF_fam eluted later than those from C (P = 0.021), and maximum width of THP peaks was higher in RCSF_fam than in C (P = 0.024). SA content was higher in specimens from RCSF_fam than from C, in native 24-h urines (207.5 ± 20.4 mg vs. 135.2 ± 16.1 mg, P = 0.013) as well as in crude salt precipitates of 24-h urines (10.4 ± 0.5 mg vs. 7.4 ± 0.9 mg, P = 0.002) and in purified THPs (75.3 ± 9.3 μg/mg vs. 48.8 ± 9.8 μg/mg THP, P = 0.043). Finally, inhibition of calcium oxalate monohydrate crystal aggregation by 40 mg/L of THP was lower in RCSF_fam (6.1 ± 5.5%, range −62.0 to +84.2%) than in C (24.9 ± 6.0%, range −39.8 to +82.7%), P = 0.022, and only 25 out of 44 (57%) THPs from RCSF_fam were inhibitory (positive inhibition value) vs. 25 out of 34 (74%) THPs from C, P < 0.05. In conclusion, severely recurrent calcium stone formers with a positive family history excrete more THP than healthy controls, and their THP molecules elute later from an analytical column and contain more SA. Such increasingly aggregated THP molecules predispose to exaggerated calcium oxalate crystal aggregation, an important prerequisite for urinary stone formation.     

Hydroxylation of dental zirconia surfaces: characterization and bonding potential

Bioinert zirconia surfaces exhibit a low chemical bonding potential to resin-based luting agents. The aim was to hydroxylate dental zirconia surfaces and to examine tensile bond strength using commercial luting agents. The measured bond strength was compared with established mechanical conditioning techniques. Five acidic and one alkaline hydroxylation pretreatments were applied and compared with air abrasion and tribochemical silica coating. For the chemical characterization of hydroxyl groups and hydroxyl value, zirconia powders were used, chemically modified, and analyzed using Fourier-transformed infrared spectroscopy and a titrimetric method according to the ISO 4629 standard. All acidic pretreatment procedures exhibited increased hydroxyl values. The highest values were recorded after treatment with phosphoric acid or Piranha solution. Tensile bond strength was examined in a universal testing machine using the commercial dual-cure luting agents Multilink (Ivoclar, Liechtenstein) and Panavia F2.0 (Kuraray, Japan). Surface hydroxylation with Piranha solution in combination with the luting agent Multilink led to a bond strength of 12.47 +/- 3.25 MPa. Tribochemical silica-coated/silanized zirconia surfaces with Multilink produced the highest bond strength of 19.33 +/- 3.65 MPa. Using the luting agent Panavia F2.0, statistically homogenous values for the untreated (11.60 +/- 1.68 MPa) and for the hydroxylated surface (12.46 +/- 3,81 MPa) were measured. Bioinert zirconia surfaces were successfully hydroxylated in terms of tensile bond strength. Resin bonding with Multilink can be strongly increased by acidic treatment with Piranha solution. Bonding with Panavia F2.0 is not affected by hydroxylation, which is likely due to the incorporation of specific functional monomers.     

In vitro cytokine responses in liver transplant recipients treated with cyclosporine A and tacrolimus

The inhibitory effect of Cyclosporine A (CsA) and Tacrolimus (Tacr) on interleukin 2 (IL-2) are well known, and the importance of Th1-type (IL-2, interferon gamma), and Th2-type (IL-4, IL-6, and IL-10) cytokine secretion in preventing allograft rejection is a controversial issue. The immunological mechanisms involved in long-term liver transplant recipients under CsA and Tacr were not studied precisely.
This study was designed to evaluate the effects of CsA and Tacr on the immune response of 62 long-term survivors following liver transplantation.
T-cell and B-cell subpopulations, the T helper (Th) cell activity, T-cell cytokine production, Staphylococcus aureus Cowan strain I (SAC-I)-stimulated B-cell responses and PWM-stimulated B-cell responses were examined.
CsA and Tacr decreased whole T-cell populations as well as CD4+T-cell IL-2 responses (p < 0.005, Tacr and p = 0.02, CsA), impaired CD4+ cell Th activity (p < 0.01, Tacr and p = 0.02, CsA) and reduced SAC-I-stimulated B-cell responses (immunoglobulin-secreting cells [ISC]: p = 0.001, Tacr and p < 0.05, CsA). A significantly impaired T-cell IL-10 secretion (p = 0.0001) and decreased Th function of whole T cells was found in Tacr-treated patients only, whereas unstimulated Th1 responses and SAC-I-stimulated B-cell IL-6 responses were reduced in CsA-treated patients.
Our data show that Tacr suppresses T-, CD4+-, and B-cell responses more effectively than CsA which may be relevant in the maintenance of long-term stable liver graft function.     

Study of biomimetic apatite formation on dentine surface

The aim of this study was investigation of the opportunity of biomimetic growth of apatite on a dentine surface at various methods of its processing. Artificial blood human plasma--simulated body fluid (SBF)--was used as a source of ions. According to the scanning electronic microscope (SEM) and confocal laser scanning microscope (CLSM) and energy dispersive X-ray analysis (EDX) they have revealed the growth of crystals of calcium--deficient apatite. The structure of the formed apatite layer differed depending on a kind of processing of a dentine surface. After acid etching the globules of the apatite are located chaotically and the part of a dentine surface remains free. The removal of the collagen fibres by NaClO promotes uniform, controllable growth of crystals, forming a monolithic layer. The hypermineralisated areas of a tooth create the best conditions for growth. The transformation hydroxyl apatite in a superficial dentine layer can be caused by increasing of pressure at preparing with diamond bur. The growth of crystals of brushite in the smear layer is possible at a storage in water at 37 degrees capital ES, Cyrillic. The crystals have lamellar form and are well integrated in a dentin surface.     

Superior 3-year kidney graft function in patients with impaired pretransplant Th2 responses

Abstract In a prospective study of 80 patients, we investigated the association of kidney graft rejection with pretransplant CD4 helpersuppressor function, B cell responses, and in vitro cytokine secretion. A pokeweed mitogen-driven allogeneic coculture system was used to assess CD4 helper/suppressor function and T cell-dependent B cell responses. SAC I was used for T celland monocyte-independent stimulation of B cell cultures. B cell differentiation was assessed in a reverse hemolytic plaque assay. ELISAs were used to determine in vitro cytokine secretion. None of the 12 patients with pretransplant CD4 helper defects (<10% helper activity) had acute rejection episodes in contrast to 32 of 68 (47%) patients with normal pretransplant CD4 helper function ( P = 0.001). Patients with pretransplant CD4 helper defects exhibited better 3-year graft function than patients without CD4 helper defects (serum creatinine of functioning grafts: 1.2 ± 0.1 mg/dl compared to 1.7 ± 0.1 mg/dl, P < 0.05). Low pretransplant IL-10 responses (<100 pg/ml; 14/80 patients) were significantly associated with a low incidence of acute rejection episodes ( P < 0.01) and good 3- year graft function ( P < 0.05). These data show that impaired pretransplant Th2 responses-CD4 help and IL-10 responses-predict a low risk of kidney graft rejection and good 3-year graft function, whereas Th1 (IL-2, IFN-γ) and B cell/monocyte responses are not of predictive value.     

Simultaneous measurements of calcium oxalate crystal nucleation and aggregation: impact of various modifiers

Rates of nucleation and aggregation of calcium oxalate crystals were derived from 20-min time course measurements of OD₆₂₀ after mixing solutions containing CaCl₂ and K₂C₂O₄ at 37°C, pH 5.7, ionic strength (IS) 0.21, with constant stirring (500 rpm); final assay concentrations were 4.25 mM calcium and 0.5 mM oxalate, respectively. The maximum increase of OD₆₂₀ with time, termed S _N, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, OD₆₂₀ progressively decreases despite ionized calcium staying constant and no new particles being formed, due to crystal aggregation. Rate of aggregation, S _A, is derived from the maximum decrease in OD₆₂₀ with time. S _N and S _A are not independent, as indicated by a positive correlation (r=0.844, P=0.0001). Among the modifiers studied, citrate at 0.5–2.5 mM lowered both S _N and S _A in a concentration-dependent manner (P<0.01 for all comparisons vs control). Chondroitin-6-sulfate at 6.25–25 mg/l moderately lowered S _N, whereas it strongly inhibited aggregation (P<0.01 vs control). At 6.8–20.4 mg/l, albumin did not affect nucleation, whereas it inhibited aggregation in a concentration-dependent manner (P<0.005 vs control for all comparisons).