CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in ...plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin.hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin.hemoglobin complexes.
Glycation reactions, such as those seen in late diabetes, can be mimicked in purely chemical systems. The glycation is time-dependent, and in in vitro systems it can continue for days. Ascorbate ...seems to enhance the reactions. The reactions are associated with free-radical formation through transformation of an Amadori product to (deoxy-)glycoson, catalysed by heavy metals. Ascorbate enhances the reaction by a factor of 5-10 compared with in vitro systems without ascorbate. In vitro systems containing bovine serum albumin retard the formation of free-radicals, because of the formation of advanced glycation products.
A previous study has shown that malnourished, clinically stable patients with liver cirrhosis are in protein and energy balance at their spontaneous dietary intake and that an improvement in ...nutritional status cannot be anticipated at this intake (Nielsen et al. 1993). In the present study we examined to what extent oral intake could be increased by nutritional support, and to what extent dietary protein would be retained with increased intake. The techniques used for balance studies were also validated since this information is not available for patients with liver cirrhosis. Fifteen malnourished patients with alcoholic liver cirrhosis were given increasing amounts of a balanced ordinary diet for 38 (SE 3) d. Intakes of protein and energy were recorded by weighing servings and leftovers on food trays. Protein intake was calculated from food tables. Total N disposal was calculated after measurement of urinary N excretion, and protein balance was calculated from the N balance. A validation study of protein balance in a subgroup of patients (analysis of N in food by the duplicate portion technique, correction for incomplete recovery of urine by measurement of urinary para-aminobenzoic acid (PABA) after administration of PABA tablets, and measurement of faecal N) did not change protein balance values. Protein intake increased from 1.0 (SE 0.1) g/kg per d to 1.8 (SE 0.1) g/kg per d. With increasing protein intake, 84 (SE 8)% of the increase in intake was retained. The rate of protein retention was not saturated at the intakes obtained in this study. Protein intolerance was only encountered in one patient. Available evidence indicates that the requirement for achieving N balance is increased in these patients but protein retention is highly efficient with increased intake. Protein retention is dependent on energy balance. Energy intake was calculated from food tables and total energy expenditure was calculated by the factorial method. A validation study was performed in a subgroup of patients. The energy contents of food sampled by the duplicate portion technique, and of urine and faeces were measured by bomb calorimetry. Resting energy expenditure (REE) was measured by indirect calorimetry before and at the end of the study, and O2 uptake during bicycle exercise was measured before and at the end of the study. The measured intake of metabolizable energy was on average 13% lower than the value given in food tables. Calculated energy expenditure was not changed by the validation study.
Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a ...sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay with a functional luciferase readout, we demonstrate that parameters such as peptide nucleic acid (PNA) charge and the method of electroporation have dramatic influence on the efficiency of productive delivery. In a suspended cell electroporation system (cuvettes), a positively charged PNA (+8) was most efficiently transferred, whereas charge neutral PNA was more effective in a microtiter plate electrotransfer system for monolayer cells. Surprisingly, a negatively charged (-23) PNA did not show appreciable activity in either system. Findings from the functional assay were corroborated by pulse parameter variations, polymerase chain reaction, and confocal microscopy. In conclusion, we have found that the charge of PNA and electroporation system combination greatly influences the transfer efficiency, thereby illustrating the complexity of the electroporation mechanism.
Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually ...accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.
Frequency-selective chemical shift magnetic resonance (MR) imaging was applied on the calf musculature and the abdomen of a patient with acquired generalized lipoatrophy (AGL; Lawrence syndrome), a ...very rare syndrome affecting selectively several types of adipose tissue accompanied by alterations in glucose and energy metabolism. In addition, (1)H-MRS was used for assessment of intra- (IMCL) and extramyocellular lipid stores (EMCL) in the skeletal musculature of the calf. Results from the AGL patient were compared with an age-matched group of five healthy volunteers. Fat-selective imaging of the calf revealed a total lack of subcutaneous adipose tissue. No EMCL signal was found in the spectra from the soleus muscle of the AGL patient. IMCL signals were present in the spectra but were clearly lower than in the controls (14% of normal value in the soleus muscle). In abdominal images, subcutaneous fat signal was not detectable, as in the calf, but nearly normal conditions were shown for visceral adipose tissue between abdominal organs. Fat-selective images showed the liver with high signal intensity, indicating hepatic steatosis combined with hepatosplenomegaly. Modern chemical shift-selective MR imaging and localized spectroscopy allow a noninvasive and quantitative assessment of tissue composition in patients with disorders of carbohydrate and lipid metabolism.
: During intravascular hemolysis hemoglobin (Hb) binds to haptoglobin (Hp) leading to endocytosis of the complex by the macrophage receptor, CD163. In the present study, we used a phage‐display Fab ...antibody strategy to explore if the complex formation between Hp and Hb leads to exposure of antigenic epitopes specific for the complex. By Hp–Hb‐affinity screening of a phage‐Fab library, we isolated a phage clone against the ligand complex. Surface plasmon resonance analyses of the Fab part expressed as a recombinant protein revealed a high affinity binding (KD = 3.9 nm) to Hp–Hb, whereas no binding was measured for non‐complexed Hp or Hb. The Fab antibody completely inhibited the binding of 125I‐labeled Hp–Hb complexes to CD163 and blocked their uptake in CD163‐transfected cells. In conclusion, we have raised a receptor‐blocking antibody specifically recognizing the Hp–Hb complex. In addition to provide new insight into the changes occurring when Hp and Hb bind, the present study provides a new potential tool for measuring and removal of Hp–Hb complexes from plasma/serum.
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