Transforming growth factor-β (TGF-β) activity in urine of patients with glomerulonephritis is related to their renal functional and structural changes

SUMMARY: Transforming growth factor-β (TGF-β) has been considered the principal cytokine involved in the pathogenesis of renal fibrosis. In the present study, we evaluated TGF-β activity in occasional samples from 22 normal individuals and 29 patients (11 with focal glomerulosclerosis, 11 with membranous nephropathy, five with Berger disease, one with type I membranoproliferative glomerulonephritis and one with postinfectious glomerulonephritis) using a CCL-64 mink lung cell growth inhibition assay.
A significantly increased urinary TGF-β activity (reported in relation to urine creatinine, Ucreat, and median) was observed in patients with glomerulonephritis compared with normal individuals ( P <0.01). the patients with Berger disease [median (Md) = 9.96/10 μg Ucreat.], membranous glomerulonephritis (Md = 7.23/10 μg Ucreat.) and focal glomerulosclerosis (Md = 16.6/10 μg Ucreat.) showed higher urinary TGF-β than normal individuals (Md = 1.09/10 μg Ucreat.) ( P <0.01). We found a positive correlation between the TGF-β activity in the urine of these patients and the incidence of segmental glomerulosclerosis ( r = 0.45, P <0.05) and their plasma creatinine levels ( r = 0.87, P <0.01). A negative correlation was observed between the TGF-β activity in the urine of these patients and their creatinine clearance ( r =−0.75, P <0.01).
Our data suggest that measurement of urinary TGF-β activity could be a useful non-invasive procedure for the evaluation of renal TGF-β production, permitting the assessment of prognosis and the evaluation of therapeutic efficacy in patients with renal disease.     

Subcellular localization of transforming growth factor-alpha in human eosinophil granulocytes

Eosinophils are involved in the inflammatory response seen in allergy and helminthic infestations. Eosinophils synthesize transforming growth factor-alpha (TGF-alpha), which may play a role in the development of the characteristic fibrosis seen in longstanding high eosinophilia. Using immunoelectron microscopic techniques, eosinophils from peripheral blood of healthy individuals and from one patient with high eosinophilia showed presence TGF-alpha in matrix of the specific crystalloid-containing granules. In cryosections, TGF-alpha was also visualized in a vesicular compartment of the cytoplasm. In double- labeling experiments, the TGF-alpha of this latter compartment did not colocalize with CD63, a marker for lysosomes, nor with albumin of secretory vesicles. In extracts from eosinophils, obtained from healthy donors, immunoreactive TGF-alpha could be detected by enzyme-linked immunosorbent assay-technique. In addition, sera from two patients with high eosinophilia showed TGF-alpha concentrations of 1.5 ng/mL and 164 pg/mL, respectively, whereas TGF-alpha could not be detected in serum from healthy controls. In conclusion, TGF-alpha is present in the specific granules, and in an additional vesicular compartment of the cytoplasm of eosinophils.     

Transplantation potential of hematopoietic cells released into the circulation during routine chemotherapy for non-Hodgkin's lymphoma [see comments]

Primitive hematopoietic cells released into the peripheral blood (PB) were studied in 50 patients with high-grade non-Hodgkin's lymphoma enrolled in a phase III trial of intensive weekly chemotherapy (VAPEC- B) alone or with granulocyte colony-stimulating factor (G-CSF). Mononuclear cells numbers were monitored and their in vitro growth potential assessed in clonogenic progenitor cell assays and in long- term culture. Total colony-forming cells (granulocyte-macrophage [GM], burst-forming unit, erythroid [BFU-E], Mix-CFC) were increased 40-fold (median) over baseline with chemotherapy alone and 106-fold with chemotherapy and G-CSF after the final dose. CD34+ cells were increased to a median of 4%, equivalent to that in normal bone marrow (BM) controls. Circulating colony-forming cell levels were maximal when the recovering total white blood cell (WBC) count reached 5 to 10 x 10(9)/L. The timing of the maximum was reproducible in individual patients. Therefore the WBC count can be used as a guide to the timing of leukapheresis. PB cells from normal controls' and patients' prechemotherapy were unable to sustain hemopoiesis in two-stage long- term cultures. In contrast, PB cells collected from patients primed with chemotherapy alone or chemotherapy with G-CSF at the time of predicted maximal colony-forming cell release were able to generate and sustain hematopoiesis in long-term cultures at a level comparable or superior to normal BM. These findings indicate that the use of G-CSF after routine outpatient chemotherapy stimulates maximal release of primitive hemopoietic cells into the circulation, including colony- forming cells and long-term culture-initiating cells. Their numbers are comparable with those in normal BM and are such that a single leukapheresis will usually yield enough cells for hemopoietic reconstitution after myeloablative chemotherapy.     

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

Background and purpose:

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A_2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine.

Experimental approach:

The trafficking of the wild type A_2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.

Key results:

The wild type A_2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln³²⁵-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu³³⁰-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln³²⁵-stop, Ser³²⁶-stop and Phe³²⁸-stop receptors. Following internalization, the wild type A_2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln³²⁵-stop receptor did not recycle.

Conclusions and implications:

Deletion of the COOH-terminus of the A_2B adenosine receptor beyond Leu³³⁰ switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A_2B adenosine receptor following prolonged agonist addition.