Elevated tryptase, nerve growth factor, neurotrophin-3 and glial cell line-derived neurotrophic factor levels in the urine of interstitial cystitis and bladder cancer patients.

PURPOSE: The 2 prominent features of interstitial cystitis are pain and increased numbers of mast cells in the bladder. In this pilot study we determined the concentration of soluble mediators associated with activation of sensory neurons and/or mast cells that were present in the urine. MATERIALS AND METHODS: The study groups included 4 interstitial cystitis patients, 7 kidney donors with no history of bladder disease as negative controls, 6 bladder cancer patients and 7 patients with urinary tract infection as reference controls. Urine samples were assayed for different soluble mediators using immunoassays for tryptase (a marker for mast cell activation), neurotrophic factors (markers of neuronal plasticity) and chemokines (markers of inflammatory cell activity). Results were normalized based on creatinine concentration. RESULTS: There was a marked increase in the average amounts of tryptase and 3 neurotrophic factors in patient urine. Interestingly, the mediator profile in the urine of bladder cancer patients was indistinguishable from that of interstitial cystitis patients with respect to these same 4 proteins. There was no difference between normal control and urinary tract infection urine samples. CONCLUSIONS: These findings may account for several clinical and pathological features found in interstitial cystitis and bladder cancer. Although preliminary due to the limited numbers of patients, they also suggest that increased levels of neurotrophin-3, nerve growth factor, glial cell line-derived neurotrophic factor and tryptase in the urine could serve as a basis for adjunct diagnosis, monitoring and treatment of interstitial cystitis.

Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme.

Human mast cell tryptase beta (EC 3.4.21.59) is a trypsin-like serine protease that is stored in and released from mast cell granules. This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome. Cells producing recombinant human tryptase (rHT) were selected by screening with antibodies. Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium. Increasing levels of enzyme were detected in the medium for up to 3 days. Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two-step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE. These multiple forms were due to differences in post-translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa. A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration. Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar Km values, but the kcat of rHT was more than twice that of HLT.

Dendritic mast cells in the human nasal mucosa.

Human mast cells can be divided into two subtypes: MCTC cells, which contain tryptase and chymase, and MCT cells, which contain tryptase only. Herein we have used a combination of histamine, tryptase and chymase immunohistochemistry as a novel approach to the study of mast cells. Using this technique, we have discovered a new type of MCTC mast cell in biopsies of the nasal mucosa from healthy subjects and allergic patients. These mast cells have histamine-positive, dendrite-like cellular processes. Some cells have only one slender process, whereas other cells have several long processes extending from different parts of the cell body. Some of the cellular processes divide into two or three terminal branches, and histamine is sometimes found in small swellings along the course of the processes. Our findings contribute new aspects to the concept of mast cell heterogeneity. Thus, human mast cells may vary not only with respect to mediator content, but also with respect to gross morphologic features such as the presence of dendrite-like cellular processes. The recognition of this extreme heterogeneity may be an important step toward a better understanding of mast cell biology.