Inhibitor regulation of tissue kallikrein activity in the synovial fluid of patients with rheumatoid athritis

Tissue kallikrein (TK) and 1-antitrypsin (AT)/TK complexes can be detected in SF from patients with RA if components of the fluids which interfere with the detection of TK are removed. 2-Macroglobulin (2-M) in SF was demonstrated to contain trapped proteases which were still active in amidase assays. Removal of 2-M from RA SF reduced their amidase activity. However, at least some of the remaining activity was due to TK because it was soya bean trypsin inhibitor resistant and trasylol sensitive and was partly removed by affinity chromatography on anti-TK sepharose. Removal of RF from the fluids reduced the values obtained for TK levels by ELISA. Addition of SF to human urinary kallikrein (HUK) considerably reduced the levels of TK detected suggesting the presence of a TK ELISA inhibitor in the fluids. Removal of components of >300 kDa from SF markedly reduced the TK ELISA inhibitory activity and increased the values for both the TK and l-AT/TK levels in fluids as measured by ELISA. It is considered this novel inhibitor does not bind to the active site of TK but rather binds to the site reactive with anti-TK antibodies

Acceptability of the live biotherapeutic LACTIN-V (Lactobacillus crispatus CTV-05) among young women at high risk of HIV acquisition in South Africa: data from the phase 2 placebo-controlled trial.

INTRODUCTION: Live biotherapeutic products (LBPs) containing Lactobacillus crispatus may optimize the vaginal microbiota, reduce genital inflammation, and protect against HIV acquisition. Determining acceptability of LBPs among African women at high risk of HIV is essential to guide product development. METHODS: The phase 2 double-blind randomized placebo-controlled trial recruited young sexually active cis-women with vaginal dysbiosis from a community-based research clinic. Following antibiotics (oral metronidazole), participants were randomized (2:1) to receive 11 doses of LACTIN-V (2 × 109 L. crispatus CTV-05) or placebo over 4 weeks. A questionnaire assessed product acceptability. RESULTS: Forty-five young Black South African women were randomized to LACTIN-V (N = 32) or placebo (N = 13). Forty-two (93.3%) had an active sexual partner. Adherence was high with 36 participants (80.0%) completing all 11 doses. Of the 43 participants who completed the acceptability questionnaire, 38 (88.4%) were satisfied using the vaginal applicator and 41 (95.5%) confirmed ease of use. For 14 (32.5%) participants, product use without the partner knowing was important. Thirty-one (72.1%) participants felt that partner approval for product use was not important. On Likert scales of 0-10 (lowest to highest), agreement with positive product attributes (effective, comfortable, easy to use) scored at means of  ≥6.7. Negative product attributes (dosing, leakage, vaginal dryness, partners disapproval) were rated less important with lower mean scores ≤3.2. Overall, 75% of participants would use the product again, with no significant difference between study arms. CONCLUSIONS: Young South African women at high risk of HIV found the LACTIN-V study product highly acceptable and easy to use. CLINICAL TRIAL REGISTRATION: [clinicaltrials.gov], identifier [NCT05022212]

Role of human tissue kallikrein in gastrointestinal stromal tumour invasion

Background: Human tissue kallikrein (hK1) generates vasodilator kinins from kininogen and promotes angiogenesis by kinin-dependent and kinin-independent mechanisms. Here, we investigate the expression and functional relevance of hK1 in human gastrointestinal stromal tumour (GIST).<p></p> Methods: Vascularisation and hK1 expression of GIST samples were assessed by immunohistochemistry. In two GIST cell lines, hK1 expression was assessed by PCR, and hK1 protein levels and activity were measured by ELISA and an amidolytic assay, respectively. The effect of hK1 silencing, inhibition or overexpression on GIST cell proliferation, migration and paracrine induction of angiogenesis was studied. Finally, local and systemic levels of hK1 were assessed in mice injected with GIST cells.<p></p> Results: Human tissue kallikrein was detected in 19 out of 22 human GIST samples. Moreover, GIST cells express and secrete active hK1. Titration of hK1 demonstrated its involvement in GIST invasive behaviour, but not proliferation. Furthermore, hK1 released by GIST cells promoted endothelial cell migration and network formation through kinin-dependent mechanisms. Gastrointestinal stromal tumour implantation in nude mice resulted in local and systemic hK1 expression proportional to tumour dimension.<p></p> Conclusions: Human tissue kallikrein is produced and released by GIST and participates in tumour invasion. Further studies are needed to validate hK1 as a diagnostic biomarker and therapeutic target in GIST

Upregulation of tissue kallikrein, kinin B(1) receptor, and kinin B(2) receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma

Background: The mitogenic kinin peptides formed by the serine protease, tissue kallikrein (TK1), stimulate the proliferation of tumour cells and, by increasing vascular permeability, enhance metastasis. Oesophageal mucosal epithelial cells are derived from the epithelial cell germ layer, which expresses the kallikrein–kinin cascade. Aim: To determine the cellular distribution of active TK1, prokallikrein, and the kinin B(1) and B(2) receptors in oesophageal carcinoma by immunocytochemistry and in situ hybridisation (ISH). Methods: Fifty oesophageal specimens (33 biopsies and 17 resections) and 10 control specimens adjacent to tumour or normal oesophageal biopsies were studied. Specific antibodies were used to determine the cellular localisation of TK1, prokallikrein, and the kinin B(1) and B(2) receptors in normal and oesophageal specimens by standard immunohistochemical techniques. The intensity of immunolabelling was quantified by image analysis. Antisense probes for TK1 and the kinin B(1) and B(2) receptors were also used to localise mRNA. Results: TK1 (active and prokallikrein) was expressed in the mucosa of normal and tumour oesophageal epithelium. In general, expression was highest in activated mast cells, followed by giant tumour cells. Immunolabelling results were confirmed by ISH experiments. Conclusions: This is the first demonstration that TK1 and kinin B(1) and B(2) receptors are expressed in oesophageal carcinoma. Because TK1 released from tumour cells enzymatically generates mitogenic kinins from its endogenous substrate, kininogen, it is possible that third generation kinin receptor antagonists, which have been shown to be cytotoxic to cancer cells, may be useful therapeutic agents in this disease