Tissue kallikrein-related peptidase8 accentuates cardiac fibrosis after myocardial ischemia-reperfusion injury via regulation of cardiac fibroblasts.

AIMS: Tissue kallikrein-related peptidase8 (KLK8) has been found to mitigate acute myocardial ischemia-reperfusion (IR) injury. However, the effect of KLK8 on cardiac remodeling in response to IR injury has not been determined. MATERIALS AND METHODS: KLK8 overexpressing transgenic rat (KLK8-TG) was used as the animal model. IR injury was induced by ligating the left anterior descending coronary artery for 1 h and subsequent reperfusion. The functional and morphological changes of the heart were examined 14 days after the injury. Neonatal rat cardiac fibroblasts (CFs) were used to investigate the molecular mechanisms in vitro. KEY FINDINGS: KLK8 overexpression enhanced cardiac diastolic dysfunction, fibrosis, and hypertrophy after IR injury, indicating that KLK8 accentuated cardiac remodeling in response to IR injury. Moreover, KLK8 overexpression increased epidermal growth factor (EGF) release and promoted the phosphorylation of EGF receptor (EGFR) and ERK1/2 in the heart after IR injury. It was interesting to find that both EGFR antagonist (AG 1478) and MEK inhibitor (PD98059) attenuated the KLK8-induced proliferation and activation of CFs in vitro, indicating that EGFR signaling might mediate the pro-fibrotic action of KLK8. SIGNIFICANCE: KLK8 plays a crucial role in cardiac remodeling after myocardial infarction. KLK8 accentuates cardiac fibrosis after IR injury, possibly mediated by EGFR signaling in CFs.

Minireview: functional roles of tissue kallikrein, kinins, and kallikrein-related peptidases in lung cancer.

Despite campaigns and improvements in detection and treatment, lung cancer continues to increase worldwide and represents a major public health problem. One approach to treating patients suffering from lung cancer is to target surface receptors overexpressed on tumor cells, such as GPCR-family kinin receptors, and proteases that control tumor progression, such as kallikrein-related peptidases (KLKs). These proteases have been visualized in recent years due to their contribution to the progression of cancers, such as prostate and ovarian cancer, facilitating the invasive and metastatic capacity of tumor cells in these tissues. In fact, KLK3 is the specific prostate antigen, the only tissue-specific biomarker used to diagnose this malignancy. In lung cancer to date, evidence indicates that KLK5, KLK6, KLK8, KLK11, and KLK14 are the major peptidases regulated and involved in its progression. The expression levels of KLKs in this neoplasm are modulated by the secretome of the different cell types present in the tumor microenvironment, the cancer subtype and the tumor stage, among others. Considering the multiple functions of kinin receptors and KLKs, this review highlights their roles, even considering the SARS-CoV-2 effects. Since lung cancer is often diagnosed in advanced stages, our efforts should focus on early diagnosis, validating for example specific KLKs, especially in high-risk populations such as smokers and people exposed to carcinogenic fumes, oil fields, and contaminated workplaces, unexplored fields to investigate. Furthermore, their modulation could be considered as a promising approach in lung cancer therapeutics.

Dysregulation of the kallikrein-kinin system in bronchoalveolar lavage fluid of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the angiotensin-converting enzyme 2 (ACE2) receptor, a critical component of the kallikrein-kinin system. Its dysregulation may lead to increased vascular permeability and release of inflammatory chemokines. Interactions between the kallikrein-kinin and the coagulation system might further contribute to thromboembolic complications in COVID-19. METHODS: In this observational study, we measured plasma and tissue kallikrein hydrolytic activity, levels of kinin peptides, and myeloperoxidase (MPO)-DNA complexes as a biomarker for neutrophil extracellular traps (NETs), in bronchoalveolar lavage (BAL) fluid from patients with and without COVID-19. FINDINGS: In BAL fluid from patients with severe COVID-19 (n = 21, of which 19 were mechanically ventilated), we observed higher tissue kallikrein activity (18·2 pM [1·2-1535·0], median [range], n = 9 vs 3·8 [0·0-22·0], n = 11; p = 0·030), higher levels of the kinin peptide bradykinin-(1-5) (89·6 [0·0-2425·0], n = 21 vs 0·0 [0·0-374·0], n = 19, p = 0·001), and higher levels of MPO-DNA complexes (699·0 ng/mL [66·0-142621·0], n = 21 vs 70·5 [9·9-960·0], n = 19, p < 0·001) compared to patients without COVID-19. INTERPRETATION: Our observations support the hypothesis that dysregulation of the kallikrein-kinin system might occur in mechanically ventilated patients with severe pulmonary disease, which might help to explain the clinical presentation of patients with severe COVID-19 developing pulmonary oedema and thromboembolic complications. Therefore, targeting the kallikrein-kinin system should be further explored as a potential treatment option for patients with severe COVID-19. FUNDING: Research Foundation-Flanders (G0G4720N, 1843418N), KU Leuven COVID research fund.

Uterine kallikrein and arterial bradykinin activities and uterine arterial proliferation in response to acute estradiol-17ß exposure in ovariectomized ewes.

Estradiol-17ß (E2) increases kallikrein in rodent and human reproductive tissues. Kallikrein specific activity is increased in the porcine uterus when conceptus E2 is secreted at maternal recognition of pregnancy. When kallikrein acts on kininogen to liberate bradykinin, angiogenic and vasoactive factors are released. The uterus of ovariectomized ewes administered E2 undergoes rapid vascular changes via different patterns of angiogenic and vasoactive factors. Our hypothesis was that E2 would increase the specific activity and protein secretion of tissue kallikrein in endometrial explants culture media (ECM) and ewes exposed to E2 would have uterine arteries that would be more sensitive to the vasodilatory effects of bradykinin. Ovariectomized ewes received 100 mg of E2 implants for 0, 12, 24, or 48 h. After treatment, uterine weights were determined, and caruncles were processed for ECM. Uterine weights and uterine weight per ewe body weight were significantly greater in the 12 and 24 h ewes compared with the 0 h ewes, with the 48 h ewes being similar to the 24 h ewes. There were no statistically significant differences in caruncular tissue kallikrein protein secretion among the treatment groups. There was a tendency (P = 0.09) for duration of E2 exposure to influence tissue kallikrein specific activity where kallikrein activity was greater (P ≤ 0.05) in the 12 and 48 h ewes compared with the 0 h ewes, with 24 h ewes being intermediate (unprotected F test). Uterine arteries from ewes with E2 for 24 and 48 h had more sensitivity to bradykinin, via the bradykinin receptor 2, than uterine arteries from ewes with 0 or 12 h E2 exposure. We fail to reject our hypothesis as E2 did elicit a positive response in tissue kallikrein specific activity and bradykinin response. Further investigations are needed to determine how kallikrein and bradykinin may be involved in vascular remodeling of the ovine uterus.

Tissue Kallikrein Protects Rat Prostate against the Inflammatory Damage in a Chronic Autoimmune Prostatitis Model via Restoring Endothelial Function in a Bradykinin Receptor B2-Dependent Way.

OBJECTIVE: The aim of this study was to investigate whether tissue kallikrein (KLK1) can protect the prostate from inflammatory damage and the mechanism involved in it. METHODS: A total of 50 male Wistar rats were used in this study. Initially, 20 rats were sacrificed to obtain the prostate antigen to induce experimental autoimmune prostatitis (EAP), and the remaining 30 rats were randomly divided into 5 experimental groups (normal control group (NC group), NC+KLK1 group (NCK group), EAP group, EAP+KLK1 group (EAPK group), and EAP+KLK1+HOE140 group (EAPKH group); n = 6). It should be explained that KLK1 mainly exerts its biological effects through bradykinin, and HOE140 is a potent and selective bradykinin receptor B2 (BDKRB2) antagonist. EAP was induced by intradermal injection of 15 mg/ml prostate antigen and complete Freund's adjuvant on days 0, 14, and 28. KLK1 was injected via tail vein at a dose of 1.5 × 10-3 PAN U/kg once a day, and HOE140 was administered by intraperitoneal injection at 20 µg/kg once every two days. Rats were sacrificed on day 42. The RNA and protein of the rat prostate were extracted to analyze the expression differences of KLK1, as well as the inflammation-, fibrosis-, and oxidative stress-related genes. The inflammatory cell infiltration and microvessel density of the prostate were also analyzed by pathological examination. In addition, pathological analysis was performed on prostate samples from patients undergoing benign prostate hyperplasia (BPH) surgery. RESULTS: The expression of KLK1 in the prostate decreased in the EAP group as well as BPH patients with obvious inflammation. KLK1 administration significantly inhibited inflammatory cell infiltration and reduced the production of inflammatory cytokines in the EAPK group. Prostate samples from the EAP group showed increased infiltration of T cells and macrophages, as well as gland atrophy, hypoxia, fibrosis, and angiogenesis. KLK1 administration upregulated endothelial nitric oxide synthase (eNOS) expression and suppressed oxidative stress, as well as transforming growth factor ß1 (TGF-ß) signaling pathways and the proangiogenic vascular endothelial growth factor (VEGF) in the EAPK group. However, in the EAPKH group in which HOE140 blocked BDKRB2, the beneficial effects of KLK1 were all cancelled. In addition, KLK1 intervention in normal rats had no obvious side effects. CONCLUSION: The KLK1 expression is inhibited in the inflamed prostates of humans and rats. Exogenous KLK1 restored endothelial function via a BDKRB2-dependent way and then played a role in improving microcirculation and exerted anti-inflammatory, antifibrotic, and antioxidative stress effects in the rat chronic-inflamed prostate.

Exogenous pancreatic kininogenase protects against tacrolimus-induced renal injury by inhibiting PI3K/AKT signaling: The role of bradykinin receptors.

BACKGROUND: Tissue kallikrein offers a wide spectrum of biological activity in the protection against various types of injury. However, information on its role in tacrolimus (TAC)-induced renal injury is limited. OBJECTIVES: This study aimed to assess the beneficial effects of pancreatic kininogenase (PK) in a rat model of chronic TAC nephrotoxicity and in vitro. METHODS: Sprague Dawley rats were treated daily with either TAC or PK or a combination of the two for four weeks. The influence of PK on renal injury was examined in terms of renal function, histopathology, cytokine expression, oxidative stress, intracellular organelles, programmed cell death, and PI3K/AKT signaling. Human kidney proximal tubular (HK-2) cells and mouse mesangial (SV40 MES13) cells treated with TAC and PK were also studied. RESULTS: PK treatment improved renal function and histopathology. This effect was paralleled by downregulation of proinflammatory and profibrotic cytokine expression. TAC-induced oxidative stress was closely associated with endoplasmic reticulum stress and mitochondrial dysfunction, resulting in excessive programmed cell death (apoptosis and autophagy) that was significantly abrogated by concurrent PK interference with PI3K/AKT signaling. PK also stimulated bradykinin receptor 1 (B1R) and B2R mRNA synthesis and increased bioactive nitric oxide (NO) and cAMP concentrations in TAC-treated kidneys. Blockade of either B1R or B2R eliminated the renoprotective effects of PK. In HK-2 and SV40 MES13 cells, PK decreased TAC-induced overproduction of intracellular reactive oxygen species and inhibited apoptotic cells, whereas cell viability was improved. Moreover, activated PI3K/AKT signaling in HK-2 cells was inhibited by PK and the PI3K inhibitor, LY294002. CONCLUSIONS: These findings indicate that PK treatment protects against chronic TAC nephrotoxicity via inhibition of PI3K/AKT signaling.

Human Tissue Kallikrein 1 Is Downregulated in Elderly Human Prostates and Possesses Potential In Vitro Antioxidative and Antifibrotic Effects in Rodent Prostates.

OBJECTIVE: The aim of the present study was to investigate the protective effects and mechanisms of KLK1 on aging-related prostate alterations and search clues about the application of KLK1 to the treatment of human BPH. METHODS: Thirty-six rats including 26 male wild-type SD rats and 10 transgenic rats were fed to 3- or 18-month-old and divided into three groups: young WTR (yWTR) as the control (n = 16), aged WTR (aWTR) (n = 10), and aged TGR (aTGR) (n = 10). The prostates of the three groups of rats (10 rats per group) were harvested to evaluate the levels of KLK1 expression, oxidative stress, fibrosis, and involved signaling pathways, such as NO/cGMP, COX-2/PTGIS/cAMP, and TGF-ß1/RhoA/ROCK1, via quantitative PCR, Western blot, histological examinations, and ELISA. Moreover, the remaining 6 yWTRs were sacrificed to obtain primary prostate fibroblast and aortic endothelial cells, and a coculture system was built with the cells for the verification of above signaling pathways in vitro. And the direct effects of bradykinin on prostate cells were detected by MTT experiment. Prostate specimens of 47 patients (age from 48 to 92 years) undergoing BPH surgery were collected after approval. Histological examinations and KLK1 IHC were preformed to analyze the relationship between KLK1 expression and age and prostate fibrosis. RESULTS: The human KLK1 gene only existed and was expressed in aTGR. The prostate of young rats expressed more KLK1 than the aged and the expression of KLK1 in prostate decreased with age in humans (r = -0.347, P = 0.018). Compared to the aWTR group, the yWTR and aTGR groups showed milder fibrosis, less oxidative stress, upregulated NO/cGMP, and COX-2/PTGIS/cAMP signaling pathways and inhibited TGF-ß1/RhoA/ROCK1 signaling pathway. In the coculture system, KLK1 suppressed TGF-ß1-mediated fibroblast-to-myofibroblast transdifferentiation via cleaving LMWK to produce the BK which upregulate eNOS expression and NO production in endothelial cells. BK not only slightly stimulated the proliferation ability of prostatic stromal cells but also upregulated iNOS and inhibited TGF-ß1 expression in them. CONCLUSION: KLK1 protects prostate from oxidative stress and fibrosis via amplified NO/cGMP signal in aged rats. The decrease of KLK1 expression with aging is laying the groundwork for the application of KLK1 to the treatment of human BPH. The current experimental data showed that the side effects of KLK1 on the prostate cell were not obvious.

Human Tissue Kallikreins in Polymorphous Adenocarcinoma: A Polymerase Chain Reaction and Immunohistochemical Study.

Polymorphous adenocarcinoma (PAC) is the second most common malignant salivary gland tumour of minor salivary glands. Human tissue kallikreins (KLKs) are a family of highly conserved serine proteases expressed by various tissues and organs. The literature demonstrates a link between KLKs and salivary gland neoplasms. The purpose of this study was to determine levels of KLK mRNA in tissue samples of PAC and to determine if KLK expression is limited to tumour cells. Nineteen cases of PAC were reviewed (1987-2013). The diagnosis was confirmed, demographic data was collected, and formalin fixed paraffin-embedded PAC and normal salivary gland tissue samples were obtained. RNA isolation was achieved, followed by conversion to complementary DNA via reverse transcription. Using PCR, the quantitative level of expression of KLKs1-15 was recorded. Samples exhibiting high and low KLK expression were selected for immunohistochemistry staining. Results revealed a statistically significant increase in mean KLK mRNA expression for KLK1, KLK4, KLK10, KLK12 and KLK15 in PAC tissue samples, compared with normal salivary gland tissue (Mann-Whitney U test, p < 0.05). Immunohistochemistry results demonstrated that KLKs were present in tumor cells. Notably, all samples demonstrating relatively higher KLK mRNA expression showed equivalent or increased staining scores relative to the low KLK mRNA expression samples. In conclusion, there appears to be aberrant kallikrein expression in polymorphous adenocarcinoma, suggesting the possibility of a kallikrein cascade influence on tumor development and progression.

Kallikrein gene family as biomarkers for recurrent prostate cancer.

AIM: To assess kallikrein (KLK) expression in recurrent and non-recurrent prostate tumors and adjacent healthy prostate tissues. METHODS: The expression levels of 15 KLK genes in 34 recurrent and 36 non-recurrent prostate cancer samples and 19 adjacent healthy prostate tissue samples was assessed with quantitative reverse-transcription polymerase chain reaction. The samples were obtained from Baylor College of Medicine, Houston, TX, USA between 2013 and 2016. RESULTS: Compared with controls, prostate cancer samples showed a strong decrease in KLK1, KLK4, KLK9, and KLK14. Recurrent samples were negative for KLK1, KLK2, and KLK14 but demonstrated higher levels of KLK3, KLK4, and KLK9 than controls. Other KLKs were not significantly expressed. CONCLUSION: This study for the first time showed a difference in the expression levels of the KLK gene family in recurrent prostate cancer. KLKs could be used as recurrence markers for prostate cancer.

Genetic signature of prostate cancer mouse models resistant to optimized hK2 targeted α-particle therapy.

Hu11B6 is a monoclonal antibody that internalizes in cells expressing androgen receptor (AR)-regulated prostate-specific enzyme human kallikrein-related peptidase 2 (hK2; KLK2). In multiple rodent models, Actinium-225-labeled hu11B6-IgG1 ([225Ac]hu11B6-IgG1) has shown promising treatment efficacy. In the present study, we investigated options to enhance and optimize [225Ac]hu11B6 treatment. First, we evaluated the possibility of exploiting IgG3, the IgG subclass with superior activation of complement and ability to mediate FC-γ-receptor binding, for immunotherapeutically enhanced hK2 targeted α-radioimmunotherapy. Second, we compared the therapeutic efficacy of a single high activity vs. fractionated activity. Finally, we used RNA sequencing to analyze the genomic signatures of prostate cancer that progressed after targeted α-therapy. [225Ac]hu11B6-IgG3 was a functionally enhanced alternative to [225Ac]hu11B6-IgG1 but offered no improvement of therapeutic efficacy. Progression-free survival was slightly increased with a single high activity compared to fractionated activity. Tumor-free animals succumbing after treatment revealed no evidence of treatment-associated toxicity. In addition to up-regulation of canonical aggressive prostate cancer genes, such as MMP7, ETV1, NTS, and SCHLAP1, we also noted a significant decrease in both KLK3 (prostate-specific antigen ) and FOLH1 (prostate-specific membrane antigen) but not in AR and KLK2, demonstrating efficacy of sequential [225Ac]hu11B6 in a mouse model.

Heparin Blocks the Inhibition of Tissue Kallikrein 1 by Kallistatin through Electrostatic Repulsion.

Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 310 helix between helix H and strand 2 of ß-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.

Kinin B1 Receptor Signaling in Skin Homeostasis and Wound Healing.

Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment.

NHBA is processed by kallikrein from human saliva.

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein of Neisseria meningitidis and a component of the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region involved in binding to heparin and heparan sulphate proteoglycans present on the surface of host epithelial cells, suggesting a possible role of NHBA during N. meningitidis colonization. NHBA has been shown to be cleaved by the meningococcal protease NalP and by human lactoferrin (hLF), a host protease presents in different body fluids (saliva, breast milk and serum). Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the only known reservoir of infection, we further investigated the susceptibility of NHBA to human proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial steps of colonization. Here we show that human saliva proteolytically cleaves NHBA, and identified human kallikrein 1 (hK1), a serine protease, as responsible for this cleavage. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and in the central nervous system. They are present in plasma, lymph, urine, saliva, pancreatic juices, and other body fluids where they catalyze the proteolysis of several human proteins. Here we report the characterization of NHBA cleavage by the tissue kallikrein, expressed in saliva and the identification of the cleavage site on NHBA both, as recombinant protein or as native protein, when expressed on live bacteria. Overall, these findings provide new insights on NHBA as target of host proteases, highlights thepotential role of NHBA in the Neisseria meningitidis nasopharyngeal colonization, and of kallikrein as a defensive agent against meningococcal infection.

Mechanisms of itching in mycosis fungoides: grade of itching correlates with eosinophil infiltration and kallikrein 5 expression.

BACKGROUND: Mycosis fungoides (MF) is the most common variant of cutaneous T-cell lymphomas (CTCL). Itching can be a major symptom for patients with CTCL, however, itching associated with MF is not relieved by conventional therapy using anti-histamines, suggesting that histamine is not the main pruritogen. Therefore, the underlying mechanisms of itching in MF patients remain unclear. OBJECTIVES: To investigate the clinical and histopathological features associated with MF-related itching. MATERIALS AND METHODS: Skin sections from MF patients and healthy subjects were used for pathophysiological analysis and evaluation of protease activity. These results were compared with the degree of itching. RESULTS: Of the MF patients, 40% did not report itching and 60% reported itching (moderate itching: 40%; strong itching: 20%). The number of eosinophils, but not mast cells, that infiltrated into skin was increased in the group with strong itching. In the skin of patients, both serine protease activity and immunoreactivity to kallikrein 5 (KLK5), a known itch mediator, increased relative to the grade of itching. CONCLUSION: These results suggest that KLK5 and eosinophil infiltration may be involved in itching in patients with MF.

Preclinical efficacy of hK2 targeted [177Lu]hu11B6 for prostate cancer theranostics.

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops. In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression. Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease. The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway. In a previous study, hu11B6 conjugated with Actinium-225 (225Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy. We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 (177Lu) and investigated whether similar tumor killing and AR-enhancement is produced. Moreover, single-photon emission computed tomography (SPECT) imaging of 177Lu is quantitatively accurate and can be used to perform treatment planning. [177Lu]hu11B6 therefore has significant potential as a theranostic agent. Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice. Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied. Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [177Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging. Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom. Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-ß-gal activity (senescence) and analyzed by digital autoradiography (DAR). Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg). Tumor accumulation of [177Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen. However, SPECT imaging with therapeutic amounts of [177Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177Lu and decreasing tumor volumes. For [177Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection. Tumor volume reduction correlated with injected activity and the absorbed dose. IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA- ß-gal activity (14 d). Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake. AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017). Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects. The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level. Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225Ac labeled hu11B6, however emerging at a later timepoint.

Tissue kallikrein regulates alveolar macrophage apoptosis early in influenza virus infection.

Host cell proteases are involved in influenza pathogenesis. We examined the role of tissue kallikrein 1 (KLK1) by comparing wild-type (WT) and KLK1-deficient mice infected with influenza H3N2 virus. The levels of KLK1 in lung tissue and in bronchoalveolar lavage (BAL) fluid increased substantially during infection. KLK1 did not promote virus infectivity despite its trypsin-like activity, but it did decrease the initial virus load. We examined two cell types involved in the early control of pathogen infections, alveolar macrophages (AMs) and natural killer (NK) cells to learn more about the antiviral action of KLK1. Inactivating the Klk1 gene or treating WT mice with an anti-KLK1 monoclonal antibody to remove KLK1 activity accelerated the initial virus-induced apoptotic depletion of AMs. Intranasal instillation of deficient mice with recombinant KLK1 (rKLK1) reversed the phenotype. The levels of granulocyte-macrophage colony-stimulating factor in infected BAL fluid were significantly lower in KLK1-deficient mice than in WT mice. Treating lung epithelial cells with rKLK1 increased secretion of this factor known to enhance AM resistance to pathogen-induced apoptosis. The recruitment of NK cells to the air spaces peaked 3 days after infection in WT mice but not in KLK1-deficient mice, as did increases in several NK-attracting chemokines (CCL2, CCL3, CCL5, and CXCL10) in BAL. Chronic obstructive pulmonary disease (COPD) patients are highly susceptible to viral infection, and we observed that the KLK1 mRNA levels decreased with increasing COPD severity. Our findings indicate that KLK1 intervenes early in the antiviral defense modulating the severity of influenza infection. Decreased KLK1 expression in COPD patients could contribute to the worsening of influenza.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery.

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Inhibitory effect of timolol on topical glucocorticoid­induced skin telangiectasia.

The aim of the present study wasto investigate the potential inhibitory effect of timolol on topical glucocorticoid­induced skin telangiectasia. In rabbits, flumethasone ointment was used to induce skin telangiectasia in the inner ear. Subsequently, timolol maleate (0.5%) eye drops (TMEDs) were administered twice daily for 4 weeks. Expression of the antibacterial peptides 37­amino acid peptide (LL­37) and kallikrein­5 (KLK5) was detected using quantitative polymerase chain reaction (PCR) and semi­quantitative reverse transcription­PCR. In patients with facial skin telangiectasia, one cheek of each patient was assigned to a treatment group and the other to a control group. For the treatment group cheeks, topical application of TMEDs was combined with 0.1% tacrolimus ointment once or twice daily for 8 weeks. The control group cheeks were administered with 0.1% tacrolimus ointment alone. Alterations in lesions were recorded by dermoscopy, and the L, a and b values of lesions were measured, based on the Commission Internationale de l'Éclairage system, with a chromameter prior to and at 1, 2, 4 and 8 weeks following treatment. The results indicated that erythema, papules and telangiectasia were significantly diminished following 4 weeks of treatment with TMEDs in rabbits. Notably, the expression of LL­37 and KLK5 mRNA was increased in the negative control group; however, it was decreased in the trial and blank groups. Clinical and dermoscopy images demonstrated that erythema was reduced in the 2 groups for 1 week, and that telangiectasia in the treatment group was markedly reduced compared with the control group at 4 weeks. The difference of the L and a values of lesions between the treatment and control group was significant (P<0.05). Overall, the present results suggested that the abnormal expression of LL­37 may be one of the mechanisms underlying the pathogenesis of facial corticosteroid addiction dermatitis (FCAD) and TMEDs may inhibit the mRNA expression of LL­37 by downregulating KLK5; in this regard, TMEDs may serve a role in attenuating telangiectasia, which may be beneficial in improving the telangiectasia symptoms of FCAD.

Overview of tissue kallikrein and kallikrein-related peptidases in breast cancer.

The kallikrein family comprises tissue kallikrein and 14 kallikrein-related peptidases (KLKs) recognized as a subgroup of secreted trypsin- or chymotrypsin-like serine proteases. KLKs are expressed in many cellular types where they regulate important physiological activities such as semen liquefaction, immune response, neural development, blood pressure, skin desquamation and tooth enamel formation. Tissue kallikrein, the oldest member and kinin-releasing enzyme, and KLK3/PSA, a tumor biomarker for prostate cancer are the most prominent components of the family. Additionally, other KLKs have shown an abnormal expression in neoplasia, particularly in breast cancer. Thus, increased levels of some KLKs may increase extracellular matrix degradation, invasion and metastasis; other KLKs modulate cell growth, survival and angiogenesis. On the contrary, KLKs can also inhibit angiogenesis and produce tumor suppression. However, there is a lack of knowledge on how KLKs are regulated in tumor microenvironment by molecules present at the site, namely cytokines, inflammatory mediators and growth factors. Little is known about the signaling pathways that control expression/secretion of KLKs in breast cancer, and further how activation of PAR receptors may contribute to functional activity in neoplasia. A better understanding of these molecular events will allow us to consider KLKs as relevant therapeutic targets for breast cancer.