Genomic and Immunologic Correlates in Prostate Cancer with High Expression of KLK2.

The identification of surfaceome proteins is a main goal in cancer research to design antibody-based therapeutic strategies. T cell engagers based on KLK2, a kallikrein specifically expressed in prostate cancer (PRAD), are currently in early clinical development. Using genomic information from different sources, we evaluated the immune microenvironment and genomic profile of prostate tumors with high expression of KLK2. KLK2 was specifically expressed in PRAD but it was not significant associated with Gleason score. Additionally, KLK2 expression did not associate with the presence of any immune cell population and T cell activating markers. A mild correlation between the high expression of KLK2 and the deletion of TMPRSS2 was identified. KLK2 expression associated with high levels of surface proteins linked with a detrimental response to immune checkpoint inhibitors (ICIs) including CHRNA2, FAM174B, OR51E2, TSPAN1, PTPRN2, and the non-surface protein TRPM4. However, no association of these genes with an outcome in PRAD was observed. Finally, the expression of these genes in PRAD did not associate with an outcome in PRAD and any immune populations. We describe the immunologic microenvironment on PRAD tumors with a high expression of KLK2, including a gene signature linked with an inert immune microenvironment, that predicts the response to ICIs in other tumor types. Strategies targeting KLK2 with T cell engagers or antibody-drug conjugates will define whether T cell mobilization or antigen release and stimulation of immune cell death are sufficient effects to induce clinical activity.

Towards a Precision Medicine Approach and In Situ Vaccination against Prostate Cancer by PSMA-Retargeted oHSV.

Prostate specific membrane antigen (PSMA) is a specific high frequency cell surface marker of prostate cancers. Theranostic approaches targeting PSMA show no major adverse effects and rule out off-tumor toxicity. A PSMA-retargeted oHSV (R-405) was generated which both infected and was cytotoxic exclusively for PSMA-positive cells, including human prostate cancer LNCaP and 22Rv1 cells, and spared PSMA-negative cells. R-405 in vivo efficacy against LLC1-PSMA and Renca-PSMA tumors consisted of inhibiting primary tumor growth, establishing long-term T immune response, immune heating of the microenvironment, de-repression of the anti-tumor immune phenotype, and sensitization to checkpoint blockade. The in situ vaccination protected from distant challenge tumors, both PSMA-positive and PSMA-negative, implying that it was addressed also to LLC1 tumor antigens. PSMA-retargeted oHSVs are a precision medicine tool worth being additionally investigated in the immunotherapeutic and in situ vaccination landscape against prostate cancers.

Phase I study of a multitargeted recombinant Ad5 PSA/MUC-1/brachyury-based immunotherapy vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: Antitumor vaccines targeting tumor-associated antigens (TAAs) can generate antitumor immune response. A novel vaccine platform using adenovirus 5 (Ad5) vectors [E1-, E2b-] targeting three TAAs-prostate-specific antigen (PSA), brachyury, and MUC-1-has been developed. Both brachyury and the C-terminus of MUC-1 are overexpressed in metastatic castration-resistant prostate cancer (mCRPC) and have been shown to play an important role in resistance to chemotherapy, epithelial-mesenchymal transition, and metastasis. The transgenes for PSA, brachyury, and MUC-1 all contain epitope modifications for the expression of CD8+ T-cell enhancer agonist epitopes. We report here the first-in-human trial of this vaccine platform. METHODS: Patients with mCRPC were given concurrently three vaccines targeting PSA, brachyury, and MUC-1 at 5×1011 viral particles (VP) each, subcutaneously every 3 weeks for a maximum of three doses (dose de-escalation cohort), followed by a booster vaccine every 8 weeks for 1 year (dose-expansion cohort only). The primary objective was to determine the safety and the recommended phase II dose. Immune assays and clinical responses were evaluated. RESULTS: Eighteen patients with mCRPC were enrolled between July 2018 and September 2019 and received at least one vaccination. Median PSA was 25.58 ng/mL (range, 0.65-1006 ng/mL). The vaccine was tolerable and safe, and no grade >3 treatment-related adverse events or dose-limiting toxicities (DLTs) were observed. One patient had a partial response, while five patients had confirmed PSA decline and five had stable disease for >6 months. Median progression-free survival was 22 weeks (95% CI: 19.1 to 34). Seventeen (100%) of 17 patients mounted T-cell responses to at least one TAA, whereras 8 (47%) of 17 patients mounted immune responses to all three TAAs. Multifunctional T-cell responses to PSA, MUC-1, and brachyury were also detected after vaccination in the majority of the patients. CONCLUSIONS: Ad5 PSA/MUC-1/brachyury vaccine is well tolerated. The primary end points were met and there were no DLTs. The recommended phase II dose is 5×1011 VP. The vaccine demonstrated clinical activity, including one partial response and confirmed PSA responses in five patients. Three patients with prolonged PSA responses received palliative radiation therapy. Further research is needed to evaluate the clinical benefit and immunogenicity of this vaccine in combination with other immuno-oncology agents and/or palliative radiation therapy. TRIAL REGISTRATION NUMBER: NCT03481816.

Generation of recombinant antibodies against human tissue kallikrein 7 to treat skin diseases.

Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.

Ultrasensitive amperometric immunosensor for the prostate specific antigen by exploiting a Fenton reaction induced by a metal-organic framework nanocomposite of type Au/Fe-MOF with peroxidase mimicking activity.

To increase the sensitivity of electrochemical sensor, Fe-MIL-88B-NH2 (Fe-MOF) with peroxidase-like activity is designed for the construction of immunoprobe. The Fe-MOF was prepared by one-step hydrothermalf method using 2-aminoterephthalic acid and iron(III) chloride. For the immunoprobe, it was fabricated by gold nanocomposite/Fe-MOF (Au/Fe-MOF) for the immobilization of labeling antibody (the antibody was used to conjuncting with label materials). The thin layer of Methylene Blue (MB) covered by reduced graphene oxide-gold nanocomposites (Au-rGO) serves as a substrate to covalently fix coating antibodies. The MB as a redox-active species was modified on the glass carbon electrode that can give a strong amperometric signal at 0.18 V (vs. Ag/AgCl). With the participation of H2O2, Fe-MOF can induce the Fenton reaction which degrades MB covered by Au-rGO on the substrate. The rest of MB on the surface of electrode becomes oxidized thereby generating a current signal. Square wave voltammetry (SWV) was used to quantify PSA. Under optimal conditions, the immunoassay is stable, specific and reproducible. It has a lower detection limit of 0.13 pg mL-1 (S/N = 3) and a wide analytical range that extends from 0.001 to 100 ng mL-1. Graphical abstractA sandwich-type amperometric immunoassay based on Fe-MOF-induced Fenton reaction was designed for sensitive determination of prostate specific antigen.

Superoxide Dismutase 3 Inhibits LL-37/KLK-5-Mediated Skin Inflammation through Modulation of EGFR and Associated Inflammatory Cascades.

The expressions of LL-37 and KLK-5 were found to be altered in various dermatoses, including atopic dermatitis, psoriasis, and rosacea. However, the downstream inflammatory effect of LL-37 and KLK-5 is not as well studied. In addition, there is little high-quality evidence for the treatment of LL-37- and KLK-5-mediated inflammation. In this study, we investigated the effect of superoxide dismutase 3 (SOD3) on LL-37- or KLK-5-induced skin inflammation in vitro and in vivo and its underlying anti-inflammatory mechanisms. Our data showed that SOD3 significantly reduced both LL-37- and KLK-5-induced expression of pro-inflammatory mediators and suppressed the activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and p38/extracellular signal-regulated kinase signaling pathways in human keratinocytes. Moreover, SOD3 suppressed LL-37-induced expression of inflammatory mediators, reactive oxygen species production, and p38/extracellular signal-regulated kinase activation in mast cells. In addition, subcutaneous injection of KLK-5 in SOD3 knockout mice exhibited erythema with increased epidermal thickness, mast cell and neutrophil infiltration, expression of inflammatory mediators, and activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and downstream mitogen-activated protein kinase pathways. However, treatment with SOD3 in SOD3 knockout mice rescued KLK-5-induced inflammatory cascades. Similarly, KLK-5-induced inflammation in wild-type mice was also ameliorated when treated with SOD3. Taken together, our data suggest that SOD3 is a potentially effective therapy for both LL-37-and KLK-5-induced skin inflammation.

A surface plasmon resonance enhanced photoelectrochemical immunoassay based on perovskite metal oxide@gold nanoparticle heterostructures.

An innovative visible light-driven photoelectrochemical (PEC) immunosensing system was reasonably established for the sensitive detection of prostate-specific antigen (PSA) by using perovskite metal oxide@gold nanoparticle heterostructures (BaTiO3/Au) as the photoactive materials. When plasmonic Au nanoparticles were directly decorated on BaTiO3, a several times surface plasmon resonance (SPR) enhancement of photocurrent density was induced via the injection of hot electrons from visible light-excited Au nanoparticles into the conduction band of BaTiO3, and the combination of BaTiO3 and Au nanoparticles was employed as a promising platform for developing a photoelectrochemical bioanalysis. As a proof of concept, PSA had been detected by the BaTiO3/Au nanocomposite-based PEC sensor. To design such an immunoassay protocol, a monoclonal anti-PSA capture antibody (cAb)-coated microplate and glucose oxidase/polyclonal anti-PSA detection antibody-modified gold nanoparticles (GOx-Au NP-dAb) were used as the immunoreaction platform and signal probe, respectively. Upon the addition of target PSA, a sandwiched immunocomplex was formed accompanying the immuno-recognition between the antigen and antibody, and then the carried GOx could oxidize glucose to produce H2O2. The photocurrent of the BaTiO3/Au nanocomposite-functionalized electrode amplified with increasing H2O2 concentration since H2O2 is considered as a good hole scavenger. On the basis of the above-mentioned mechanisms and the optimized conditions, the assembled PEC immunosensor was linear with the logarithm of the PSA concentration in the range of 0.01-40 ng mL-1 with a detection limit of 4.2 pg mL-1. It afforded rapid response, good precision, and high stability and specificity, implying its great promise in photoelectrochemical immunoassays. More generally, this system sets up an ideal PEC immunosensing system based on the BaTiO3/Au nanocomposites and represents an innovative and low-cost "signal-on" assay scheme for the practical quantitative screening of low-abundance proteins.

Highly sensitive piezoelectric immunosensors employing signal amplification with gold nanoparticles.

We present a quartz crystal microbalance (QCM) immunosensor for highly sensitive detection of prostate-specific antigen (PSA) in a human serum immunoassay. In particular, in this study, we employed signal amplification using and enlarging gold nanoparticles. Because QCM measures the change of resonance frequency according to the mass change occurring on the sensor surface, we could quantitatively analyze PSA based on a tremendous increase in mass by sandwich immunoassay using AuNP-conjugated anti-PSA-detecting antibody enhanced with subsequent gold staining. The limit of detection of the PSA immunoassay in human serum without gold staining enhancement was 687 pg ml-1 but was 48 pg ml-1 with the gold staining-mediated signal amplification. That is, amplifying the signal resulted in increased sensitivity and reproducibility of immunoassay in a human serum.

A proinflammatory role of KLK6 protease in Netherton syndrome.

BACKGROUND: Netherton syndrome (NS) is a rare but severe type of ichthyosis characterized by atopy, allergies, and potentially lethal skin overdesquamation associated with highly elevated proteolytic activities in LEKTI-deficient epidermis. NS symptoms are recapitulated in Spink5-/- mouse where the gene encoding Lekti has been invalidated. Spink5-/- mice die within 5h from birth due to their severe skin barrier defect leading to dehydration. Spink5-/- mice also serve as a model for atopic dermatitis. The KLK6 protease is expressed by epidermal keratinocytes and shown in vitro to cleave desmosomal components. OBJECTIVE: To investigate in vivo whether KLK6 is implicated in epidermal overdesquamation and/or inflammation associated with NS. METHODS: The role of KLK6 was evaluated by generating Spink5-/-Klk6-/- double knockout mice. The phenotype was assessed by macroscopic observation, immunohistochemistry for differentiation markers, in situ zymography for proteolysis, and quantification of proinflammatory cytokines. RESULTS: Elimination of Klk6 in Spink5-/- remarkably suppresses the expression of Tslp, a major itching-inducing factor and driver of allergic reactions. Tnfα and the Th17 promoting cytokine Il-23 were also suppressed. Spink5-/-Klk6-/- mice display normalized keratinocyte differentiation, nevertheless, epidermal proteolytic activities and the associated overdesquamation were not ameliorated, and Spink5-/-Klk6-/- still died from a severe epidermal barrier defect as the Spink5-/-. CONCLUSIONS: Ablation of Klk6 largely suppresses epidermal inflammation but cannot rescue overdesquamation leading to the lethal NS phenotype. Nonetheless, our findings demonstrate for the first time that KLK6 is implicated in skin inflammation and may represent a novel druggable target for NS and other inflammatory conditions e.g. atopic dermatitis.

Vertical flow assays based on core-shell SERS nanotags for multiplex prostate cancer biomarker detection.

Rapid, simultaneous, and sensitive quantification of multiplex prostate biomarkers plays an important role in early diagnosis, especially for obese men and patients. Herein, a surface-enhanced Raman scattering (SERS)-based vertical flow assay (VFA) is presented for simultaneous detection of multiplex prostate cancer biomarkers, such as prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) on a single test spot. In practice, Raman dyes (RDs) encoded core-shell SERS nanotags instead of conventional gold colloids used in the colorimetric assay are employed in the sensing membrane of SERS based VFAs for multiplex protein detection. Because of the enhanced Raman signal of the core-shell nanostructure and the high surface area to volume ratio (SVR) of the porous sensing membrane, this proposed biosensor shows a wide linear dynamic range (LDR) with detection limits of 0.37, 0.43, and 0.26 pg mL-1 for PSA, CEA, and AFP, respectively, suggesting that this approach can be a good candidate in point of care testing (POCT) for rapid and sensitive biomarker detection and has a huge potential in multiplex analysis and cancer diagnosis.

Nanopore blockade sensors for ultrasensitive detection of proteins in complex biological samples.

Nanopore sensors detect individual species passing through a nanoscale pore. This experimental paradigm suffers from long analysis times at low analyte concentration and non-specific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis. Here, we address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). The (anti-PSA)-MNPs are magnetically driven to block an array of nanopores rather than translocate through the nanopore. Specificity is obtained by modifying nanopores with anti-PSA antibodies such that PSA molecules captured by (anti-PSA)-MNPs form an immunosandwich in the nanopore. Reversing the magnetic field removes (anti-PSA)-MNPs that have not captured PSA, limiting non-specific effects. The combined features allow detecting PSA in whole blood with a 0.8 fM detection limit. Our 'magnetic nanoparticle, nanopore blockade' concept points towards a strategy to improving nanopore biosensors for quantitative analysis of various protein and nucleic acid species.

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA+ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.

Rolling circle amplification integrated with suspension bead array for ultrasensitive multiplex immunodetection of tumor markers.

Multiplex detection of ultra-low abundant tumor markers is extremely important for early diagnosis and therapy evaluation. Herein, an ultrasensitive multiplex immunoassay was developed by combination of rolling circle amplification (RCA) and suspension bead array (SBA) technology. Based on a conventional sandwich-type immunoreaction on beads, the detection antibodies were conjugated with DNA primers, so RCA could be implemented to generate long-stranded DNA with abundant repeated sequences allowing for hybridization with fluorochrome-labeled oligonucleotide probes. Thus the fluorescence signal of immunocomplexes on the encoded beads can be greatly enhanced. Using the as-developed immuno-RCA suspension bead array (iRCA-SBA), simultaneous analysis of multiple tumor markers was achieved with the limits of detection of 3.1 pg/mL (∼0.1 pM) for prostate specific antigen (PSA), 9.1 pg/mL (∼50 fM) for carcinoembryonic antigen (CEA), and 0.66 pg/mL (∼9 fM) for α-fetoprotein (AFP), which are two to three orders of magnitude lower than those obtained by the conventional SBA method. The dynamic range were 4.5, 4.7, and 5.5 orders of magnitude for PSA, CEA, and AFP, respectively. Tests on clinical serum samples demonstrate that the tumor marker concentrations measured by the newly developed iRCA-SBA assay agreed well with those obtained by the conventional SBA method. These results indicate that the iRCA-SBA assay significantly increased the detection sensitivity and dynamic range without sacrificing the reliability and accuracy of conventional SBA. Upon the integration with iRCA, SBA could find more applications in the detection of low abundance protein biomarkers for early diagnosis of cancer and other diseases.

Neutrophil α-defensins promote thrombosis in vivo by altering fibrin formation, structure, and stability.

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.

High-sensitivity lateral flow immunoassay with a fluorescent lanthanide nanoparticle label.

Lateral flow (LF) immunoassays are commonly used for point-of-care testing and typically incorporate visually read reporters, such as gold particles. To improve sensitivity and develop quantitative LF immunoassays, visual reporters can be replaced by fluorescent reporters detected by an instrument. In this study, we used fluorescent europium(III) chelate doped nanoparticle (Eu-np) reporters to develop a quantitative high-sensitivity LF immunoassay for free prostate specific antigen (fPSA). Furthermore, we tested different simplified formats of the assay and the effect of different modifiable parameters on the detection limit of the assay: dynamic range, assay duration and number of assay steps. The molar detection limits of the different assay formats were compared with published detection limits of LF immunoassays with different reporters. The cutoff was calculated from 11 female serum samples. The detection limit of the sensitivity optimized fPSA assay with fPSA spiked into pooled female serum was 0.01 ng/ml, which is approximately 100-fold lower than the most sensitive gold particle LF assays and 10-fold lower than other Eu-np and carbon nanoparticle based LF immunoassays. Thus, Eu-np reporters can be used to develop highly sensitive and quantitative LF immunoassays.

Subverting bradykinin-evoked inflammation by co-opting the contact system: lessons from survival strategies of Trypanosoma cruzi.

PURPOSE OF REVIEW: During Chagas disease, Trypanosoma cruzi alternates between intracellular and extracellular developmental forms. After presenting an overview about the roles of the contact system in immunity, I will review experimental studies showing that activation of the kallikrein-kinin system (KKS) translates into mutual benefits to the host/parasite relationship. RECENT FINDINGS: T. cruzi trypomastigotes initiate inflammation by activating tissue-resident innate sentinel cells via the TLR2/CXCR2 pathway. Following neutrophil-evoked microvascular leakage, the parasite's major cysteine protease (cruzipain) cleaves plasma-borne kininogens and complement C5. Tightly regulated by angiotensin-converting enzyme (ACE), kinins and C5a in turn further propagate inflammation via iterative cycles of mast cell degranulation, contact system activation, bradykinin release and activation of endothelial bradykinin B2 receptors (B2R). Recently, studies in the intracardiac model of infection revealed a dichotomic role for bradykinin and endothelin-1: generated upon contact activation (mast cell/KKS pathway), these pro-oedematogenic peptides reciprocally stimulate trypomastigote invasion of heart cells that naturally overexpress B2R and endothelin receptors (ETaR/ETbR). SUMMARY: Studies focusing on the immunopathogenesis of Chagas disease revealed that the contact system plays a dual role in host/parasite balance: T. cruzi co-opts bradykinin-induced plasma leakage as a strategy to increment heart parasitism and increase immune resistance by upregulating type-1 effector T-cell production in secondary lymphoid tissues.

Sandwich pair nanobodies, a potential tool for electrochemical immunosensing serum prostate-specific antigen with preferable specificity.

Prostate-Specific Antigen (PSA) is a crucial biomarker for screening prostate cancer, but a sensitive and selective immunosensor for rapid quantification of serum PSA remains to be developed. In this study, a sandwich pair of nanobodies (Nbs) (i.e., Nb2 and Nb40) against PSA surface antigen was obtained from an alpaca-derived immune phage display library. A sandwich-type immunosensor for the sensitive and selective detection of PSA in serum samples was ingeniously designed based on the pair of Nbs. The small size of Nb40 allowed high capture densities on the surface of reduced graphene oxide (rGO) nanocomposed with massive Au nanoparticles (rGO@AuNPs), which significantly improved the conductivity and provided a large area to anchor many primary antibodies. The secondary antibody Nb2 fused with streptavidin -binding peptide (SBP) cooperated with Nb40 for PSA sandwiching. Accompanying introduction of horseradish peroxidase-streptavidin (HRP-SA) coupled with Nb2-SBP, the faradaic current was linearly correlated with the logarithm of PSA concentration in a range of 0.1-100 ng mL-1. More importantly, this immunosensor exhibited excellent selectivity, stability, and reproducibility due to the sandwich pair Nbs. The proposed immunosensor was successfully applied in determining PSA in serum samples and could be used for the sensitive and specific detection of PSA.

Hemostasis in Allergy.

The involvement of the hemostatic system in immune-mediated inflammation is widely reported. Many coagulation factors play a role in the pathogenesis of autoimmune diseases, such as systemic vasculitis and systemic lupus erythematosus. Hemostatic disorders are also involved in asthma and chronic spontaneous urticaria (CSU). Factor XIIa (FXIIa) was one of the first coagulation factors implicated in inducing both humoral and cellular responses and is therefore considered a prime new therapeutic target in immune-mediated inflammation. The involvement of coagulation factors, such as tissue factor and fibrinogen, in the pathogenesis of asthma has been reported. The finding of platelet activation in asthma also indicates a link between bronchial inflammation and hemostasis. The pathogenesis of mast cell degranulation and CSU was also shown to be associated with the activation of hemostatic factors such as fibrinogen and FXIIa. Increased plasma levels of D-dimer have been widely reported as a biological marker for the duration and severity of CSU. In addition, endothelial-induced cell activation by the kallikrein-high molecular weight complex and the release of heat shock protein 90 was shown to be involved in mast cell degranulation disorders. In this narrative review, the authors aim to summarize the role of hemostasis in inflammation, asthma, and CSU by focusing on the increasing information linking hemostatic factors and immune-mediated disorders.

Multifunctional Serine Protease Inhibitor-Coated Water-Soluble Gold Nanoparticles as a Novel Targeted Approach for the Treatment of Inflammatory Skin Diseases.

The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme-such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody-in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.