Beneficial effects of alpha-1 antitrypsin therapy in a mouse model of colitis-associated colon cancer.

BACKGROUND: It is widely accepted that chronic inflammatory bowel diseases significantly higher a risk for colorectal cancer development. Among different types of treatments for patients with colon cancer, novel protein-based therapeutic strategies are considered. AIM: To explore the effect of human plasma alpha-1 antitrypsin (AAT) protein in the chemically induced mouse model of colorectal cancer. METHODS: BALB/c mice with azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colitis-associated colorectal cancer (CAC), we intraperitoneally treated with commercial preparation of human plasma AAT (4 mg per mouse). Effects of this therapy were evaluated histologically, and by immunohistochemical and gene expression assays. RESULTS: When compared with non-treated controls, AOM/DSS mice receiving AAT therapy exhibited significantly longer colons, and less anal bleeding. Concurrently, AAT-treated mice had significantly fewer polyps, and lower numbers of large colon tumors. Immunohistochemical examinations of colon tissues showed significantly lower neutrophil counts, more granzyme B-positive but fewer MMP9 (gelatinase B)-positive cancer cells and lower numbers of apoptotic cells in mice receiving AAT therapy. The expression levels of IL4 were significantly higher while TNFA was slightly reduced in tumor tissues of AOM/DSS mice treated with AAT than in AOM/DSS mice. CONCLUSION: Human AAT is an acute phase protein with a broad-protease inhibitory and immunomodulatory activities used as a therapeutic for emphysema patients with inherited AAT deficiency. Our results are consistent with previous findings and support an idea that AAT alone and/or in combination with available anti-cancer therapies may represent a new personalized approach for patients with colitis-induced colon cancer.

Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase.

The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1ß gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60-80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.

The development of an electrochemical immunosensor utilizing chicken IgY anti-spike antibody for the detection of SARS-CoV-2.

This paper introduces a novel approach for detecting the SARS-CoV-2 recombinant spike protein combining a label free electrochemical impedimetric immunosensor with the use of purified chicken IgY antibodies. The sensor employs three electrodes and is functionalized with an anti-S IgY antibody, ELISA and immunoblot assays confirmed the positive response of chicken immunized with SARS-CoV2 S antigen. The developed immunosensor is effective in detecting SARS-CoV-2 in nasopharyngeal clinical samples from suspected cases. The key advantage of this biosensor is its remarkable sensitivity, and its capability of detecting very low concentrations of the target analyte, with a detection limit of 5.65 pg/mL. This attribute makes it highly suitable for practical point-of-care (POC) applications, particularly in low analyte count clinical scenarios, without requiring amplification. Furthermore, the biosensor has a wide dynamic range of detection, spanning from 11.56 to 740 ng/mL, which makes it applicable for sample analysis in a typical clinical setting.

SARS-CoV-2 Antinucleocapsid Antibody Response of mRNA and Inactivated Virus Vaccines Compared to Unvaccinated Individuals.

Comparative studies of SARS-CoV-2 antinucleocapsid (anti-N) antibody response in the context of inactivated virus vaccines versus natural infection are limited. This study aims to determine and compare the anti-N antibody levels in people vaccinated with Sinopharm's (Wuhan, China) inactivated virus vaccine in comparison with naturally infected unvaccinated and Pfizer's spike (S) mRNA-based vaccinated subjects. Two hundred ninety-nine Jordanian adults participated in the study including unvaccinated COVID-19-infected patients (n = 99), Pfizer-vaccinated (n = 100), and Sinopharm-vaccinated recipients (n = 100). Serum samples were assayed for anti-N IgG, anti-N IgM, and anti-S IgG. Sera of 64.6% of naturally infected unvaccinated participants had positive anti-S IgG (median = 36.35 U/mL; range: 0.04−532.5 U/mL) compared to 88% of Pfizer-vaccinated (Manhattan, NY, USA) (median = 26.52 U/mL; range: 0.39−1265 U/mL) and 58% of Sinopharm-vaccinated subjects (median = 14.35 U/mL; range: 0.39−870.17 U/mL). Samples of 60.6% of naturally infected unvaccinated people had positive anti-N IgG (median = 15.03 U/mL; range: 0−265.1 U/mL) compared to 25% of Pfizer-vaccinated (median = 0.02 U/mL; range: 0−68 U/mL) and 48% of Sinopharm-vaccinated subjects (median = 0.8 U/mL; range: 0−146.3 U/mL). Anti-N titers among the three groups were significantly different (p < 0.05). Anti-N IgM antibodies appeared in 23.2% of the naturally infected unvaccinated group (median = 0.29 U/mL; range: 0−15 U/mL) compared to only 9.0% of Pfizer-vaccinated (median = 018 U/mL; range: 0−33 U/mL) and 7.0% of Sinopharm-vaccinated subjects (median = 0.2 U/mL; range: 0−12.02 U/mL). A significant negative correlation was found between anti-S and age for both vaccines and between anti-S and the presence of chronic disease in Sinopharm-vaccinated subjects. A significant positive correlation between anti-N and anti-S titers was found among the three groups. This study shows that the inactivated virus vaccine, Sinopharm, induces an anti-N response that can boost that of natural infection or vice versa. On the other hand, the Pfizer mRNA-based vaccine induces a significantly stronger anti-S Ab response.

Review of current in vitro COVID-19 diagnostics methods.

The diagnosis of COVID-19 is considered a significant step in the management of the disease that is causing a major worldwide public health challenge from the time of its emergence in December 2019. Since it has been established that SARS-CoV-2 spreads rapidly, timely detection of the positive cases and isolation of such individuals and their contacts helps in containing viral transmission. In this paper, we review the in vitro technology platforms for testing and diagnosing COVID-19 patients: molecular tests, rapid antigen tests, and serology tests. As part of our review of each category of tests, we discuss the commercialized testing platforms, their analyzing systems, specimen collection protocols, and testing methodologies. Moreover, the efficacy and limitations of each technique are also discussed. The key structural components of the virus are presented to provide an understanding of the scientific principles behind the testing tools.

Acute-phase protein α1-antitrypsin inhibits neutrophil calpain I and induces random migration.

A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a better understanding of the mechanisms underlying neutrophil influx and activation is of critical importance. We have previously shown that the acute phase protein α1-antitrypsin (AAT) inhibits neutrophil chemotaxis. In this study, we examine mechanisms related to the effect of AAT on neutrophil responses. We report a previously unknown function of AAT to inactivate calpain I (µ-calpain) and to induce a rapid cell polarization and random migration. These effects of AAT coincided with a transient rise in intracellular calcium, increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extra-cellular signal-regulated kinase (ERK1/2). Furthermore, AAT caused a significant inhibition of nonstimulated as well as formyl-met-leu-phe (fMLP)-stimulated neutrophil adhesion to fibronectin, strongly inhibited lipopolysaccharide-induced IL-8 release and slightly delayed neutrophil apoptosis. The results presented here broaden our understanding of the regulation of calpain-related neutrophil functional activities, and provide the impetus for new studies to define the role of AAT and other acute phase proteins in health and disease.

Production of monoclonal antibodies against the 8 kDa subunit of Echinococcus granulosus Antigen B (EgAgB8/2) using DNA immunization.

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.