Nuha Khalid Alekhmimi

Matrix metalloproteinases (MMPs) are zinc-dependent proteinases that are capable of cleavage of extracellular matrix (ECM) proteins and enzymes and play an important role in lung dysfunction. Specifically, MMP-2 is produced in the lung by alveolar epithelial and endothelial cells and other immune cells, such as macrophages. MMP-2 regulatory pathway is initiated in alveolar macrophages during acute lung injury (ALI), which may increase pulmonary inflammation. Therefore, there is a critical need for fast and reliable techniques to track the acute respiratory distress syndrome (ARDS). Here, we describe near-infrared fluorescence resonance energy transfer (NI-FRET) MMP-2-based probe for the in vivo detection of ALI induced by lipopolysaccharides (LPS). LPS-induced MMP-2 was measured using near-infrared (NIR) imaging after 1, 2, 4, 5, and 24 h of LPS exposure. Our results were compared with the data obtained from ELISA and Western blotting, demonstrating that MMP-2 fluorescence probe provide a promising in vivo diagnostic tool for ALI/ARDS in infected mice.

Sepsis is an immune response to a microbial invasion that causes organ injury and dysfunction due to a systemic inflammatory response. Sepsis is a serious, life-threatening condition and a widely recognized global health challenge. Given its high death rate, it is critical to diagnose sepsis and start treatment as early as possible. There is an urgent need for a sensitive and rapid screening method for detecting sepsis. In this study, we investigated the use of MMP-9 as a biomarker for sepsis. A colorimetric paper-based biosensor was used for the detection of MMP-9 utilizing peptide-magnetic nanoparticle conjugates. The method is based on the cleavage of the MMP-9-specific peptide by the protease leading to the detaching of the magnetic beads from the sensor surface and changing of color. A fecal intraperitoneal (FIP) challenge was used to induce sepsis in mice, and an MMP-9 secretion was measured by taking blood and Bronchoalveolar Lavage (BAL) fluid samples at 1 h, 2 h, 4 h, and 20 h (early sepsis) post-challenge intervals. The results of the paper-based sensor for the detection of MMP-9 levels in blood samples and BAL samples were compared with ELISA and Western Blot. We found that both blood and BAL levels of MMP-9 increased immediately and could be detected as early as 1 h in FIP mice post-challenge. Our work adds evidence to the assertion that MMP-9 is a reliable biomarker for the detection of sepsis at early stages.

Acute respiratory distress syndrome (ARDS) is a worldwide health concern. The pathophysiological features of ALI/ARDS include a pulmonary immunological response. The development of a rapid and low-cost biosensing platform for the detection of ARDS is urgently needed. In this study, we report the development of a paper-based multiplexed sensing platform to detect human NE, PR3 and MMP-2 proteases. Through monitoring the three proteases in infected mice after the intra-nasal administration of LPS, we showed that these proteases played an essential role in ALI/ARDS. The paper-based sensor utilized a colorimetric detection approach based on the cleavage of peptide–magnetic nanoparticle conjugates, which led to a change in the gold nanoparticle-modified paper sensor. The multiplexing of human NE, PR3 and MMP-2 proteases was tested and compared after 30 min, 2 h, 4 h and 24 h of LPS administration. The multiplexing platform of the three analytes led to relatively marked peptide cleavage occurring only after 30 min and 24 h. The results demonstrated that MMP-2, PR3 and human NE can provide a promising biosensing platform for ALI/ARDS in infected mice at different stages. MMP-2 was detected at all stages (30 min–24 h); however, the detection of human NE and PR3 can be useful for early- (30 min) and late-stage (24 h) detection of ALI/ARDS. Further studies are necessary to apply these potential diagnostic biosensing platforms to detect ARDS in patients.

Objectiv e While the role of CD4⁺ regulatory T cells in the resolution of lung inflammation is well documented, their potential contribution to the onset of the inflammatory response has been largely neglected. Herein, we assessed the impact of germline CD4 deficiency on the alveolar macrophage (AMΦ)-neutrophil (PMN) axis in initiating the LPS-induced lung inflammation. Methods In vivo, The levels of AMΦ and PMN in bronchoalveolar lavage fluid (BALF) and tissue histopathology were compared in wild type (WT) and CD4-deficient (CD4^−/−) mice at 4 hours after local LPS challenge (2–3 µg/g: intranasally). CD4 expression (flow cytometry) and representative functions of AMΦ (phagocytosis, cytokine secretion) and PMN (migration) of WT and CD4^−/− mice were compared ex vivo. Results LPS increased PMN infiltration of alveoli in WT mice; a response severely blunted in CD4^−/− mice. AMΦ isolated from WT mice captured more microbeads, but secreted less tumor necrosis factor-α, than AMΦ from CD4^−/− mice. Notably, as compared to PMN of WT mice, PMN of CD4^−/− mice had a diminished motility response to a relevant chemotactic stimulus (cell-free BALF). PMN expressed CD4 while AMΦ did not. Conclusions The motility defect of PMN derived from CD4^−/− mice is most likely due to an intrinsic loss of CD4, whereas the quantitative and qualitative shift of the AMΦ pool of CD4^−/− mice cannot.

MMP2 probe based on in vivo imaging technique for tracking ALI/ARDS in mice Matrix metalloproteinases (MMPs) are zinc-dependent proteinase which is capable of cleavage extracellular matrix proteins and enzymes that has an important role in lung dysfunction, MMP2 are produced by the lung alveolar epithelial and endothelial cells and other immune cells such macrophages. Therefore, MMP2 regulatory pathway are activated in alveolar macrophages during acute lung injury, which may exacerbate the pulmonary inflammation. Thus, there is an urgent need for the development of a fluorescence imaging for the tracking of ARDS. In this Study, we are modifying MMP2 probe based on in vivo in live mice treated with LPS intranasally. Our data demonstrated that MMP2 fluorescence probe, can provide a promising in vivo diagnostic for ALI/ARDS in infected mice at all stages (1h to 24h). Further studies are warranted to determine whether immune cells secretions of other proteases are affected by COVID-19 which lead to ARDS.

Sepsis is an inflammatory and immune response to microbial invasion that produces organs injury developed to systemic inflammatory respon seyndrome due to organs dysfunction. Sepsis is the indirect risk factor which can developed to ARDS. The most critical point for best prognosis is to diagnose the infection at its early stage, therefore, we need a novel sensitive and rapid method for screening is necessary for sepsis detection. In this study we designed a paper-based sensing platform for the detection of a number of expecting sepsis biomarkers such as, MMP9, α9β6 integrins, integrons RI Class1 and R2 Class 2 secreted during treated mice with FIP for 1h, 2h, 4h, and 20h (early /severe sepsis). Later on, we developed a multiplexing paper-based biosensor after adding BAL collected from septic mice on it, in order to target sepsis biomarker by cleavage of the four multiplex paper based peptides with comparison blood cultures of septic mice samples.