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Schematic representation of a microscope glass slide with AbMAs printed. Twenty-four AbMAs with two spots of rabbit IgG anti-human MMP-9 at 200 µg/mL in SIVG 0.05% were immobilized onto treated slides. Image created with BioRender.com.
Source publication
Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using...
Contexts in source publication
Context 1
... glass slides of 76 × 26 mm with 45 • frosted ends purchased from LineaLAB (#1053057, Badalona, Spain) were preactivated with an acid treatment carried out following different washing steps to make the surface hydrophobic (EP2048534A4, IMG Pharma Biotech S.L., Derio, Spain). Twenty-four antibodymicroarrays (AbMAs) were printed onto each slide using a four-column-six row format (Figure 7). Each AbMA had two replicate spots of rabbit IgG anti-human MMP-9 (#10327-R043, Sino Biological, Beijing, China) immobilized at 200 µg/mL onto SIVG printing solution at 0.05% (IMG Pharma Biotech S.L., Derio, Spain). ...
Context 2
... glass slides of 76 × 26 mm with 45° frosted ends purchased from LineaLAB (#1053057, Badalona, Spain) were preactivated with an acid treatment carried out following different washing steps to make the surface hydrophobic (EP2048534A4, IMG Pharma Biotech S.L., Derio, Spain). Twenty-four antibody-microarrays (AbMAs) were printed onto each slide using a four-column-six row format (Figure 7). Each AbMA had two replicate spots of rabbit IgG anti-human MMP-9 (#10327-R043, Sino Biological, Beijing, China) immobilized at 200 µg/mL onto SIVG printing solution at 0.05% (IMG Pharma Biotech S.L., Derio, Spain). ...
Citations
... Additionally, the process can become quite sluggish due to the symptoms and damage not commonly manifesting at the onset of the disease; they may gradually appear over time or occur intermittently [41]. In this context, a tool like CMMAs to identify reactive antibodies in the serum of patients with autoimmune disorders during the initial phases of the disease could pave the way for improved monitoring and control of such patients, even in asymptomatic periods [38]. ...
... Furthermore, ELISAs require a higher volume of reagents and samples, making it a less cost-effective technique than CMMA, given the type and origin of samples that are used as antigen fingerprints [33]. Consequently, having tools that overcome these drawbacks, like the CMMA technology, would enhance the prognosis for these patients and streamline the management of the disease and its treatment [38,80]. ...
... Aspects to improve in future studies include the exclusive fabrication of CMMA using only animal tissues and HEK 293 cells. This approach could enhance result reproducibility and address challenges associated with obtaining human tissues, as demonstrated in previous studies with CMMAs [38,80,81]. However, it is essential to consider that an optimal approach for monitoring and evaluating the immune system response would involve a test crafted with the patient's own tissue. ...
Immune disorders arise from complex genetic and environmental factors, which lead to dysregulation at the cellular and inflammatory levels and cause tissue damage. Recent research highlights the crucial role of reactive antibodies in autoimmune diseases and graft rejection, but their complex determination poses challenges for clinical use. Therefore, our study aimed to ascertain whether the presence of reactive antibodies against membrane antigens in tissues from both animal models and humans could serve as biomarkers in patients with autoimmune disorders. To address this issue, we examined the binding profile of serological antibodies against a diverse panel of cell membranes from the spleen, liver, and kidney tissues of monkeys, rats, and humans. After developing the cell membrane microarrays, human sera were immunologically assayed. The study was first conducted on sera from two groups, healthy subjects and patients with inflammatory and autoimmune disorders, and then optimized for kidney transplant patient sera. A significant increase in antibody reactivity against specific monkey kidney and spleen membranes was observed in the serum of patients with lupus nephritis, while kidney transplant patients showed a significant enhancement against human tissues and human embryonic kidney 293 cells. These results show the potential importance for clinical and basic research purposes of studying the presence of specific IgG against membrane antigens in patients’ serum as potential biomarkers of immune disorders. However, it is important to note that these results need to be verified in further studies with a larger sample size to confirm their relevance.
... To overcame this limitation, the use of whole cells or membrane fractions can be a potential alternative, as membrane antigens are presented together with the other characteristic compounds of those membranes such as lipids. The printing of whole cells or membrane fractions on glass slides has been used to study membrane proteins and lipids by radioligand binding studies, enzymatic analyses or immunoassays [19][20][21], which have demonstrated that not only the structure and functionality of membrane proteins, but also their antigenic profile seem to be preserved in these cell membrane microarrays (CMMAs). Thus, this research focused first on the development of a methodology for the identification of reactive antibodies in the serum of patients with autoimmune disorders, using CMMAs containing kidney, spleen and liver tissues from rats, monkeys, and humans. ...
... Additionally, the process can become quite sluggish due to the symptoms and damage not commonly manifesting at the onset of the disease; they may gradually appear over time or occur intermittently [25]. In this context, a tool like CMMAs to identify reactive antibodies in the serum of patients with autoimmune disorder during the initial phases of the disease could pave the way for improved monitoring and control of such patients even in asymptomatic periods [21]. This could involve early intervention with immunosuppressants or alternative therapies, preventing potential long-term damage to other tissues or organs before it manifests. ...
... Furthermore, they require higher volume of reagents and sample, making it less cost-effective techniques than CMMA [24]. Consequently, having tools that overcome these drawbacks like the CMMAs technology, would enhance the prognosis for these patients and streamline the management of the disease and its treatment [21,64]. CMMAs are cost-effective approach which also opens the possibility of creating useful POCT tools, since they require less sample, less processing times and they are less complex to carry out, with the possibility of multiplexing. ...
Immune disorders, characterized by dysregulation at cellular and inflammatory levels, result from a complex interplay of genetics and environmental factors that lead to an abnormal immune response against autoantigens, triggering tissue damage. Recent research highlights reactive antibodies as key players in autoimmune diseases and graft rejection, but the complexity of their determination limits their use in clinic. Hence, we studied the specific binding profile of serological antibodies against a panel of cellular membranes in order to determine whether this antigenic panel could be used to diagnose specific immune disorders in humans. For this purpose, cell membrane microarrays of spleen, liver, and kidney tissues of monkey, rat, and human were developed, and human sera were analyzed, including healthy controls, patients with autoimmune disorders, and kidney transplant patients. A significant increase in antibody reactivity against specific monkey kidney and spleen membranes was observed in the serum of patients with lupus nephritis, while kidney transplant patients showed a significant enhancement against human tissues and Human embryonic kidney 293 cells. These results show the potential importance for clinical and basic research purposes of studying the presence of specific IgG against membrane antigens in the serum of patients as potential biomarkers of immune disorders.
... Biological biomarkers such as MMP-9 can be utilized to accurately diagnose and monitor DED patients. 15 B-cell epitope mapping was used to select the ideal MMP-9 epitope peptide sequence for preparing the appropriate antibodies that are both reactive and specific. 7 The propensity of aas is most important for prediction of linear epitopes on B-lymphocyte cells. ...
Purpose
To obtain a reactive and specific antibody against truncated matrix metalloproteinase 9 (MMP-9), that has reactivity toward the native protein. Precision, accuracy, specificity, and sensitivity were evaluated using a point-of-care test.
Methods
An in silico study was used to confirm the anti peptide truncated MMP-9 is against native MMP-9. After an antibody titer assessment, purification, and characterization, the anti MMP-9 was assessed. The cut-off value was determined using the purified gelatinases of the supernatant HCT 116 cell line. The supernatant was purified by preparative native-polyacrylamide gel electrophoresis based on charge and size of the proteins. Furthermore, quality control (QC) of the results were performed following standard densitometry methods.
Results
A truncated MMP-9 is the major epitope peptide that can trigger the immune system to scavenge for a specific and reactive antibody against the native MMP-9. The MMP-9 native protein is purified from the supernatant of the HCT 116 cell line and the commercially available, full-length MMP-9. The cut-off value was estimated at 30 μg/mL. QC results indicated that the specificity was 80%, sensitivity was 96.7%, accuracy was 91%, and precision was 91.66%. The area under curve was 0.827 (P < 0.001). The positive predictive value was 83%, and the negative predictive value was 96%.
Conclusions
The antibody against the synthetic epitope peptide can detect the native MMP-9. Native MMP-9 is considered the main biomarker in an immunoassay POCT and is used to diagnose dry eye disease (DED). In accordance with QC results, MMP-9 point of care test can be utilized for screening patients suffering from DED.
... Finally, the protein expression level of MMP9 was increased to 898.77% by BAC treatment. MMP9 is a new diagnostic biomarker for DE, and our results showing an increase in MMP9 expression agree with published results that have led to the development of the biomarker [36,37]. HY7302 treatment significantly reduced the expression level of MMP9 by 46.02% compared with BAC-damaged cells. ...
... showing an increase in MMP9 expression agree with published results that have led to the development of the biomarker [36,37]. HY7302 treatment significantly reduced the expression level of MMP9 by 46.02% compared with BAC-damaged cells. ...
1) We investigated the effects of the Lactobacillus fermentum HY7302 (HY7302) in a mouse model of benzalkonium chloride (BAC)-induced dry eye, and the possibility of using HY7302 as a food supplement for preventing dry eye. (2) The ocular surface of Balb/c mice was exposed to 0.2% BAC for 14 days to induce dry eye (n = 8), and the control group was treated with the same amount of saline (n = 8). HY7302 (1 × 10 9 CFU/kg/day, 14 days, n = 8) was orally administered daily to the mice, and omega-3 (200 mg/kg/day) was used as a positive control. To understand the mechanisms by which HY7302 inhibits BAC-induced dry eye, we performed an in vitro study using a human conjunctival cell line (clone-1-5c-4). (3) The probiotic HY7302 improved the BAC-induced decreases in the corneal fluorescein score and tear break-up time. In addition, the lactic acid bacteria increased tear production and improved the detached epithelium. Moreover, HY7302 lowered the BAC-induced increases in reactive oxygen species production in a conjunctival cell line and regulated the expression of several apoptosis-related factors, including phosphorylated protein kinase B (AKT), B-cell lymphoma protein 2 (Bcl-2), and activated caspase 3. Also, HY7302 alleviated the expression of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and IL-8, and also regulated the matrix metallopeptidase-9 production in the conjunctival cell line. (4) In this study, we showed that L. fermentum HY7302 helps prevent dry eye disease by regulating the expression of pro-inflammatory and apoptotic factors, and could be used as a new functional food composition to prevent dry eye disease.
... Microarray data sets have long been valuable in identifying genes involved in cancer 20 and neuroinflammation. 21,22 Our findings revealed 12 differently upregulated genes. ...
Background
Levodopa (L‐DOPA) is considered the most reliable drug for treating Parkinson's disease (PD) clinical symptoms. Regrettably, long‐term L‐DOPA therapy results in the emergence of drug‐induced abnormal involuntary movements (AIMs) in most PD patients. The mechanisms underlying motor fluctuations and dyskinesia induced by L‐DOPA (LID) are still perplexing.
Methods
Here, we first performed the analysis on the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs) using linear models for microarray analysis (Limma) R packages from the Bioconductor project. 12 genes (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, Lypd1) were found to be upregulated. Six genes were validated on quantitative polymerase chain reaction and subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Areg LV_shRNA was used to knock down Areg to explore its therapeutic role in the LID model.
Results
Western blotting and immunofluorescence results show that AREG is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. Moreover, Areg knockdown reduced the protein expression of P‐ERK. In order to ascertain whether the inhibition of the ERK pathway (a common pathway known to mediate levodopa‐induced dyskinesia) could also impede Areg, the animals were injected with an ERK inhibitor (PD98059). Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the control group. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group.
Conclusion
Taken together, our results indicate unequivocal involvement of Areg in levodopa‐induced dyskinesia, thus a target for therapy development.
... Based on our previous research [27,35,36] and the use of previously characterized AbMAs [37,38], we analyzed the S100A6, MMP-9 and CST4 proteins in tear samples from a group of 32 individuals, control individuals (CTs) and SS patients. The use of AbMAs to analyze tear protein biomarkers was first validated by comparing MMP-9 tear biomarker quantification using an AbMA and ELISA [37]. ...
... Based on our previous research [27,35,36] and the use of previously characterized AbMAs [37,38], we analyzed the S100A6, MMP-9 and CST4 proteins in tear samples from a group of 32 individuals, control individuals (CTs) and SS patients. The use of AbMAs to analyze tear protein biomarkers was first validated by comparing MMP-9 tear biomarker quantification using an AbMA and ELISA [37]. The microarrays were fabricated on glass 76 × 26 mm microscope slides with 45 • frosted ends (#1053057, LineaLAB, Badalona, Spain), preactivated with an acid treatment involving different washing steps to make the surface hydrophobic (EP2048534A4, IMG Pharma Biotech S.L., Derio, Spain). ...
... To assess the diagnostic capacity of these analytes, tear biomarker concentrations were compared among the distinct sample types and the Cliff's value was calculated as a useful complementary analysis to test the corresponding hypothesis [52]. In short, tear S100A6 and MMP-9 are elevated in SS patients, albeit to a different extent, which ratifies the diagnostic potential of these biomarkers as their presence is enhanced in conditions of ocular surface inflammation, as described previously [28,37,38]. Moreover, CST4 is downregulated in SS, again in concordance with earlier data [30,38]. ...
Ocular diseases have a strong impact on individuals, the effects of which extend from
milder visual impairment to blindness. Due to this and to their prevalence, these conditions constitute
important health, social and economic challenges. Thus, improvements in their early detection and
diagnosis will help dampen the impact of these conditions, both on patients and on healthcare systems
alike. In this sense, identifying tear biomarkers could establish better non-invasive approaches to
diagnose these diseases and to monitor responses to therapy. With this in mind, we developed a solid
phase capture assay, based on antibody microarrays, to quantify S100A6, MMP-9 and CST4 in human
tear samples, and we used these arrays to study tear samples from healthy controls and patients
with Sjögren’s Syndrome, at times concomitant with rheumatoid arthritis. Our results point out that
the detection of S100A6 in tear samples seems to be positively correlated to rheumatoid arthritis,
consistent with the systemic nature of this autoinflammatory pathology. Thus, we provide evidence
that antibody microarrays may potentially help diagnose certain pathologies, possibly paving the
way for significant improvements in the future care of these patients.
... The obtained results confirmed the reliability of the AbMAs test for the quantification of the MMP-9 concentration in human tear samples. Thus, the authors concluded that the use of biomarker detection technologies is also advantageous for the evaluation of the prognosis and render the work of the ophthalmologist easier, thus leading to greater improvements in patients' health [2]. ...
This is the first part of a Special Issue on enzymes and enzymes inhibitors and their applications in medicine and diagnosis [...]
... [20][21][22][23] And its role is under investigation not only in cardiovascular pathology, but also in chronic Chagas disease, ocular pathology, ischemic stroke, in diabetes. [24][25][26][27][28] It is worth mentioning the work studying MMP-9 in renal diseases. For example, O. Zakiyanov et al. examined the dynamics of MMPs in patients with kidney disease. ...
... Several cytokine studies have identified increased levels of many proinflammatory cytokines in tears of treated glaucoma patients, including interleukins (IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL15, IL1B), TNF, C-C motif chemokine ligand 2 (CCL2), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and IFNG [47,76,77]. In studies focusing on prostaglandin analogue-treated glaucoma patients, IL1B, IL6, and matrix metallopeptidases 1, 3, and 9 (MMP1, MMP3, and MMP9) were seen to be increased, while TIMP metallopeptidase inhibitors 1 and 2 (TIMP1 and TIMP2) were decreased [78,79]. Instead of cytokine measurements, Wong et al. [80] performed a comparison using MS and discovered increased levels of S100A8, S100A9, secretoglobin family 2A member 1 (SCGB2A1), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). ...
Tear fluid forms the outermost layer of the ocular surface and its characteristics and composition have been connected to various ocular surface diseases. As tear proteomics enables the non-invasive investigation of protein levels in the tear fluid, it has become an increasingly popular approach in ocular surface and systemic disease studies. Glaucoma, which is a set of multifactorial diseases affecting mainly the optic nerve and retinal ganglion cells, has also been studied using tear proteomics. In this condition, the complete set of pathophysiological changes occurring in the eye is not yet fully understood, and biomarkers for early diagnosis and accurate treatment selection are needed. More in-depth analyses of glaucoma tear proteomics have started to emerge only more recently with the implementation of LC-MS/MS and other modern technologies. The aim of this review was to examine the published data of the tear protein changes occurring during glaucoma, its topical treatment, and surgical interventions.
Neuroinflammation has a significant impact on different pathologies, such as stroke or spinal cord injury, intervening in their pathophysiology: expansion, progression, and resolution. Neuroinflammation involves oxidative stress, damage, and cell death, playing an important role in neuroplasticity and motor dysfunction by affecting the neuronal connection responsible for motor control. The diagnosis of this pathology is performed using neuroimaging techniques and molecular diagnostics based on identifying and measuring signaling molecules or specific markers. In parallel, new therapeutic targets are being investigated via the use of bionanomaterials and electrostimulation to modulate the neuroinflammatory response. These novel diagnostic and therapeutic strategies have the potential to facilitate the development of anticipatory patterns and deliver the most beneficial treatment to improve patients’ quality of life and directly impact their motor skills. However, important challenges remain to be solved. Hence, the goal of this study was to review the implication of neuroinflammation in the evolution of motor function in stroke and trauma patients, with a particular focus on novel methods and potential biomarkers to aid clinicians in diagnosis, treatment, and therapy. A specific analysis of the strengths, weaknesses, threats, and opportunities was conducted, highlighting the key challenges to be faced in the coming years.
