Donor Serum Research Articles

Allogeneic Serum Eye Drops: A Randomized Clinical Trial to Evaluate the Clinical Effectiveness of Two Drop Sizes.

Allogeneic serum from blood donors is starting to be used to treat patients with dry eye disease (DED). However, the optimal dose is not known. We therefore aimed to evaluate the clinical efficaciousness and user-friendliness of micro-sized versus conventional-sized allogeneic serum eye drops (SEDs). In a randomized trial, patients with DED first receive micro-sized SEDs (7µl/unit) for 1month, followed by a 1-month washout, before receiving conventional-sized SEDs (50µl/unit) for 1month; or vice versa. The primary endpoint was the Ocular Surface Disease Index (OSDI) score. Secondary endpoints were tear break-up time (TBT), tear production (TP), and presence of corneal punctate lesions (CP). The user-friendliness of both application systems was also compared. A linear mixed model for cross-over design was applied to compare both treatments. Forty-nine patients completed the trial. The mean OSDI score significantly improved from 52 ± 3 to 41 ± 3 for micro-sized SEDs, and from 54 ± 3 to 45 ± 3 for conventional-sized SEDs. Non-inferiority (margin = 6) of micro-sized SEDs was established. We demonstrate a significant improvement for TBT in case of conventional-sized SEDs and for CP in both treatment groups. TP trended towards an improvement in both treatment groups. The user-friendliness of the conventional drop system was significantly higher. For the first time, non-inferiority of micro-sized allogeneic SEDs was established. The beneficial effect of both SED volumes was similar as measured by the OSDI score. Although user-friendliness of the micro drop system was significantly lower, it is an attractive alternative as it saves valuable donor serum. ClinicalTrials.gov (NCT03539159).

FRI485 High Interlaboratory Variability In Free Thyroxine Measurement Indicates Need For Method Standardization

Abstract Disclosure: A. Ribera: None. U. Danilenko: None. O. Sugahara: None. L. Collins: None. T. Buchannan: None. N. Vazquez: None. F. Pokuah: None. D. Wirtz: None. L. Zhang: None. A.N. Lyle: None. H.W. Vesper: None. Thyroid function tests are critical to diagnose diseases of the thyroid. Concerns about the accuracy and reliability of thyroid function tests, especially free thyroxine (FT4), have been stated by the clinical laboratory community for many years. In response to these concerns, the CDC Clinical Standardization Program (CSP) has created a standardization program for FT4 based on the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference system and is partnering with IFCC to standardize FT4 and harmonize TSH testing globally. As part of this program, CDC developed an accurate and sensitive reference measurement procedure (RMP) to assign reference values to individual donor sera. Assay manufacturers and research and clinical laboratories can use these sera to assess the analytical performance of their measurements, and monitor analytical performance over time. To obtain initial information about the analytical performance of FT4 and TSH measurements, an interlaboratory study among commercial assays and laboratory developed test was conducted. A panel of 40 blinded, fresh-frozen single donor patient serum samples with FT4 concentration ranging between 11-32 pmol/L concentrations was measured by participants and compared to the results obtained by CDC RMP for FT4. In addition, commercial assay manufacturers were asked to measure thyroid-stimulating hormone (TSH). Results from sixteen commercial assays and four laboratory developed test (LDT) participated. Preliminary results indicate a negative mean bias for all commercial FT4 assays ranging from -34.46% to -5.08%. Some of the assays agreed more closely to the RMP than others (four assays had mean bias over -25%). An equilibrium dialysis mass spectrometry-based assays had mean biases ranging from -8.86% to - 1.72% agreeing well with RMP. The data suggest that the observed bias can be effectively minimized through recalibration to the RMPs operated by the CDC CSP and the IFCC reference laboratory network. The mean TSH concentrations ranged from 0.34 to 21.56 uIU/mL with mean %CV of 11.42% (95%CI 8.07%-14.76%) suggesting good agreement between various platforms. Findings from this study will be used to customize CDC’s protocols and procedures for standardizing FT4 measurements. Presentation: Friday, June 16, 2023

A-032 Comparative Serum Free Thyroxine Measurements between Immunoassays and ID-LC/MS/MS Procedure Based on Equilibrium Dialysis

Abstract Background Serum free thyroxine (FT4) measurement is one of the most important clinical laboratory tests for diagnosis and classification of thyroid disorders. Currently serum FT4 is commonly measured with Immunoassays (IAs) in patient care. However, there are concerns about the accuracy of FT4 IAs. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for standardization of routine FT4 IAs. As FT4 RMP is not intended for routine clinical application, in the current study, we aim to develop a high-throughput routine FT4 method based on ED-isotope dilution liquid chromatography tandem mass spectrometry (ED-ID-LC/MS/MS). FT4 RMP was used as a reference to evaluate the measurement accuracy of our ED-ID-LC/MS/MS method and an IA method, respectively. Methods FT4 in dialysate was isolated from protein-bound T4 in serum by using a commercially available micro-ED plate after 18-h ED. The 13C6 labeled T4 was added into dialysate as an internal standard. FT4 in dialysate was purified with 96-well C18 SPE plate. FT4 in the dialysate samples was analyzed by using LC/MS/MS. The T4 certified primary reference material (IRMM-468) was used for assay calibration. FT4 IA measurement was conducted in clinical analyzers (Roche e411). A set of 40 single donor sera, covering a FT4 concentration range of 11.2–32.1 pmol/L were measured by IA and our ED-ID-LC/MS/MS, respectively. Deming regression analysis was performed to compare the results obtained by these two methods with the reference values assigned by FT4 RMP. Results The described high-throughput method based on ED-ID-LC/MS/MS could simultaneously quantified T4, T3, reverse triiodothyronine (rT3) in the dialysate within 4.5 min. The linear range of the routine FT4 assay covered 1–100 pg/mL. The assay sensitivity allowed detection of 0.3 pg/mL FT4 in serum, which is sufficient for FT4 measurement in clinically relevant ranges including hypothyroid patient samples. The throughput of the ED-ID-LC/MS/MS method could be further improved by application of automated liquid handling system. Deming regression analysis showed good agreement between RMP and the routine ED-ID-LC/MS/MS method with a slope close to 1 (P > 0.05) and an intercept close to 0 (P > 0.05), indicating that the results generated from the platform of high-throughput ED-ID-LC/MS/MS could be tracible to FT4 RMP. However, proportional bias (P < 0.05) with slope of 0.74 and constant bias (P < 0.05) with intercept of 2.6 was observed between RMP and IA tested, indicating calibration bias should be addressed to standardize IA. Conclusion In summary, the accuracy, sensitivity, and throughput of ED-ID-LC/MS/MS method are appropriate for FT4 measurements in clinical laboratories and in large epidemiologic studies to establish accurate reference intervals for FT4. Population data generated by FT4 ED-ID-LC/MS/MS method will support the use of standardized FT4 measurements.

SOLUBLE FORMS OF THE NKG2D RECEPTOR AND LIGAND NKG2DL1 IN PATIENTS WITH COLORECTAL CANCER

Relevance. Natural killer (NK) cells are considered to be effective cytotoxic cells of the tumor microenvironment, whose function is to effectively eliminate tumor cells. Their activation can occur when the NKG2D receptor interacts with the NKG2DL ligand expressed, for example, on tumor cells, which leads to the death of the latter. The aim of the study was a comparative study of the content of soluble forms of the sNKG2D receptor and its ligand sNKG2DL1 in the blood serum of healthy donors and patients with colorectal cancer (CRC), taking into account the clinical and morphological characteristics of the disease and prog-nosis. Material and methods. We examined 65 patients with CRC (32 women and 33 men), who were treated at the “National Medical Research Center of Oncology named after N.N. Blokhin” of the Ministry of Health of Russia aged (median 60 years) and 30 healthy donors (20 women and 10 men) aged (median 47 years). The clinical diagnosis in all patients was confirmed by the data of the morphological study of the tumor according to the Interna-tional Histological Classification of Tumors of the Digestive System (WHO, 2019), all had adenocarcinoma. The concentration of NKG2D and NKG2DL proteins was determined by ELISA in blood serum before treatment with Human B7-H3 Quantikine ELISA Kit reagents firm R&D (USA) in accordance with the manufacturer's instructions. The measurements were carried out on an automatic enzyme immunoassay analyzer BEP 2000 Advance (Siemens Healthcare Diagnostics, Germany). Statistical analysis of the obtained results was carried out using GraphPad Prizm v. 9. Overall survival analysis was performed by constructing survival curves using the Kaplan-Meier method. Results. In patients with colorectal cancer, a significant increase in the content of sNKG2DL in blood serum was found compared to the control group. sNKG2D concentrations did not differ between controls and CRC patients. No significant associations were found between the content of sNKG2D and sNKG2DL1 proteins in the blood serum of CRC patients and the main clinical and morphological characteristics of the disease. A decrease in the con-centration of sNKG2D was noted with the progression of the disease, and the highest content of sNKG2D was found in the localization of the tumor in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the cae-cum. At the same time, the levels of sNKG2D and sNKG2DL1 in the control group directly and significantly correlated with each other (r=0.453; p=0.014), while no such pattern was found in the group of CRC patients (r=0.014; p=0.935). sNKG2DL1 concentrations are not a prognostic factor in CRC, while high levels of sNKG2D tended to lead to a favorable prognosis of the disease, which indirectly confirms the positive significance of tumor in-filtration by NK cells. Conclusions. The mechanisms of the relationship between CRC avoidance of immune surveillance and the expression of the sNKG2D receptor and the sNKG2DL ligand are discussed as one of the directions in the study of immune checkpoints. We believe that the effect of CRC immunotherapy can be achieved by increasing the expression of NKG2D on immune cells and inducing the expression of NKG2DL in tumor cells, which is the basis of the con-cept of this line of research.

A cell-based, SARS-CoV-2 spike protein interaction assay to inform the neutralising capacity of recombinant and patient sera antibodies

IntroductionThe engagement of the SARS-CoV-2 spike protein with ACE2 is a critical step for viral entry to human cells, and, therefore, blocking this interaction is a major determinant of the efficacy of monoclonal antibody therapeutics and vaccine elicited serum antibodies. The emergence of SARS-CoV-2 variants has necessitated the development of adaptable assays that can be applied to assess the effectiveness of antibody-based therapeutics.MethodsThrough the testing of a range of recombinant spike proteins, we have developed a cell-based, ACE2/spike protein interaction assay that characterises monoclonal anti-spike protein antibodies and neutralising antibodies in donor serum. The assay uses high-content imaging to quantify cell-bound spike protein fluorescence.ResultsUsing spike proteins from the original “Wuhan” SARS-CoV-2 strain and the Delta and Omicron variants, we identified differential blocking activity of three monoclonal antibodies directed against the spike receptor-binding domain. Importantly, biological activity in the spike interaction assay translated to efficacy in a SARS-CoV-2 infection assay.DiscussionThe spike protein interaction assay can be used to monitor anti-spike antibodies against the major known SARS-CoV-2 variants and is readily adaptable for quantification of the impact of antibodies against new and emerging SARS-CoV-2 variants.

IL8 derived from macrophages inhibits CD8+ T-cell function by downregulating TIM3 expression through IL8-CXCR2 axis in patients with advanced colorectal cancer.

T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is a vital immune checkpoint that regulates the immune response. However, the specific role of TIM3 in patients with colorectal cancer (CRC) have rarely been studied. In this study, we investigated the effect of TIM3 on CD8+ T cells in CRC and explored the mechanism of TIM3 regulation in tumor microenvironment (TME). Peripheral blood and tumor tissues of patients with CRC were collected to evaluate TIM3 expression using flow cytometry. Cytokines in the serum of healthy donors and patients with early- and advanced-stage CRC were screened using a multiplex assay. The effects of interleukin-8 (IL8) on TIM3 expression on CD8+ T cells were analyzed using cell incubation experiments in vitro. The correlation between TIM3 or IL8 and prognosis was verified using bioinformatics analysis. TIM3 expression on CD8+ T cells was obviously reduced in patients with advanced-stage CRC, whereas a lower TIM3 expression level was associated with poorer prognosis. Macrophage-derived IL8, which could inhibit TIM3 expression on CD8+ T cells, was significantly increased in the serum of patients with advanced CRC. In addition, the function and proliferation of CD8+ and TIM3+CD8+ T cells were inhibited by IL8, which was partly depending on TIM3 expression. The inhibitory effects of IL8 were reversed by anti-IL8 and anti-CXCR2 antibodies. In summary, macrophages-derived IL8 suppresses TIM3 expression on CD8+ T cells through CXCR2. Targeting the IL8/CXCR2 axis may be an effective strategy for treating patients with advanced CRC.

The content of sEMMPRIN/CD147 in the blood serum of patients with bone tumors and its relationship with the clinical and morphological characteristics of the disease

Introduction. Enzyme-linked immunosorbent assay of biochemical markers is one of the most important methods for diagnosing tumors. One of these markers is an inducer of expression of matrix metalloproteases EMMPRIN/CD147. Changes in its expression are associated with the progression of some tumors. This study is the first work devoted to the study of the content of the soluble form of the transmembrane glycoprotein EMMPRIN (sEMMPRIN) in the blood serum of patients with various bone tumors.Aim. To study the content of sEMMPRIN in the blood serum of patients with malignant bone tumors, its relationship with the clinical and morphological characteristics of neoplasms and prognosis.Materials and methods. The study included 88 patients with malignant tumors (osteosarcoma – 37 cases, chondrosarcoma – 39, chordoma – 5, Ewing’s sarcoma – 7) and borderline (11 cases) bone neoplasms, of which 14 patients were under the age of 18 years. The control group consisted of 29 healthy donors, 8 of which were under the age of 18 years. The concentration of EMMPRIN was determined in the serum of patients and donors with reagents for direct enzyme immunoassay “Human EMMPRIN” (R&D, USA) in accordance with the manufacturer’s instructions and expressed in nanograms (ng) per 1 ml of blood serum. The obtained data were processed using the GraphPad Prizm 9.4 program. When comparing indicators and analyzing their relationships, we used the nonparametric Mann–Whitney and Kruskal–Wallis tests. Overall survival was analyzed using the Kaplan–Meier method.Results. Our analysis of the sEMMPRIN content in the blood serum of patients with bone tumors did not reveal statistically significant differences between the control group and patients with borderline and malignant tumors, both in adults and in children. At the same time, a trend towards a decrease in the level of sEMMPRIN in the blood serum was noted in the presence of a malignant neoplasm of the bone compared with the corresponding control group. Additionally, we found that the content of sEMMPRIN is associated with age and higher in the group of patients under 18 years of age, both among healthy donors and oncological patients. An analysis of the association of sEMMPRIN content with clinical and morphological characteristics did not reveal statistically significant patterns, however, a trend towards an increase in the level of the marker with disease progression in both studied age groups was observed, which is consistent with other studies conducted on other solid tumors.Conclusion. ELISA revealed the marker sEMMPRIN in the blood serum of all examined children and adults with borderline malignant bone tumors and healthy donors. At the same time, the levels of sEMMPRIN did not differ between the above groups, however, there was a tendency for a decrease in the concentration of the marker in patients with bone sarcomas compared with the control group, regardless of age.

Dried serum spots on pre-punched filter paper discs are ready-to-use storage and shipping devices for blood-borne antigens and antibodies

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the “Submerge and Dry” protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.

Comparison of human immunodeficiency virus and syphilis results in pooled sera versus individual samples of blood donors attending suez canal university hospital

Egypt is one of the countries where sexually transmitted diseases like human immunodeficiency virus (HIV) and syphilis are least prevalent. HIV and syphilis count less than one percent of total Egyptian population. An ELISA protocol for pooling serum samples is simple and may provide a way to reduce the cost and time needed for analysis. This study aimed to investigate the applicability and reliability of testing pooled sera of blood donors for HIV and syphilis compared to testing their individual sera and to assess the cost-effectiveness of this procedure. The study included 75 sera from randomly selected blood donors attending Suez Canal University hospital. Sera were screened by two ELISA kits, HIV Ag-Ab ELISA kit, and syphilis total antibody ELISA kit. Screening protocols were done by two sequential steps. At first, samples in pools of five were screened for both HIV and syphilis then, samples in positive pools were individually retested. There was no significant difference between the mean optical density for samples tested HIV and syphilis positive either individually or in pooled sera. There was no difference between the number of individual sera, tested positive for both HIV and syphilis and their pooled sera results (100 % positivity). There was significant decrease of the mean cost in one pool of 5 samples (16.5 L. E) in comparison to 5 individual samples (82.5 L. E) by HIV ELISA. Also, there was significant decrease of the mean cost in one pool of 5 samples (16 L.E) in comparison to 5 individual samples (80 L.E) by syphilis ELISA. In conclusion, the studied pooling protocol appeared reliable and can save up to 80 % of the cost for testing either HIV or syphilis by regular procedures.

A comprehensive evaluation of serum tRF-29-R9J8909NF5JP as a novel diagnostic and prognostic biomarker for gastric cancer.

Gastric cancer (GC) is a common malignant digestive system tumor. Since the early symptoms of GC are usually vague and the positive rate of common biomarkers of GC is low, it is of urgent need to find new biomarkers with good sensitivity and specificity to screen and diagnose GC patients. The tRNA-derived small RNAs (tsRNAs) are emerging small noncoding RNAs that play an essential role in cancer progression. In this study, we explored whether novel tsRNAs have the potential to serve as biomarkers for GC. Three tsRNAs significantly upregulated in GC were screened by the tsRFun database. The expression level of tRF-29-R9J8909NF5JP was detected by real-time fluorescence quantitative polymerase chain reaction. Agarose gel electrophoresis and Sanger sequencing were used to verify the characteristics of tRF-29-R9J8909NF5JP. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of tRF-29-R9J8909NF5JP. The χ2 test was used to analyze the correlation between tRF-29-R9J8909NF5JP expression level and clinicopathological parameters. Kaplan-Meier survival curves were used to analyze the correlation between tRF-29-R9J8909NF5JP expression levels and survival time of GC patients. In this study, the expression level of tRF-29-R9J8909NF5JP was significantly increased in GC tissues. The expression level of tRF-29-R9J8909NF5JP was considerably higher in the serum of GC patients than in the serum of gastritis patients and in the serum of healthy donors, and the expression level of tRF-29-R9J8909NF5JP was significantly decreased in the serum of GC patients after surgery. In addition, the χ2 test showed that the expression level of tRF-29-R9J8909NF5JP in GC serum was correlated with differentiation grade, T-stage, lymph node metastasis, tumor node metastasis stage, and neurological/vascular invasion. The results of the survival curve showed that the high expression of serum tRF-29-R9J8909NF5JP was associated with a low survival rate. ROC analysis showed that serum tRF-29-R9J8909NF5JP had higher diagnostic efficiency than common GC biomarkers, and the diagnostic efficiency was further improved by combining them. At the end of the study, we predicted the downstream of tRF-29-R9J8909NF5JP. The expression level of tRF-29-R9J8909NF5JP in the serum of GC patients can effectively identify GC patients and has higher efficacy than conventional biomarkers. In addition, serum tRF-29-R9J8909NF5JP can monitor the postoperative condition of GC patients, suggesting that it has the potential to become a biomarker for GC.

#5930 ENDOTHELIAL CELL PROTEOME AND SECRETOME ALTERATION UNDER CHRONIC UREMIC EXPOSURE

Abstract Background and Aims Dialysis therapies including peritoneal dialysis (PD) are described to lead to premature vascular aging, atherosclerosis and associated cardiomyopathies, which are associated with high cardiovascular morbidity and mortality. Chronic inflammation, incomplete clearance of uremic toxins, and dyslipidaemia, are factors affecting the vasculature system. However, how these factors affect the endothelium, the fist cell layer in contact with these molecules, and the molecular mechanisms triggered by uremic toxins remain poorly understood. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PD fluids reduce peritoneal damage in mesothelial and endothelial (EC) cells. Their potential systemic effects were not studied so far. Here, we demonstrate, in a newly developed model system, the changes induced by chronic exposure to serum of PD patients in the proteome and secretome of endothelial cells, and the effects of cytoprotective additives. Method For modelling systemic conditions of PD patients, primary EC were cultured for 5 passages in medium containing 10% serum collected from PD patients (n = 26) during regular PET tests, or from healthy donors (n = 12). Cytoprotective additives were added in parallel experiments. Cells were stably labelled (SILAC) to differentiate cell and donor serum proteins and the cellular proteome and secretome profiles were analysed by quantitative mass spectrometry. Prior to analysis of the secretome, equalizer beads, to enrich low abundant and deplete high abundant proteins, were used. ECIS (Electric Cell-substrate Impedance Sensing), was used to measure barrier function, growth rate, and permeability. Results Proteome analysis revealed perturbation of major cellular processes by serum of PD patients including inflammatory related processes such TLR regulation and complement activation, as well membrane related processes such as extracellular matrix interactions and junctions, and plasma lipoprotein remodelling. Addition of LiCl counteracted cell-adhesion related proteins. ECIS analysis showed that in uremic conditions the EC monolayer has a decreased barrier function compared to healthy conditions, and LiCl partially restores the tightness of the membrane. Secretome analysis showed differentially regulated proteins related to oxidative stress, senescence-associated secretory phenotype (SASP), and apoptosis. Interestingly, in the secretome, LiCl addition counter regulated INHBA and tissue factor pathway inhibitor 2, both proteins related to vascular calcification. Conclusion Surprisingly few studies have analysed uremic effects on EC using proteomics approaches, and no reports of chronic settings, modelling the patient situation, are available. Our data demonstrates that EC react to serum factors of PD patients with increased inflammation, permeability and a secretory profile. Interestingly, our model reflects many of the known effects on the vasculature, but unravels the molecular mechanism that may induce those processes. We have also identified potential mechanisms by which the addition of cytoprotective additives may counteract some of the uremic effects systemically. Secretomics data identified several proteins secreted by EC that are regulated in uremia with potential for cellular crosstalk with other cells of the vasculature, showing the potential to identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.

MiR-3960 inhibits bladder cancer progression via targeting of DEXI

PurposeMicroRNAs (miRNAs) are dominant cargo in exosomes and act as master regulators of cell function, inhibiting mRNA translation and affecting gene silencing. Some aspects of tissue-specific miRNA transport in bladder cancer (BC) and its role in cancer progression are not fully understood. Materials and methodsA microarray was used to identify miRNAs in mouse bladder carcinoma cell line MB49 exosomes. Real-time reverse transcription polymerase chain reaction was used to examine the expression of miRNAs in BC and healthy donor serum. Western blotting and immunohistochemical staining were used to examine the expression of dexamethasone-induced protein (DEXI) in patients with BC. CRISPR-Cas 9 was used to knock out Dexi in MB49, and flow cytometry was performed to test cell proliferation ability and apoptosis under chemotherapy. Human BC organoid culture, miR-3960 transfection, and 293T-exosome-loaded miR-3960 delivery were used to analyze the effect of miR-3960 on BC progression. ResultsThe results showed that miR-3960 levels in BC tissue were positively correlated with patient survival time. Dexi was a major target of miR-3960. Dexi knockout inhibited MB49 cell proliferation and promoted cisplatin- and gemcitabine-induced apoptosis. Transfection of miR-3960 mimic inhibited DEXI expression and organoid growth. In parallel, 293T-exosome-loaded miR-3960 delivery and Dexi knockout significantly inhibited subcutaneous growth of MB49 cells in vivo. ConclusionOur results demonstrate the potential role of miR-3960-mediated inhibition of DEXI as a therapeutic strategy against BC.

Mapping early serum proteome signatures of liver regeneration in living donor liver transplant cases.

The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.

Detection of serum M-protein in acetonitrile pecipitates by MALDI-TOF mass spectrometry: A novel, low-cost methodology.

Several studies have demonstrated the analytical sensitivity of MALDI-TOF mass spectrometry (MALDI-TOF MS) by immunoenrichment for M-protein analysis. We report the results of a novel, low-cost, reagent-based extraction process using acetonitrile (ACN) precipitation to enrich for κ and λ light chains which can be analysed by MALDI-TOF MS. Institutional Ethics committee approval was obtained. Serum samples from patients with monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasmacytoma, AL amyloidosis and Waldenström macroglobulinemia (WM) underwent ACN precipitation. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range: m/z- [M + 2H]2+: 11,550-12,300Da and λ m/z- [M + 2H]2+: 11,100-11,500Da. Images were acquired at a m/z range of 10,000-29,000Da. Corresponding serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE) and serum free light chain (sFLC) assay by nephelometry were performed for all the samples. Two-hundred-and-two serum samples were included in the study: MM- 184 (91%); AL amyloidosis- 2 (1%); plasmacytoma- 8 (4%); MGUS- 6 (3%) and WM- 2 (1%). All the SPEP positive samples were identified by MALDI-TOF MS. Out of 179 samples positive for M-protein by IFE, MALDI-TOF MS was positive in 176 samples (98%). Compared to IFE, the sensitivity and specificity of M-protein identification by MALDI-TOF MS were 98.3% and 52.2%, respectively. This study demonstrates the feasibility of qualitatively identifying M-protein without the need for antibody-based immunoenrichment, making the technique cost-effective.

Values of Donor Serum Lipids and Calcium in Predicting Graft Function after Kidney Transplantation: A Retrospective Study.

Delayed graft function (DGF) and early graft loss of renal grafts are determined by the quality of the kidneys from the deceased donor. As "non-traditional" risk factors, serum biomarkers of donors, such as lipids and electrolytes, have drawn increasing attention due to their effects on the postoperative outcomes of renal grafts. This study aimed to examine the value of these serum biomarkers for prediction of renal graft function. The present study consecutively collected 306 patients who underwent their first single kidney transplantation (KT) from adult deceased donors in our center from January 1, 2018 to December 31, 2019. The correlation between postoperative outcomes [DGF and abnormal serum creatinine (SCr) after 6 and 12 months] and risk factors of donors, including gender, age, body mass index (BMI), past histories, serum lipid biomarkers [cholesterol, triglyceride, high-density lipoprotein (HDL) and low-density lipoprotein (DL)], and serum electrolytes (calcium and sodium) were analyzed and evaluated. (1) Donor age and pre-existing hypertension were significantly correlated with the incidence rate of DGF and high SCr level (≥2 mg/dL) at 6 and 12 months after KT (P<0.05); (2) The donor's BMI was significantly correlated with the incidence rate of DGF after KT (P<0.05); (3) For serum lipids, merely the low level of serum HDL of the donor was correlated with the reduced incidence rate of high SCr level at 12 months after KT [P<0.05, OR (95% CI): 0.425 (0.202-0.97)]; (4) The serum calcium of the donor was associated with the reduced incidence rate of high SCr level at 6 and 12 months after KT [P<0.05, OR (95% CI): 0.184 (0.045-0.747) and P<0.05, OR (95% CI): 0.114 (0.014-0.948), respectively]. The serum HDL and calcium of the donor may serve as predictive factors for the postoperative outcomes of renal grafts after KT, in addition to the donor's age, BMI and pre-existing hypertension.

The changing profile of SARS-CoV-2 serology in Irish blood donors

BackgroundThe present study aimed to investigate the progression of the SARS-CoV-2 pandemic in Ireland over the first three waves of infection. MethodA selection of blood donor serum samples collected between February 2020 and December 2021 were analysed by various commercially available serological assays for antibodies to SARS-CoV-2 (n = 15,066). ResultsAn increase in seropositivity was observed between wave 1 (February to September 2020) and wave 2 (November and December 2020) of 2.20% to 3.55%. A large increase in estimated seroprevalence to 11.89% was observed in samples collected in February and March 2021 (wave 3 of infection).The rate of seropositivity varied by age group, with the highest rate observed in the youngest donors (18–29 years) peaking at 18.79% in wave 3. The results of spike antibody (anti-S) testing indicated that 44/1009 (4.36%) of seroreactive donors in wave 3 had a serological profile consistent with vaccination. By November 2021, we detected an overall seropositivity of 97.04%. ConclusionsThe present study provides a comprehensive estimation of the level of circulating SARS-CoV-2 antibodies in Irish blood donors, enabling differentiation between vaccination and natural infection, as well as real-time monitoring of the progression of the COVID-19 pandemic in Ireland. Seroepidemiology has a role in determining reliable estimates of transmission, infection fatality rates and vaccine uptake. The continued screening of blood donors for this purpose has the potential to generate important data to assist with the management of future waves of SARS-CoV-2.

Complement activation by AAV-neutralizing antibody complexes.

Treatment of monogenetic disorders using adeno-associated viral vectors (AAV) is an area of intense interest. AAV is a human pathogen and pre-existing capsid antibodies are prevalent in the population posing a challenge to safety and efficacy of AAV-mediated gene therapies. Here we investigated the risk of AAV-mediated complement activation when sera from a cohort of human donors was exposed to AAV9 capsid. Seropositive donor sera carrying neutralizing antibodies from a previous environmental exposure activated complement when admixed with AAV9 capsids and complement-activation was associated with donors who had higher levels of ant-AAV IgG1 antibodies. These findings were consistent with Mass spectrometry analysis that identified increased binding of immunoglobulins and complement factors when AAV9 capsids were admixed with seropositive sera. Finally, complement activation was abrogated after IgG-depletion using affinity columns or serum pre-treatment with an IgG degrading enzyme. Overall, these results demonstrate an important role of pre-existing neutralizing antibodies in activating complement; a risk that can be mitigated by employing adequate immunosuppression strategies when dosing seropositive patients with vector.

Review and assessment of the donor safety among plasma donors.

Review and assessment of the donor safety among plasma donors.

Donor Liver Blood Tests and Liver Transplant Outcomes: UK Registry Cohort Study.

Safely increasing organ utilization is a global priority. Donor serum transaminase levels are often used to decline livers, despite minimal evidence to support such decisions. This study aimed to investigate the impact of donor "liver blood tests" on transplant outcomes. This retrospective cohort study used the National Health Service registry on adult liver transplantation (2016-2019); adjusted regressions models were used to assess the effect of donor "liver blood tests" on outcomes. A total of 3299 adult liver transplant recipients were included (2530 following brain stem death, 769 following circulatory death). Peak alanine transaminase (ALT) ranged from 6 to 5927 U/L (median = 45). Donor cause of death significantly predicted donor ALT; 4.2-fold increase in peak ALT with hypoxic brain injury versus intracranial hemorrhage (adjusted P < 0.001). On multivariable analysis, adjusting for a wide range of factors, transaminase level (ALT or aspartate aminotransferase) failed to predict graft survival, primary nonfunction, 90-d graft loss, or mortality. This held true in all examined subgroups, that is, steatotic grafts, donation following circulatory death, hypoxic brain injury donors, and donors, in which ALT was still rising at the time of retrieval. Even grafts from donors with extremely deranged ALT (>1000 U/L) displayed excellent posttransplant outcomes. In contrast, donor peak alkaline phosphatase was a significant predictor of graft loss (adjusted hazard ratio = 1.808; 1.016-3.216; P = 0.044). Donor transaminases do not predict posttransplant outcomes. When other factors are favorable, livers from donors with raised transaminases can be accepted and transplanted with confidence. Such knowledge should improve organ utilization decision-making and prevent future unnecessary organ discard. This provides a safe, simple, and immediate option to expand the donor pool.

Abstract 601: Serological responses to tumor-associated adducts in pancreatic cancer

Abstract The incidence of pancreatic cancer has increased over the past several decades and is now the fourth leading cause of cancer death in the USA. Only 8.5% of the patients live beyond 5 years after their initial diagnosis. This dismal survival rate is partly explained by the fact that most pancreatic cancer cases are detected late when treatment options are limited. Novel biomarkers are crucially needed. It has long been recognized that cancer cells abnormally accumulate chemical groups covalently linked to proteins, DNA and other molecules. Humoral responses to these “adducts” have not been thoroughly explored. To investigate these responses, we developed an ELISA platform for the detection of serum IgG reactive to 93 adducts, including post-translational modifications known to contribute to tumorigenesis, progression, and metastasis. Our panel also includes oxidation-related modification, advanced glycation end products, and certain co-enzymes that qualify as adducts based on their binding properties. Using this assay, we measured anti-adduct antibodies in the serum of adult healthy donors (N=24; age range 40-80) as well as patients with pancreatic cancer (N=31; age range 50-90). Importantly, all patient specimens were collected before any treatment. Reactivity to all adducts was analyzed using a random forest predictive model with 10,000 bootstrapped decision trees to distinguish between the two groups. Results revealed a distinctive anti-adduct IgG reactivity profile for pancreatic cancer patients when compared to controls. Applying feature selection using the Boruta algorithm identified a reduced set of 19 target adducts recognized by IgG that most efficiently discriminated between healthy donors and pancreatic cancer cases. All 19 adducts were confirmed as IgG targets in cancer patients using multiple unpaired t-tests (p≤0,05). Aside from their predictive value, we reasoned that the detection of adduct-specific antibodies in cancer patients reflected their accumulation in the tumor cells. To test this hypothesis, we assessed the level of two representative target adducts by immunofluorescence staining in tumor tissue compared to control adjacent non-tumoral tissue or healthy tonsil. Results unequivocally show higher levels of these two adducts in cancer cells compared to non-cancer cells. Taken together, these preliminary findings support our hypothesis of the development of anti-adduct antibodies in pancreatic cancer patients. Furthermore, the specific antibody signature detected in these patients is consistent with the abnormal presence of corresponding adducts in the transformed cells. Our findings pave the way for the development of diagnostic tests based on the detection of serum IgG specific to tumor-associated adducts. Citation Format: Talita Aguiar, Shunya Mashiko, Poulomi Roy, Max Dietzel, Kesava Asam, Bradley Aouizerat, Jeanine Genkinger, Helen Remotti, Emmanuel Zorn. Serological responses to tumor-associated adducts in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 601.