Anti-HLA-B7/HLA-B44 strong cross immunoreactivity observed in flow cytometry HLA-B27 immunotyping.

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies.

FRET causing misleading signal from fluorescein excited by the violet laser in flow cytometry.

Multiple immunolabeling introduces high risks of interferences between fluorescences. As an example, in analyzing T cell clonality, we recently reported a fluorescence resonance energy transfer (FRET) effect providing an unexpected signal on B770 (PE-Cy7) detector, on the Vß-PE positive CD3 APC-Alexa750+ T cell subsets. Here, we report another FRET effect produced by the violet laser in Vß-FITC positive CD3-Pacific Blue (PB) T cells providing signal on V550 (Krome Orange; KrO) detector. The study was performed on fresh whole blood, labeled with anti-CD3-PB, CD8-KrO, Vbeta FITC, Vbeta PE, CD4 AA750 then fixed, treated for erythrolysis, and washed before analysis on DxFlex cytometer from Beckman Coulter. Data were analyzed using Kaluza software. Using this panel, we repeatedly observed an added CD8dim-KrO (V550) cell population on all Vß FITC positive T cells. The unexpected green signal excited by the violet laser was still observed after removing anti-CD8-KrO (FMO) but disappeared where either anti-CD3-PB or anti-Vß-FITC was removed. The effect was also observed with an anti-TCR gamma delta-FITC labeling, but not with another FITC labeled antibody targeting a protein out of the CD3-TCR complex. The analysis of fluorochrome spectra confirms that PB emission and FITC excitation spectra partly overlap. This observation clearly reminds users that FRET can give misleading results in case of labeling of very close markers with complementary fluorochromes. This risk has to be considered in panel design. These observations clearly highlight the potential for FRET to give misleading results in cases where very close markers are labeled with complementary fluorochromes. This risk must be considered when designing panels. To our knowledge, this is the first description of a FRET between PB and FITC as acceptor thus excited by the violet laser.

Diagnostic Significance of hsa-miR-21-5p, hsa-miR-192-5p, hsa-miR-155-5p, hsa-miR-199a-5p Panel and Ratios in Hepatocellular Carcinoma on Top of Liver Cirrhosis in HCV-Infected Patients.

Early hepatocellular carcinoma (HCC) diagnosis is challenging. Moreover, for patients with alpha-fetoprotein (AFP)-negative HCC, this challenge is augmented. MicroRNAs (miRs) profiles may serve as potential HCC molecular markers. We aimed to assess plasma homo sapiens-(hsa)-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p-expression levels as a panel of biomarkers for HCC in chronic hepatitis C virus (CHCV) patients with liver cirrhosis (LC), especially AFP-negative HCC cases, as a step toward non-protein coding (nc) RNA precision medicine. SUBJECTS AND METHODS: 79 patients enrolled with CHCV infection with LC, subclassified into an LC group without HCC (n = 40) and LC with HCC (n = 39). Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p. RESULTS: Plasma hsa-miR-21-5p and hsa-miR-155-5p demonstrated significant upregulation, while hsa-miR-199a-5p demonstrated significant downregulation in the HCC group (n = 39) when compared to the LC group (n = 40). hsa-miR-21-5p expression was positively correlated with serum AFP, insulin, and insulin resistance (r = 0.5, p < 0.001, r = 0.334, p = 0.01, and r = 0.303, p = 0.02, respectively). According to the ROC curves, for differentiating HCC from LC, combining AFP with each of hsa-miR-21-5p, hsa-miR-155-5p, and miR199a-5p improved the diagnostic sensitivity to 87%, 82%, and 84%, respectively, vs. 69% for AFP alone, with acceptable specificities of 77.5%, 77.5%, and 80%, respectively, and AUC = 0.89, 0.85, and 0.90, respectively vs. 0.85 for AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios discriminated HCC from LC at AUC = 0.76 and 0.71, respectively, with sensitivities = 94% and 92% and specificities = 48% and 53%, respectively. Upregulation of plasma hsa-miR-21-5p was considered as an independent risk factor for HCC development [OR = 1.198(1.063-1.329), p = 0.002]. CONCLUSIONS: Combining each of hsa-miR-21-5p, hsa-miR-155-5p, and hsa-miR-199a-5p with AFP made it possible to identify HCC development in the LC patients' cohort with higher sensitivity than using AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios are potential HCC molecular markers for AFP-negative HCC patients. hsa-miR-21-5p was linked, clinically and via in silico proof, to insulin metabolism, inflammation, dyslipidemia, and tumorigenesis in the HCC patients' group as well as for an upregulated independent risk factor for the emergence of HCC from LC in the CHCV patients.

Association of gut-specific non-inflammatory T lymphocytes with chronic anorexia nervosa and constitutional thinness.

OBJECTIVE: Previous studies of AN showed low-grade inflammation. Are low-grade inflammation and circulating lymphocytes associated with chronic conditions? METHOD: Peripheric blood cytokines were measured using Luminex™ technology in a chronic AN cohort (mean = 67.42 months), compared to Constitutional Thinness (CT), Constitutional Obesity (CO), and Healthy Controls (HC). Secondarily a prospective cohort of chronic AN (mean = 54.11 months) was recruited to compare the functional lymphocyte profile in blood by flow cytometry to CT and HC. RESULTS: In the AN group, most cytokine concentrations were lower than in CT and HC groups. The IL-23 (98.02 pg/ml) was elevated related to HC and CO, and the IL-10 (4.178 pg/ml) was elevated versus CO. In the CT group, IL-9 (0.06216 pg/ml) was elevated compared to AN. The AN group had high Treg (9.259% of CD4+ ) and CD8+ Integrinß7+ (9.552% of CD3+ ) versus HC for lymphocyte populations. In CT group, elevated Treg (9.7% of CD4+ ) elevated percentage of CD4+ CCR9+ (5.867% of CD3+ ) and CD8+ Integrinß7+ (10.21% of CD3+ ) were found versus HC. CONCLUSIONS: The chronic state of AN and CT is surprisingly non-inflammatory with elevated Treg cells. These results suggest that maintaining a dysregulated response to intestinal antigens may contribute to maintaining AN.

Fluorescent energy transfer causing misleading signal in multicolor flow cytometry.

Multiple immunolabeling introduces high risks of interferences between fluorochromes. In an intend to analyze T cell clonality using CD3-APC Alexa750, CD4-Pac Blue, CD8-Krome Orange, CD56-PE-Cy7 and Vbeta clonotypes FITC and PE, we repeatedly observed a clear, unexpected signal on B770 (PE-Cy7) detector on the Vb subset mimicking a lymphoproliferative disorder. The aim of this study was to identify and prevent this source of artifact. The study was performed on a seven color panel performed on fresh whole blood, labeled, fixed, lyzed and analyzed on Navios Cytometer Beckman Coulter. Data were reanalyzed using Kaluza. Eleven tubes tested two clonotypes each with the same T cell backbone. Only one representative combination is presented. Using this panel, we observed repeatedly a strong CD56 PE-Cy7 (B755 LP) on all Vbeta1 T cell subsets but not on Vbeta 2-FITC T cells. The effect was still observed after removing CD56-PE-Cy7 (Full Minus One). Changing anti-CD3 APC-Alexa 750 with CD3APC, the B755 LP signal disappeared but a B695/30 signal appeared. Shifting to CD3-FITC abolished any unexpected red signal. This demonstrates a fluorescent energy transfer (FRET) between PE excited by the blue laser and Alexa750 to be excited by the red laser. Accordingly, the Vbeta PE fluorescence intensity was reduced when FRET happened and clearly increased when CD3-FITC was used instead. This observation clearly reminds that FRET can give misleading results in case of labeling of very close markers with complementary fluorochromes. This risk has to be considered in panel design.

Basophil activation test: Implementation and standardization between systems and between instruments.

The basophil activation test (BAT) is a good ex vivo alternative for measuring hypersensitivity to an allergen in sensitized patients but still lacks standardization. In this present study, we have implemented one of the systems and proposed inter-systems, inter-instrument standardization. Our method for basophil activation and labeling on whole blood: EDTA in one step using BasoflowEx® and FlowCast® . Setup on Navios and fluorescence targets converted to set up FACSCanto™ instrument. Our results: 1) A CD203c/CD63 (BasoflowEx) method was adapted for EDTA samples and simplified. 2) Final washing and concentration and use of time parameter help acquiring as many basophils as possible, spare acquisition time and noise. 3) The modified method was validated according to ISO15189 with a precision at 5.1% RCV, linearity between 1 and 1/8 of anti-IgE stimulation. Results were very close with CCR3/CD63 system (FlowCast). 4) Standardization, between systems and even between instruments. Mean Fluorescence Intensity targets are proposed using standard beads (Cytocal® ) middle peak: FITC = 19.4; PE = 28.8 on Navios® corresponding to FITC = 4,966; PE = 7,373 for FACSCanto. Data analyzed on common software (Kaluza® ) were very closely correlated. 5) Co-labeling of B cells (CD20+) gives the possibility to monitor a significant drop of basophils under stimulation that could explain some underestimation in case of strong hypersensitivity. In conclusion, BAT would strongly benefit from easy implementation [EDTA, one step stimulation/labeling, wash, full sample analysis over time parameter, B cell relative basophil count] and standardization of instrument settings on MFI targets whatever system or instrument is used. © 2017 International Society for Advancement of Cytometry.

Major influence of CD4 count at the initiation of cART on viral and immunological reservoir constitution in HIV-1 infected patients.

BACKGROUND: A persistent immune activation is observed in gut during HIV-1 infection, which is not completely reversed by a combined antiretroviral therapy (cART). The impact of the time of cART initiation may highly influence the size of the viral reservoir and the ratio of CD4(+)/CD8(+) T cells in the gut. In this study, we analyzed the characteristics of HIV rectal reservoir of long-term treated patients, regarding their blood CD4(+) T cells count at the time of cART initiation. RESULTS: Twenty-four consenting men were enrolled: 9 exhibiting a CD4(+) T cells count >350/mm(3) ("high-level CD4 group") and 15 < 350/mm(3) ("low-level CD4 group") in blood, at the start of cART. An immunophenotypical analysis of T and B cells subpopulations was performed in blood and rectal biopsies. HIV cell-associated DNA loads and qualitative intra-cellular RNA were determined in both compartments. The ratio of CD4(+)/CD8(+) T cells was significantly decreased in the blood but not in the rectum of the "low-level CD4 group" of patients. The alteration in ß7(+) CD4(+) T cells homing was higher in this group and was correlated to a low ratio of CD4(+)/CD8(+) T cells in blood. An initiation of cART in men exhibiting a low-level CD4 count was also associated with an alteration of B cells maturation. HIV blood and gut DNA reservoirs were significantly lower in the "high-level CD4 group" of men. A high HIV DNA level was associated to a detectable intracellular HIV RNA in rectum. CONCLUSIONS: An early initiation of cART could significantly preserve gut immunity and limit the viral reservoir constitution.

A Difficult and Rare Diagnosis of Autoimmune Enteropathy in a Patient Affected by Down Syndrome.

Patients with Down syndrome are more susceptible to autoimmune pathologies, in particular endocrine or digestive diseases such as celiac disease. Autoimmune enteropathy is another form of digestive autoimmune disease, non-gluten-dependant, more often diagnosed in male neonates with immunodysregulation and polyendocrinopathy such as the Immunodysregulation, Polyendocrinopathy, Enteropathy, X-linked syndrome. It also exists in the adult, but this pathology is less known and therefore frequently under-diagnosed. Clinical manifestations are similar to celiac disease, but not improved after a gluten-free diet. Autoimmune enteropathy is frequently associated with other autoimmune diseases, such as thyroiditis, myasthenia gravis, lupus or immune deficiencies, as Common Variable Immunodeficiency. Pathological analysis of intestinal biopsies can frequently distinguish autoimmune enteropathy and celiac disease. Autoimmune enteropathy usually has an important lymphoplasmacytic infiltration of the mucosa and a lack of intraepithelial lymphocytes in the gastrointestinal mucosal surface, while celiac disease usually has a polymorph infiltration of the mucosa and an important intraepithelial lymphocytes infiltration. Nevertheless, the two pathological patterns may overlap. Here we report the first case of a patient with Down syndrome associated to autoimmune enteropathy (initially diagnosed as celiac disease), chronic pancreatitis and cutaneous lupus erythematosus. Even if autoimmune pathologies are much more common in patients with Down syndrome, we would like to report on this rare and original association found in our patient.

Cytokine and cellular responses to human herpesvirus-6B in patients with B-acute lymphoblastic leukemia.

Primary infection with human herpesvirus-6 (HHV-6), is followed by its lifelong persistence in the host. Most T-cell responses to HHV-6 have been characterized using peripheral blood from healthy adults; however, the role of HHV-6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV-6 infection in patients with B-acute lymphoblastic leukemia (B-ALL) was analyzed. HHV-6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti- and pro-inflammatory cytokines (IL-4, IL-1, IL-6, IL-8, IL-12p70, IL-17a, TNF-α and IFN-γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV-6 virus loads in blood. Characterization of T-cell responses to HHV-6 showed low specific T-cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN-γ-producing T cells were detected in 0.03%-0.23% and in 0%-0.2% of CD4+T cells, respectively. Strong production of IL-6 was detected in medium supernatants of challenged T-cells whatever the HHV-6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF-α and IFN-γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV-6 in blood was identified, suggesting that HHV-6 is not strongly associated with development of B-ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV-6B specific immune responses may be below the detection threshold of the assays used.

Hsa-miR-21-5p reflects synovitis and tenosynovitis components of musculoskeletal ultrasonography Seven-joint scores in rheumatoid arthritis disease and predicts the disease flare.

Rheumatoid arthritis (RA) is characterized by progressive joint destruction with subsequent serious disability. Objective biomarkers of RA course progression are lacking, which necessitates the discovery of activity indicators and predictors of the disease outcome. Musculoskeletal Ultrasound Seven-joint Score (MSUS7) is proposed as a reliable technique to evaluate radiographic RA progression. Homo sapiens-microRNA-21-5p (hsa-miR-21-5p) plays an important role during joint remodeling and the pro-inflammatory process driving RA progression. We aimed to evaluate plasma hsa-miR-21-5p as a noninvasive RA activity biomarker and to investigate if hsa-miR-21-5p is linked to MSUS7 components in the context of RA activity. This cross-sectional study included 71 RA patients classified into inactive (n = 36) and active (n = 35) groups according to the Disease Activity Score 28-joint count with ESR (DAS28-ESR). Joints were assessed by MSUS7. Gray-scale ultrasound (GSUS) and power Doppler ultrasound (PDUS) were used to rate the synovitis, tenosynovitis, and erosion in the joints. Plasma hsa-miR-21-5p expression was measured by real-time PCR. The absolute count of regulatory T cell (Treg) was calculated after Treg frequency was assessed by flow cytometry. Results: Hsa-miR-21 expression was significantly up-regulated in the active RA group with a median fold change of 51.6 in comparison to the inactive cases with a median fold change of 7.7 (p < 0.001). Hsa-miR-21-5p was positively correlated with DAS28-ESR, C reactive protein (CRP), and rheumatoid factor (r = 0.7, p < 0.001, r = 0. 0.6, p < 0.001, and r = 0.4, p = 0.002, respectively), while negatively correlated with Treg absolute count (r = -0.4, p < 0.001). Hsa-miR-21-5p levels were correlated with synovitis and tenosynovitis in GSUS (r = 0.4, p < 0.001, r = 0.3, p = 0.025, respectively) and in PDUS (r = 0.5, p < 0.001 and 0.4, p = 0.001, respectively). The hsa-miR-21-5p accurately distinguished RA activity [AUC 0.933, 94.3% sensitivity, and 86.1% specificity]. Logistic regression analysis revealed hsa-miR-21-5p as an independent predictor for RA flare (OR = 1.228, p = 0.004). Hsa-miR-21-5p was linked to synovitis and tenosynovitis components of the MSUS7. Up-regulated hsa-miR-21-5p can be utilized as a predictor for RA disease flare.

Monitoring immune modulation by nutrition in the general population: identifying and substantiating effects on human health.

Optimal functioning of the immune system is crucial to human health, and nutrition is one of the major exogenous factors modulating different aspects of immune function. Currently, no single marker is available to predict the effect of a dietary intervention on different aspects of immune function. To provide further guidance on the assessment and interpretation of the modulation of immune functions due to nutrition in the general population, International Life Sciences Institute Europe commissioned a group of experts from academia, government and the food industry to prepare a guidance document. A draft of this paper was refined at a workshop involving additional experts. First, the expert group defined criteria to evaluate the usefulness of immune function markers. Over seventy-five markers were scored within the context of three distinct immune system functions: defence against pathogens; avoidance or mitigation of allergy; control of low-grade (metabolic) inflammation. The most useful markers were subsequently classified depending on whether they by themselves signify clinical relevance and/or involvement of immune function. Next, five theoretical scenarios were drafted describing potential changes in the values of markers compared with a relevant reference range. Finally, all elements were combined, providing a framework to aid the design and interpretation of studies assessing the effects of nutrition on immune function. This stepwise approach offers a clear rationale for selecting markers for future trials and provides a framework for the interpretation of outcomes. A similar stepwise approach may also be useful to rationalise the selection and interpretation of markers for other physiological processes critical to the maintenance of health and well-being.

A specific technique of immunolabelling of urinary small extracellular vesicle biomarkers for the diagnostic of renal cancer.

Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoural pathologies. Clear cell renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have set up a specific protocol for the preparation, the immunostaining characterization and the transmission electron microscopy observation of small EVs isolated from the urine of CCRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EV biomarkers for the detection and the monitoring of cancer diseases.

Effects of isolation and confinement on gastrointestinal microbiota-a systematic review.

Purpose: The gastrointestinal (GI) microbiota is a complex and dynamic ecosystem whose composition and function are influenced by many internal and external factors. Overall, the individual GI microbiota composition appears to be rather stable but can be influenced by extreme shifts in environmental exposures. To date, there is no systematic literature review that examines the effects of extreme environmental conditions, such as strict isolation and confinement, on the GI microbiota. Methods: We conducted a systematic review to examine the effects of isolated and confined environments on the human GI microbiota. The literature search was conducted according to PRISMA criteria using PubMed, Web of Science and Cochrane Library. Relevant studies were identified based on exposure to isolated and confined environments, generally being also antigen-limited, for a minimum of 28 days and classified according to the microbiota analysis method (cultivation- or molecular based approaches) and the isolation habitat (space, space- or microgravity simulation such as MARS-500 or natural isolation such as Antarctica). Microbial shifts in abundance, alpha diversity and community structure in response to isolation were assessed. Results: Regardless of the study habitat, inconsistent shifts in abundance of 40 different genera, mainly in the phylum Bacillota (formerly Firmicutes) were reported. Overall, the heterogeneity of studies was high. Reducing heterogeneity was neither possible by differentiating the microbiota analysis methods nor by subgrouping according to the isolation habitat. Alpha diversity evolved non-specifically, whereas the microbial community structure remained dissimilar despite partial convergence. The GI ecosystem returned to baseline levels following exposure, showing resilience irrespective of the experiment length. Conclusion: An isolated and confined environment has a considerable impact on the GI microbiota composition in terms of diversity and relative abundances of dominant taxa. However, due to a limited number of studies with rather small sample sizes, it is important to approach an in-depth conclusion with caution, and results should be considered as a preliminary trend. The risk of dysbiosis and associated diseases should be considered when planning future projects in extreme environments. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42022357589.

Transmission electron microscopy assessment of a novel method for isolating pure exosomes from serum.

Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

Proinflammatory role of monocytes in SARS-CoV-2 infection in chronic hemodialysis patients.

Background: Fully mature monocytes that express CD14, but not CD16, undergo phagocytosis within tissues, whereas non-classical monocytes, CD14-low CD16+, represent <11% of peripheral monocytes and have primary pro-inflammatory functions. Inflammation plays a major role in Covid-19 disease and adds to the inflammation caused by chronic hemodialysis. The aim of our study was to monitor monocyte subsets in five patients with end-stage kidney disease (ESKD) over a 1-year period after a mild Covid-19 infection. Five ESKD patients with a mild Covid-19 infection were monitored using CD14, CD16, CD300e, HLA-DR, CD64, and CD45 panels using a BD FACS Canto flow cytometer. Results: CD14-low CD16+ was dramatically (p=0,001) decreased in patients during Covid-19 infection, as previously described for patients without chronic renal failure. In addition, CD14-low CD16+ monocytes remained decreased for 10 months after recovery from Covid. Intermediate monocytes increased during Covid-19 infection and decreased 10 months after infection but this subtype of monocytes retained their inflammatory activity with a significant increase in HLA-DR expression after recovery from Covid infection. Conclusion: Our study shows that ESKD patients had a pro-inflammatory profile induced by Covid 19, but this status was prolonged significantly over a 10-month period. Thus, advanced renal failure treated by hemodialysis did not dramatically change the inflammatory response against to SARS Covid 2. It seems that monocytes retain their inflammatory status for many months in ESKD patients after a Covid-19 infection.

Monocytes subsets altered distribution and dysregulated plasma hsa-miR-21-5p and hsa-miR-155-5p in HCV-linked liver cirrhosis progression to hepatocellular carcinoma.

PURPOSE: The authors aim to investigate the altered monocytes subsets distribution in liver cirrhosis (LC) and subsequent hepatocellular carcinoma (HCC) in association with the expression level of plasma Homo sapiens (has)-miR-21-5p and hsa-miR-155-5p. A step toward non-protein coding (nc) RNA precision medicine based on the immune perturbation manifested as altered monocytes distribution, on top of LC and HCC. METHODS: Seventy-nine patients diagnosed with chronic hepatitis C virus (CHCV) infection with LC were enrolled in the current study. Patients were sub-classified into LC group without HCC (n = 40), LC with HCC (n = 39), and 15 apparently healthy controls. Monocyte subsets frequencies were assessed by flow cytometry. Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p and hsa-miR-155-5p expression. RESULTS: Hsa-miR-21-5p correlated with intermediate monocytes (r = 0.30, p = 0.007), while hsa-miR-155-5p negatively correlated with non-classical monocytes (r = - 0.316, p = 0.005). ROC curve analysis revealed that combining intermediate monocytes frequency and hsa-miR-21 yielded sensitivity = 79.5%, specificity = 75%, and AUC = 0.84. In comparison, AFP yielded a lower sensitivity = 69% and 100% specificity with AUC = 0.85. Logistic regression analysis proved that up-regulation of intermediate monocytes frequency and hsa-miR-21-5p were independent risk factors for LC progression to HCC, after adjustment for co-founders. CONCLUSION: Monocyte subsets differentiation in HCC was linked to hsa-miR-21-5p and hsa-miR-155-5p. Combined up-regulation of intermediate monocytes frequency and hsa-miR-21-5p expression could be considered a sensitive indicator of LC progression to HCC. Circulating intermediate monocytes and hsa-miR-21-5p were independent risk factors for HCC evolution, clinically and in silico proved.

Flow Cytometry, a Unique Biotechnology in Medical Applications.

It is good to see that the Journal of Clinical Medicine has recently published several good papers, highlighting the special input of cell analysis using flow cytometry [...].