Higher serum ß2-microglobulin is a predictive biomarker for cognitive impairment in spinal cord injury.

Objective: Recent studies have suggested that high levels of ß2-microglobulin are linked to cognitive deterioration; however, it is unclear how this connects to spinal cord injury (SCI). This study sought to determine whether there was any association between cognitive decline and serum ß2-microglobulin levels in patients with SCI. Methods: A total of 96 patients with SCI and 56 healthy volunteers were enrolled as study participants. At the time of enrollment, specific baseline data including age, gender, triglycerides (TG), low-density lipoprotein (LDL), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), smoking, and alcohol use were recorded. Each participant was assessed by a qualified physician using the Montreal cognitive assessment (MoCA) scale. Serum ß2-microglobulin levels were measured using an enzyme-linked immunosorbent assay (ELISA) reagent for ß2-microglobulin. Results: A total of 152 participants were enrolled, with 56 in the control group and 96 in the SCI group. There were no significant baseline data differences between the two groups (p > 0.05). The control group had a MoCA score of 27.4 ± 1.1 and the SCI group had a score of 24.3 ± 1.5, with the difference being significant (p < 0.05). The serum ELISA results revealed that the levels of ß2-microglobulin in the SCI group were considerably higher (p < 0.05) than those in the control group (2.08 ± 0.17 g/mL compared to 1.57 ± 0.11 g/mL). The serum ß2-microglobulin level was used to categorize the patients with SCI into four groups. As serum ß2-microglobulin levels increased, the MoCA score reduced (p < 0.05). After adjustment of baseline data, further regression analysis showed that serum ß2-microglobulin level remained an independent risk factor for post-SCI cognitive impairment. Conclusions: Patients with SCI had higher serum levels of ß2-microglobulin, which may be a biomarker for cognitive decline following SCI.

α1- and ß2-Microglobulin reduction ratios and survival in patients on predilution online haemodiafiltration.

AIM: ß2-Microglobulin (ß2-MG) and α1-microglobulin (α1-MG) have molecular weights of 11,800 and 33,000 Da, respectively. We studied the α1-MG and ß2-MG reduction ratios (RRs) and survival in patients on predilution online haemodiafiltration (Pre-OL-HDF). METHODS: Participants were 247 Pre-OL-HDF patients. α1-MG and ß2-MG RRs were assessed at baseline. Kaplan-Meier survival and Cox proportional hazard analyses were used. RESULTS: In 247 patients, the median age was 67 (56-73) years, the dialysis duration was 77 (46-150) months, and the diabetes prevalence was 47.4%. Twenty-two patients died over the 450-day study period. The mortality cut-off values using receiver-operating characteristic curves for the α1-MG and ß2-MG RRs were 20% and 80%, respectively. Survival rates were significantly (p < 0.05) higher in patients with α1-MG RRs ≥20% (n = 134) compared with patients with α1-MG RRs <20% (n = 113) and in patients with ß2-MG RRs ≥80% (n = 87) compared with patients with ß2-MG RRs <80% (n = 160). Cox models adjusting for diabetes and dialysis duration showed that α1-MG RR, ß2-MG RR, and pre- and postdialysis ß2-MG were risk factors for all-cause mortality; however, after additional adjustment for age, sex, and serum albumin, only ß2-MG RR and pre- and postdialysis ß2-MG were significant predictors of mortality (p < 0.05). α1-MG RRs were significantly correlated with ß2-MG RRs (ρ = 0.73, p < 0.0001) and serum albumin levels (ρ = 0.13, p < 0.05). CONCLUSION: In patients on Pre-OL-HDF, α1-MG RRs ≥20% and ß2-MG RRs ≥80% were associated with better survival, ß2-MG RR ≥80% and pre-and postdialysis ß2-MG levels were significant predictors of all-cause mortality, and α1-MG RR ≥20% may predict mortality.

Pathological review of cardiac amyloidosis using autopsy cases in a single Japanese institution.

AIM: Amyloidosis is a systemic or localized disease of protein deposition characterized by amorphous eosinophilic morphology and positivity of Congo Red staining. The typing of amyloidosis is becoming increasingly important because therapeutic agents for each amyloidosis type have been developed. Herein, the authors review the autopsy cases at an institution to reveal the putative Japanese characteristics of each amyloidosis type and evaluate the clinicopathological significance of each type. MATERIALS AND METHODS: A total of 131 autopsy cases of systemic and localized amyloidosis were retrieved for classification by immunohistochemistry. Immunohistochemistry for transthyretin, amyloid A (AA), immunoglobulin light-chain kappa and lambda, and ß2-microglobulin was performed for all cases. RESULTS: The 131 amyloidosis cases were classified as follows: 71 cases (54.2%) of transthyretin amyloidosis, 32 cases (24.4%) of AA amyloidosis, 8 cases (6.1%) of light-chain amyloidosis, and 5 cases (3.8%) of ß2-microglobulin amyloidosis, along with 15 equivocal cases (11.5%). All cases showed myocardial involvement of amyloidosis. Histopathologically, the transthyretin type was significantly associated with the interstitial and nodular patterns, and with the absence of the perivascular and endocardial patterns. The AA type was significantly associated with the perivascular and endocardial patterns, and with the absence of the nodular pattern. CONCLUSION: The authors revealed the putative characteristics of cardiac amyloidosis in Japan by using autopsy cases. About 90% of amyloidosis cases were successfully classified using only commercially available antibodies.

Development of a protein assay with copper chelator chromeazurol B, based on the biuret reaction.

This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, ß-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (ε) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (ε = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins.

Bilateral Renal Hypoplasia with High ß2-Microglobulinuria in the Neonatal Period.

Urinary ß2 microglobulin (ß2-MG) is a low-molecular-weight protein that is filtered by the glomerular basement membrane and absorbed by the proximal tubule epithelial cells. In perinatal management, urinary ß2-MG levels are used to assess intrauterine inflammation in newborns, since urinary excretion increases during inflammation. Furthermore, ß2-MG levels in fetal blood and urine are also used for predicting fetal renal function because ß2-MG is not transferred to the placenta. Herein, we reported a patient with persistent high urinary ß2-MG levels since neonatal period, who was later diagnosed with bilateral renal hypoplasia. If a newborn presents persistent hyper ß2-microglobulinuria even without hematuria or proteinuria, congenital renal malformations should be considered.

Circulating low CD4+/CD8+ ratio is associated with poor prognosis in Waldenstrom macroglobulinemia patients.

Waldenstrom macroglobulinemia (WM) is a rare type of non-Hodgkin lymphoma with great heterogeneity, and the data of peripheral blood T-lymphocyte subsets in WM are limited. This study aimed to investigate the clinical correlation and distribution of circulating T-lymphocyte subsets in newly diagnosed WM patients. We retrospectively searched medical records for 86 newly diagnosed WM patients. Comparisons of the absolute CD3+ T-lymphocyte count (ACD3C), CD4+ T-lymphocyte count (ACD4C), CD8+ T-lymphocyte count (ACD8C), and CD4+/CD8+ T-lymphocyte ratio (CD4+/CD8+) as continuous parameters in different groups were calculated. Univariate and multivariate analyses were used to assess prognostic factors for overall survival (OS) and progression-free survival (PFS). Young patients (<65 years) had lower ACD8C levels and a higher CD4+/CD8+ ratio. And the lower level of ß2-microglobulin (<3 mg/L) was associated with a higher CD4+/CD8+ ratio. With a median follow-up of 25 months, the univariate survival analysis showed that CD4+/CD8+ ratio inversion (CD4+/CD8+<1.5) was associated with shorter OS and PFS, and multivariate analysis confirmed that inverted CD4+/CD8+ ratio could be an independent adverse prognostic factor for OS and PFS. Additionally, initial treatment with rituximab or bortezomib significantly improved the PFS and OS of CD4+/CD8+ inversion patients but did not affect normal CD4+/CD8+ patients. We show that low circulating CD4+/CD8+ ratio at diagnosis is an adverse prognostic factor in WM patients and that first-line therapy which included rituximab or bortezomib significantly improved PFS and OS for patients with CD4+/CD8+ ratio less than 1.5.

Investigation of D76N ß2-Microglobulin Using Protein Footprinting and Structural Mass Spectrometry.

NMR studies and X-ray crystallography have shown that the structures of the 99-residue amyloidogenic protein ß2-microglobulin (ß2m) and its more aggregation-prone variant, D76N, are indistinguishable, and hence, the reason for the striking difference in their aggregation propensities remains elusive. Here, we have employed two protein footprinting methods, hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), in conjunction with ion mobility-mass spectrometry, to probe the differences in conformational dynamics of the two proteins. Using HDX-MS, a clear difference in HDX protection is observed between these two proteins in the E-F loop (residues 70-77) which contains the D76N substitution, with a significantly higher deuterium uptake being observed in the variant protein. Conversely, following FPOP-MS only minimal differences in the level of oxidation between the two proteins are observed in the E-F loop region, suggesting only modest side-chain movements in that area. Together the HDX-MS and FPOP-MS data suggest that a tangible perturbation to the hydrogen-bonding network in the E-F loop has taken place in the D76N variant and furthermore illustrate the benefit of using multiple complementary footprinting methods to address subtle, but possibly biologically important, differences between highly similar proteins.

Biomarker Discovery in Patients with Immunotherapy-Treated Melanoma with Imaging Mass Cytometry.

PURPOSE: Imaging mass cytometry (IMC) is among the first tools with the capacity for multiplex analysis of more than 40 targets, which provides a novel approach to biomarker discovery. Here, we used IMC to characterize the tumor microenvironment of patients with metastatic melanoma who received immunotherapy in efforts to find indicative factors of treatment response. In spite of the new power of IMC, the image analysis aspects are still limited by the challenges of cell segmentation. EXPERIMENTAL DESIGN: Here, rather than segment, we performed image analysis using a newly designed version of the AQUA software to measure marker intensity in molecularly defined compartments: tumor cells, stroma, T cells, B cells, and macrophages. IMC data were compared with quantitative immunofluorescence (QIF) and digital spatial profiling. RESULTS: Validation of IMC results for immune markers was confirmed by regression with additional multiplexing methods and outcome assessment. Multivariable analyses by each compartment revealed significant associations of 12 markers for progression-free survival and seven markers for overall survival (OS). The most compelling indicative biomarker, beta2-microglobulin (B2M), was confirmed by correlation with OS by QIF in the discovery cohort and validated in an independent published cohort profiled by mRNA expression. CONCLUSIONS: Using digital image analysis based on pixel colocalization to assess IMC data allowed us to quantitively measure 25 markers simultaneously on formalin-fixed, paraffin-embedded tissue microarray samples. In addition to showing high concordance with other multiplexing technologies, we identified a series of potentially indicative biomarkers for immunotherapy in metastatic melanoma, including B2M.

Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure.

Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.

New Potential Biomarkers for Chronic Kidney Disease Management-A Review of the Literature.

The prevalence of chronic kidney disease (CKD) is increasing worldwide, and the mortality rate continues to be unacceptably high. The biomarkers currently used in clinical practice are considered relevant when there is already significant renal impairment compromising the early use of potentially successful therapeutic interventions. More sensitive and specific biomarkers to detect CKD earlier on and improve patients' prognoses are an important unmet medical need. The aim of this review is to summarize the recent literature on new promising early CKD biomarkers of renal function, tubular lesions, endothelial dysfunction and inflammation, and on the auspicious findings from metabolomic studies in this field. Most of the studied biomarkers require further validation in large studies and in a broad range of populations in order to be implemented into routine CKD management. A panel of biomarkers, including earlier biomarkers of renal damage, seems to be a reasonable approach to be applied in clinical practice to allow earlier diagnosis and better disease characterization based on the underlying etiologic process.

Síndrome de hiperviscosidade em paciente com mieloma múltiplo / Hyperviscosity syndrome in multiple myeloma patients

O mieloma múltiplo é uma neoplasia progressiva e incurável de células B, caracterizado pela proliferação desregulada e clonal de plasmócitos na medula óssea. A síndrome de hiperviscosidade é uma das complicações relacionadas às gamopatias monoclonais, sendo considerada emergência oncológica. O objetivo deste estudo foi descrever o quadro clínico de um paciente diagnosticado com mieloma múltiplo que apresentou síndrome de hiperviscosidade, avaliando a prevalência de sinais e sintomas, bem como características fisiopatológicas dessa entidade clínica. Foi revisado o prontuário de um paciente internado na enfermaria da Clínica Médica do Hospital Regional do Cariri (CE) no período de junho a julho de 2018. Além disso, foi realizada revisão de literatura em base de dados (PubMed®) direcionada ao tema proposto. O diagnóstico de mieloma múltiplo foi comprovado por mielograma, sendo prontamente iniciada a corticoterapia e avaliada a resposta clínica após essa terapêutica. Apesar de incomum e menos frequentemente relacionada ao mieloma múltiplo, a síndrome de hiperviscosidade está relacionada a uma grande taxa de mortalidade quando apresenta diagnóstico tardio. A terapia de primeira linha indicada para a síndrome de hiperviscosidade foi a plasmaferese, no entanto, as condições clínicas (instabilidade hemodinâmica) impossibilitaram sua realização. O desfecho deste caso foi o óbito do paciente. Concluiu-se que o diagnóstico precoce e a intervenção terapêutica estão diretamente relacionados à ocorrência de menor incidência de complicações relacionadas ao mieloma múltiplo e à síndrome de hiperviscosidade.

Multiple myeloma is a progressive and incurable B-cell neoplasm characterized by unregulated and clonal proliferation of plasmocytes in the bone marrow. Hyperviscosity syndrome is one of the complications related to monoclonal gammopathies and is considered an oncological emergency. The aim of this study was to describe the clinical condition of a patient diagnosed with multiple myeloma who presented hyperviscosity syndrome, evaluating the prevalence of symptoms and signs, as well as the pathophysiological characteristics of this clinical entity. The medical records of a patient admitted to the Internal Medicine ward of the Hospital Regional do Cariri (CE) from June to July of 2018 were reviewed. In addition, we conducted a literature review in a database (PubMed®) directed to the theme proposed. The diagnosis of multiple myeloma was confirmed by myelogram, and corticosteroid therapy was promptly initiated and the clinical response was evaluated after this therapy. Although uncommon and less frequently related to multiple myeoloma, hyperviscosity syndrome is related to a high mortality rate when diagnosed late. The first line therapy indicated to hyperviscosity syndrome was plasmapheresis; however, the clinical conditions (hemodynamic instability) precluded its performance. The outcome of this case was the patient's death. Thus, it was concluded that early diagnosis and therapeutic intervention are directly related to the occurrence of lower incidence of complications related to multiple myeloma and hyperviscosity syndrome.

Association between renal function and co-infection with Clonorchis sinensis and Helicobacter pylori: a cross-sectional study.

BACKGROUND: Studies have shown that liver fluke infections may be associated with kidney injury and that Helicobacter pylori (Hp) may be involved in the pathogenesis of kidney diseases. However, no studies have reported the relationship between co-infection with Clonorchis sinensis (Cs) and Hp and renal function. The aim of this study was to examine the relationship between co-infection with Cs and Hp and estimated glomerular filtration rate (eGFR) in a general population, and gender-related differences were also investigated. METHODS: In the cross-sectional study, 4122 subjects from the Health Examination Center of Guangdong Provincial Hospital of Integrated Traditional Chinese and Western Medicine from January 2017 to December 2018 were enrolled. All participants underwent stool examination for the diagnosis of Cs infection and 13C-urea breath test (UBT) for the diagnosis of Hp infection. Participants were categorized into four groups: (1) co-infection with Cs and Hp group comprising 207 cases (Hp(+) + Cs(+) group), (2) Cs infection group comprising 1392 cases (Hp(-) + Cs(+)group), (3) Hp infection group comprising 275 cases (Hp(+) + Cs(-) group), and (4) non-infection group comprising 2248 cases (Hp(-) + Cs(-) group). Multiple linear regression analysis was performed to evaluate the relationship between co-infection with Cs and Hp and eGFR. RESULTS: Hp infection without Cs infection was present in 6.67% (275/4122) of subjects, while Cs infection without Hp infection was present in 33.77% (1392/4122) of subjects. Co-infection with Hp and Cs were present in 5.02% (207/4122) of subjects. Median age of the participants was 43 years (IQR 35-51). Most of the participants were male (2955/4122, 71.69%). Median eGFR was 96.61 ml/min/1.73 m2 (IQR 85.05-106.24). Co-infection with Cs and Hp was negatively associated with eGFR after full adjusting (ß = - 1.89, 95% CI: - 3.33 to - 0.45, p = 0.01). The relationship remained significant in females (ß = - 9.37, 95% CI: - 11.60 to - 7.1, p < 0.001), but not in males. CONCLUSION: Our findings suggest that co-infection with Cs and Hp may be associated with reduced renal function in females, but not in males.

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models.

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and ß2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and ß2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and ß2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic ß2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and ß2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and ß2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.

A nomogram to predict cadmium-induced renal tubular dysfunction.

Cadmium-induced renal dysfunction varies between individuals. It would be valuable to figure out those susceptible individuals or predict the risk of cadmium induced renal dysfunction. In the present study, we used a nomogram model to identify high-risk of cadmium-induced renal tubular dysfunction. 342 subjects living in low and moderately cadmium polluted areas were included in this study. The daily cadmium intake from food (FCd) was estimated using food survey. The cadmium in blood (BCd) and urine (UCd) were detected by using flame atomic absorption spectrometry. Urinary ß2Microglobulin (UBMG) was chosen as indicator of renal dysfunction. Logistic regression was used to select the independent risk factors for renal dysfunction. Bootstrap self-sampling and calibration curves were performed to quantify our modeling strategy. Age, sex, BCd and TCd were used to construct the nomogam in total population; age, BCd and TCd were adopted in women; age and BCd were used in men. The internal validation showed the C-index was 0.76 (95% 47 confidence interval (CI): 0.71-0.82) in total population, 0.74 (95% CI: 0.69-0.79) in men and 0.78 (95% CI: 0.72-0.84) in women. The area under the curve of the nomogram was 0.77 (95% CI: 0.71-0.83) in total population, 0.82(95% CI: 0.74-0.90) in women and 0.74(95% CI: 0.66-0.82) in men. Nomogram may be a rapid and simple risk assessment tool for predicting high-risk of renal tubular dysfunction in subjects exposed cadmium.

Removal of Urea, ß2-Microglobulin, and Indoxyl Sulfate Assessed by Absorbance and Fluorescence in the Spent Dialysate During Hemodialysis.

In this study, simultaneous removal assessment of marker molecules from three uremic toxin groups was performed during different hemodialysis treatment modalities using optical characteristics of spent dialysate. Results from optical measurements were compared with the results from chemical laboratory. Ten chronic dialysis patients, mean age 59 ± 15 years, were included in the study during 40 hemodialysis sessions. Low-flux hemodialysis (HD), high-flux hemodialysis (HF), and postdilutional online hemodiafiltration (HDF) with different settings were used. The reduction ratio (RR) and total removed solute (TRS) of three uremic solutes were determined: small molecular weight urea, middle molecular ß2-microglobulin (B2M), and protein-bound indoxyl sulfate (IS). Concentrations of these solutes in the spent dialysate were measured by laboratory (lab) and optical (opt) methods, in the serum by laboratory methods, and calculated RR values in percentage were compared accordingly. Total removed solute was obtained from the total dialysate collection (TDC) using lab and opt methods. The highest RR values were found for urea and B2M, and the lowest for IS. The difference between RR of lab and opt results estimated as mean accuracy (BIAS) was ≤8.1% for all three solutes. Good correspondence between TRS lab vs. opt was achieved, resulting in strong linear correlation values R from 0.727 for urea to 0.971 for IS. Accuracy for TRS values as BIAS ± standard error (SE), comparing lab vs. opt, showed no statistical difference for any of the observed uremic solutes (P > 0.05). The accuracy of the optical method was not influenced by the dialysis modality (HD, HF, and HDF).

The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/ß2m overexpression.

We previously demonstrated that HLA-E/ß2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, ß2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/ß2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/ß2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/ß2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αß T cells, and  to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/ß2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

Sensitive immunosensor based on high effective resonance energy transfer of lucigenin to the cathodic electrochemiluminescence of tris(bipyridine) Ru(II) complex.

Electrochemiluminescence resonance energy transfer (ECL-RET) has been attracting much focus as an effective approach for great ECL enhancement. Here, we found that lucigenin (Luc) could serve as a new energy transfer donor and greatly improve the cathodic ECL of bis(2,2'-bipyridyl)(4'-methyl-[2,2']bipyridinyl-4-carboxylicacid) ruthenium(II) (Ru(Bpy)2(Mcbpy)2+, acceptor). Then, both Luc and Ru(Bpy)2(Mcbpy)2+ were largely co-immobilized onto the PdCu nanocrystals and polyethyleneimine (PEI) modified single-walled carbon nanohorns (SWCNHs-PdCuNCs-PEI) through π-π stacking and crosslinking reaction, respectively. By this way, the excellent electrocatalytic behavior and high loading capability for both Luc and Ru(Bpy)2(Mcbpy)2+ of SWCNHs-PdCuNCs-PEI effectively facilitated the ECL reaction. Particularly, the co-immobilization strategy making the donor (Luc)/acceptor (Ru(Bpy)2(Mcbpy)2+) pairs co-exist in the same nano-composite could obviously increase the ECL-RET efficiency by shortening the electron-transfer path and reducing energy loss, further significantly improving the ECL signal. Combining the obtained nano-composite (Luc-SWCNHs-PdCuNCs-PEI-Ru(Bpy)2(Mcbpy)2+) with sandwiched immunoreaction, an ECL immunosensor was constructed for ß2-microglobulin (ß2-M) measurement. And as a result, it exhibited excellent performance in sensitivity, stability and selectivity. The establishment of the new effective donor/acceptor pairs for ECL-RET and the co-immobilization strategy of making those donor/acceptor pairs largely co-exist in the same nano-composite would greatly improve the ECL efficiency and motivate the wider application of ECL technology.