Fallacies of a purely virtual platform: Virtual plus reality versus virtual reality - A case study.

Pretransplant immunological assessment of a transplant donor has evolved significantly over the last few decades with the advent of testing platforms with enhanced sensitivity and varying formats. The single antigen bead assay (SAB) assay, a virtual crossmatch (vXM) is used extensively and considered the gold standard for defining donor-specific antibodies (DSA) in many parts of the World. A country like India, is however challenged by the lack of adequate representation of locally frequent HLA alleles and hence in our institution, we continue to perform a physical crossmatch (pXM) on the Complement Dependent Cytotoxicity and flow cytometry platforms alongside the SAB. We report here a case report where the discrepancy between platforms of testing have raised certain pertinent questions in our interpretation of the vXM.

Anti-Hla Donor-Specific Antibodies Are Associated to Infection and Not to the Engraftment Rate in Outpatient Haploidentical Hematopoietic Cell Transplantation.

Background: Recipients of a related haploidentical stem cell transplant (haplo-SCT) can have preformed antibodies to HLA donor's antigens. Objective: The aim of the study was to evaluate the engraftment rate and major clinical associations of anti-HLA donor-specific antibodies (DSA) at two mean fluorescence intensity (MFI) thresholds in recipients of an outpatient haplo-SCT. Methods: Seventy haplo-HCT recipients were analyzed. A virtual crossmatch was performed using the donor HLA typing and the recipient's anti-HLA DSA test results. Data for anti-HLA-A, -B, -C, and -DR were analyzed. Recipients with DSA ≥ 500 MFI were considered positive, and those with < 500 were considered negative; the same was adopted for MFI ≥ 1000. Results: Post-transplant infection was higher in recipients with DSA ≥ 500 MFI (84.6%, p = 0.041). First-year mortality was higher in DSA-positive patients ≥ 500 MFI, p = 0.004, and DSA ≥ 1000 MFI, p = 0.022, than in DSA-negative recipients. Graft failure in the first 100 days was not associated with DSA ≥ 500 or ≥ 1000 MFI. There was no difference in acute (a-GVHD) or chronic (c-GVHD) graft versus host disease between DSA-positive and negative patients. Conclusions: There was no association of anti-HLA DSA at MFI ≥ 500 and ≥ 1000 with graft failure, however, increased infection and 1st-year mortality were documented in related haplo-HCT at the MFI cutoffs studied.

Anti-Hla Donor-Specific Antibodies Are Associated to Infection and Not to the Engraftment Rate in Outpatient Haploidentical Hematopoietic Cell Transplantation

ABSTRACT Background: Recipients of a related haploidentical stem cell transplant (haplo-SCT) can have preformed antibodies to HLA donor's antigens. Objective: The aim of the study was to evaluate the engraftment rate and major clinical associations of anti-HLA donor-specific antibodies (DSA) at two mean fluorescence intensity (MFI) thresholds in recipients of an outpatient haplo-SCT. Methods: Seventy haplo-HCT recipients were analyzed. A virtual crossmatch was performed using the donor HLA typing and the recipient's anti-HLA DSA test results. Data for anti-HLA-A, -B, -C, and -DR were analyzed. Recipients with DSA ≥ 500 MFI were considered positive, and those with < 500 were considered negative; the same was adopted for MFI ≥ 1000. Results: Post-transplant infection was higher in recipients with DSA ≥ 500 MFI (84.6%, p = 0.041). First-year mortality was higher in DSA-positive patients ≥ 500 MFI, p = 0.004, and DSA ≥ 1000 MFI, p = 0.022, than in DSA-negative recipients. Graft failure in the first 100 days was not associated with DSA ≥ 500 or ≥ 1000 MFI. There was no difference in acute (a-GVHD) or chronic (c-GVHD) graft versus host disease between DSA-positive and negative patients. Conclusions: There was no association of anti-HLA DSA at MFI ≥ 500 and ≥ 1000 with graft failure, however, increased infection and 1st-year mortality were documented in related haplo-HCT at the MFI cutoffs studied. (REV INVEST CLIN. 2023;75(5):249-58)

Umbilical Cord Blood Transplantation for Myelodysplastic Syndromes with Donor-Specific Anti-HLA Antibodies against HLA-DP.

The presence of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) against anti-HLA-A, -B, -C, and -DRB1 in HLA-mismatched hematopoietic stem cell transplantation (HSCT) is associated with graft failure. DSAs against HLA-A, -B, -C, and -DRB1 with a mean fluorescence intensity (MFI) of greater than > 1,000 was shown to increase the risk of graft failure in single-unit umbilical cord blood transplantation (UCBT). Nevertheless, the impact of DSAs against HLA-DP or -DQ on transplantation outcomes is not fully understood. In this report, we present a case of UCBT in a patient with myelodysplastic syndrome who was positive for DSAs against HLA-DP with MFI of 1,263 before UCBT but successfully achieved neutrophil engraftment. If HLA-DP or -DQ is mismatched in UCBT, evaluating DSAs against HLA-DP or -DQ is crucial to avoid graft failure. However, the criteria for DSAs against HLA-A, -B, -C, and -DRB1 may not be directly applicable to those against HLA-DP or -DQ.

A retrospective observational study to estimate the risk of HLA alloimmunization with blood transfusion: Can the risk be reduced by leucodepletion?

BACKGROUND: In this retrospective study, our aim was to find the effect of leucodepleted (LD) blood transfusions on the formation of anti-HLA-antibodies when compared to non-leucodepleted (non-LD) transfusions using Luminex-based method. METHODS: In this study, Luminex single antigen bead assay (L-SAB) and HLA typing were performed on 310 patients. Test positivity rates (as MFI - Mean florescence intensity) were analyzed according to the different sensitization events and gender. RESULTS: Of the 310 patients included in the study, 58.06% (180) patients were male and 41.93% (130) were female. The average age of the patients was 42.86 (±12.37) years. In this study, test positivity rates were significantly lower in the patients who received LD RBC units than in those who received non-LD RBC units (28.43% = 29 of 102 Vs 55.22% = 74 of 134, p < 0.05). In our study, transfusion combined with a history of pregnancy had higher number of significant HLA antibodies compared to cases where transfusion was the only sensitization event (81.81% = 18/22 Vs 39.71% = 85/214, p < 0.05). In addition, anti-HLA-antibodies-MFI were significantly (p < 0.01) higher in non-LD patients compared to LD patients. CONCLUSION: Patients who received LD RBC units had a significantly lower rate of transfusion-associated alloimmunization compared to those who received non-LD RBC units. Multiparous women had a high risk for transfusion-related alloimmunization compared to both nulliparous women and male patient. Furthermore, class I-anti-HLA-antibodies (HLA-B and HLA-A + B) were significantly associated with pregnancy sensitization and/or blood transfusion as a single sensitization.

Transcriptomic landscapes of effective and failed liver regeneration in humans.

Background & Aims: Although extensive experimental evidence on the process of liver regeneration exists, in humans, validation is largely missing. However, liver regeneration is critically affected by underlying liver disease. Within this project, we aimed to systematically assess early transcriptional changes during liver regeneration in humans and further assess how these processes differ in people with dysfunctional liver regeneration. Methods: Blood samples of 154 patients and intraoperative tissue samples of 46 patients undergoing liver resection were collected and classified with regard to dysfunctional postoperative liver regeneration. Of those, a matched cohort of 21 patients were used for RNA sequencing. Samples were assessed for circulating cytokines, gene expression dynamics, intrahepatic neutrophil accumulation, and spatial transcriptomics. Results: Individuals with dysfunctional liver regeneration demonstrated an aggravated transcriptional inflammatory response with higher intracellular adhesion molecule-1 induction. Increased induction of this critical leukocyte adhesion molecule was associated with increased intrahepatic neutrophil accumulation and activation upon induction of liver regeneration in individuals with dysfunctional liver regeneration. Comparing baseline gene expression profiles in individuals with and without dysfunctional liver regeneration, we found that dual-specificity phosphatase 4 (DUSP4) expression, a known critical regulator of intracellular adhesion molecule-1 expression in endothelial cells, was markedly reduced in patients with dysfunctional liver regeneration. Mimicking clinical risk factors for dysfunctional liver regeneration, we found liver sinusoidal endothelial cells of two liver disease models to have significantly reduced baseline levels of DUSP4. Conclusions: Exploring the landscape of early transcriptional changes of human liver regeneration, we observed that people with dysfunctional regeneration experience overwhelming intrahepatic inflammation. Subclinical liver disease might account for DUSP4 reduction in liver sinusoidal endothelial cells, which ultimately primes the liver for an aggravated inflammatory response. Impact and implications: Using a unique human biorepository, focused on liver regeneration (LR), we explored the landscape of circulating and tissue-level alterations associated with both functional and dysfunctional LR. In contrast to experimental animal models, people with dysfunctional LR demonstrated an aggravated transcriptional inflammatory response, higher intracellular adhesion molecule-1 (ICAM-1) induction, intrahepatic neutrophil accumulation and activation upon induction of LR. Although inflammatory responses appear rapidly after liver resection, people with dysfunctional LR have exaggerated inflammatory responses that appear to be related to decreased levels of LSEC DUSP4, challenging existing concepts of post-resectional LR.

Current Status of Biomarkers and Molecular Diagnostic Tools for Rejection in Liver Transplantation: Light at the End of the Tunnel?

Strategies to minimize immune-suppressive medications after liver transplantation are limited by allograft rejection. Biopsy of liver is the current standard of care in diagnosing rejection. However, it adds to physical and economic burden to the patient and has diagnostic limitations. In this review, we aim to highlight the different biomarkers to predict and diagnose acute rejection. We also aim to explore recent advances in molecular diagnostics to improve the diagnostic yield of liver biopsies.

Detection of donor-specific HLA antibodies: A retrospective observation in 350 renal transplant cases.

BACKGROUND: The main objective of this study was to determine the results of the cell-based assay (CDC-XM and FC-XM), and correlate with the results of solid phase assay (L-SAB). METHODS: In this retrospective study, 350 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, FC-XM and L-SAB screening with their corresponding donor. RESULTS: T-cell-FC-XM showed a sensitivity of 71.43% and a specificity of 91.50% for detecting class I L-SAB (+), while B-cell-FCXM showed a sensitivity of 94.94% and a specificity of 61.99% for detecting class II L-SAB (+). On the other hand, T-CDC-XM showed a sensitivity of 32.14% and a specificity of 98.64% for detecting class I L-SAB (+), while B-CDC-XM showed a sensitivity of 44.30% and a specificity of 94.83% for detecting class II L-SAB (+). In this study, the results indicated that DSA class I MFI value of 2845 and above significantly (p ≤0.001) correlated with T-cell-FC-XM positivity, while MFI value of 4585 and above (p ≤0.001) showed strong predictive accuracy of a positive T-cell-CDC-XM. However, DSA class II MFI cut-off of 1988 and above significantly (p ≤0.001) correlated with B-cell-FC-XM positivity, while MFI value of 5986 and above (p ≤0.001) showed strong predictive accuracy of a positive B-cell-CDC-XM. CONCLUSIONS: Our study showed that CDC-XM has poor sensitivity, while FC-XM has poor specificity to detect DSA. L-SAB has good correlation with T-cell-FC-XM (p < 0.0001) but not with B-cell-FC-XM (P = 0.31). DSA strength >2845 and > 1988 significantly correlated with T-cell-FC-XM positivity and B-cell-FC-XM positivity, respectively. While, a MFI value of >4585 and > 5986 significantly correlated with T-cell-CDC-XM positivity and B-cell-CDC-XM positivity, respectively. These MFI cut-off values could serve as a surrogate marker for CDC-XM and FC-XM tests and may help in resolving the limitations of cell-based techniques. In conclusion, we found that L-SAB is more sensitive and specific than CDC-XM and FC-XM and therefore may be used as a test of choice.

In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis among children worldwide, yet there is no vaccine for RSV disease. This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles (CNP) to modulate reactive oxygen (ROS) and nitrogen (RNS) species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo. Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods, respectively. Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential. In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages. Specifically, cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels. Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFα and IL-12p70, while simultaneously decreasing M2 CD206 expression. Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice. Notably, cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation, avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils, which are associated with RSV-mediated inflammation. In conclusion, we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.

TNFR1 Contributes to Activation-Induced Cell Death of Pathological CD4+ T Lymphocytes During Ischemic Heart Failure.

CD4+ T cells turn pathological during heart failure (HF). We show that the expression of tumor necrosis factor (TNF)-α and tumor necrosis factor receptor (TNFR1) increases in HF-activated CD4+ T cells. However, the role of the TNF-α/TNFR1 axis in T-cell activation/proliferation is unknown. We show that TNFR1 neutralization during T-cell activation (ex vivo) or the loss of TNFR1 in adoptively transferred HF-activated CD4+ T cells (in vivo) augments their prosurvival and proliferative signaling. Importantly, TNFR1 neutralization does not affect CD69 expression or the pathological activity of HF-activated TNFR1-/- CD4+ T cells. These results show that during HF TNFR1 plays an important role in quelling prosurvival and proliferative signals in CD4+ T cells without altering their pathological activity.

The natural history of de novo donor-specific HLA antibodies after kidney transplantation.

Background: De novo donor-specific HLA antibodies (dnDSA) are key factors in the diagnosis of antibody-mediated rejection (ABMR) and related to graft loss. Methods: This retrospective study was designed to evaluate the natural course of dnDSA in graft function and kidney allograft survival and to assess the impact of mean fluorescence intensity (MFI) evolution as detected by annual Luminex® screening. All 400 kidney transplant recipients with 731 dnDSA against the last graft (01/03/2000-31/05/2021) were included. Results: During 8.3 years of follow-up, ABMR occurred in 24.8% and graft loss in 33.3% of the cases, especially in patients with class I and II dnDSA, and those with multiple dnDSA. We observed frequent changes in MFI with 5-year allograft survivals post-dnDSA of 74.0% in patients with MFI reduction ≥ 50%, 62.4% with fluctuating MFI (MFI reduction ≥ 50% and doubling), and 52.7% with doubling MFI (log-rank p < 0.001). Interestingly, dnDSA in 168 (24.3%) cases became negative at some point during follow-up, and 38/400 (9.5%) patients became stable negative, which was associated with better graft survival. Multivariable analysis revealed the importance of MFI evolution and rejection, while class and number of dnDSA were not contributors in this model. Conclusion: In summary, we provide an in-depth analysis of the natural course of dnDSA after kidney transplantation, first evidence for the impact of MFI evolution on graft outcomes, and describe a relevant number of patients with a stable disappearance of dnDSA, related to better allograft survival.

New evidence for dietary fatty acids in the neutrophil traffic between the bone marrow and the peripheral blood.

Chronic administration of a high-fat diet in mice has been established to influence the generation and trafficking of immune cells such as neutrophils in the bone marrow, the dysregulation of which may contribute to a wide range of diseases. However, no studies have tested the hypothesis that a short-term, high-fat diet could early modulate the neutrophil release from bone marrow at fasting and at postprandial in response to a high-fat meal challenge, and that the predominant type of fatty acids in dietary fats could play a role in both context conditions. Based on these premises, we aimed to establish the effects of different fats [butter, enriched in saturated fatty acids (SFAs), olive oil, enriched in monounsaturated fatty acids (MUFAs), and olive oil supplemented with eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids] on neutrophil navigation from bone marrow to blood in mice. The analysis of cellular models for mechanistic understanding and of postprandial blood samples from healthy volunteers for translational purposes was assessed. The results revealed a powerful effect of dietary SFAs in promotion the neutrophil traffic from bone marrow to blood via the CXCL2-CXCR2 axis. Dietary SFAs, but not MUFAs or EPA and DHA, were also associated with increased neutrophil apoptosis and bone marrow inflammation. Similar dietary fatty-acid-induced postprandial neutrophilia was observed in otherwise healthy humans. Therefore, dietary MUFAs might preserve bone marrow health and proper migration of bone marrow neutrophils early in the course of high-fat diets even after the intake of high-fat meals.

The effects of acute exercise and inflammation on immune function in early-stage prostate cancer.

Background: The immune system plays a vital role in cancer development and progression. Strategies mobilizing cytotoxic cells of the immune system to combat immunosuppression in cancer may help to improve the treatment response of patients. To this end, we aimed to characterize the anti-cancer effect of acute exercise, including the involvement of inflammatory signals. Patients and methods: Twenty patients with early-stage prostate cancer (PCa) scheduled to undergo prostatectomy performed one bout of acute exercise consisting of a watt-max test and four high-intensity intervals. Natural Killer (NK), NKT-like and T cell phenotype, NK cell cytotoxic activity (NKCA), and NKCA per-cell against cell lines of leukemia (K562) and prostate cancer origin (LNCaP and PC-3) were assessed. Inflammatory markers (TNF-α, IL-6, and CRP) were measured in plasma. Results: Exercise increased NK, NKT-like, and CD8 T cell concentration in the circulation. Furthermore, exercise shifted immune cells towards a mature and cytotoxic phenotype e.g., NK cells exhibited higher CD57 as well as lower NKG2A expression. NKT-like and CD8 cells exhibited elevated CD57, TIGIT and Granzyme-B expression. Exercise significantly improved NKCA against K562 (+16% [5%; 27%]; p = 0.002) and LNCaP (+24% [14%; 34%]; p < 0.001) but not PC-3. NKCA per NK cell decreased during exercise and increased 1-h post exercise compared to baseline in K562, LNCap, and PC-3 cell lines. Baseline IL-6 correlated with lymphocyte, monocyte and T cell concentration pre-exercise and inversely correlated with the fold-change of mobilized lymphocytes and CD8 T cells during exercise. Furthermore, baseline IL-6 and TNF-α inversely correlated with NKCA against PC-3 cells during exercise. Conclusions: Acute exercise mobilized cytotoxic immune cells and improved NKCA in patients with PCa whereas low-grade inflammation might impair the response. Whether the observed improvements impact long-term outcomes warrant further investigation. Clinical trial number: NCT03675529.

Cumulative mean fluorescent intensities of HLA specific antibodies predict antibody mediated rejections after kidney transplantation.

It is still not fully elucidated which pretransplant donor-specific HLA antibodies (DSA) are harmful after kidney transplantation. In particular, it needs to be clarified whether cumulative mean fluorescence intensities (MFI) against multiple HLA specificities have a predictive value for allograft function. Our retrospective single centre study analyzed preformed HLA antibodies determined by Luminex™ Single Antigen Bead (SAB) assay, including C1q addition, in relation to rejection and clinical outcome in 255 cross match negative kidney allograft recipients. Only 33 recipients (13%) of the total cohort showed early AMR during the first year posttransplant, but in patients with pre-transplant DSA the rate was increased to 15 out of 40 (38%). Three year graft survival was significantly shorter in patients with histological signs of AMR compared with patients without AMR or with no biopsy (74%, 92%, and 97%, respectively, p < 0.0001). In patients with HLA-DSA, a cumulative MFI value of all HLA antibodies of more than 103.000 indicated the highest risk for AMR posttransplant (p = 0.01). In conclusion, in patients with HLA-DSA, the cumulative MFI value may help to further stratify the risk of AMR after kidney transplantation.

Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement.

T cell engaging bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3+ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

Effects of different sensitization events on HLA alloimmunization in renal transplant cases; a retrospective observation in 1066 cases.

BACKGROUND: Patients awaiting solid organ transplantation may develop anti-HLA antibodies after sensitization events such as transfusions, pregnancies, or previous transplantations. However, the effects of a particular sensitization event on HLA alloimmunization have not been well studied in parallel using cell-based assays and solid-phase assays. In this study, we evaluated and compare how different sensitization events affect the HLA antibody screening (HLA-Ab) and donor specific antibody (DSA) status in solid renal organ transplantation patients. METHODS: HLA antibody (HLA-Ab) screening tests like complement-dependent cytotoxicity crossmatch (CDC-XM), flow cytometry crossmatch (FC-XM) and Luminex panel-reactive antibody (L-PRA) were performed in all 1066 patients (635 males and 431 females). If any of these tests turned out to be positive, a Luminex single antigen bead (L-SAB) assay was performed for DSA identification. Test positive rates and antibody strengths were analyzed according to the different sensitization events and gender. RESULTS: In this study, HLA-Ab screening tests positive rates (L-PRA, FC-XM and CDC-XM) were significantly higher in patients with previous transplantation (73.91%, 100% and 56.52% p < 0.001), previous pregnancy (57.46%, 70.14% and 18.85% p < 0.001) or blood transfusion (27.33%, 35.55% and 7.33% p < 0.001) compared with patients without a sensitizing event (6.17%, 13.58% and 1.09). In this study, re-transplantation group showed significantly stronger antibody strength (DSA) than non sensitized group (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 2659, 3329; P < 0.001) and those with single sensitization events of transfusion (class I and II MFI 11418.04, 17,837.78 vs class I and II MFI 5790.26, 6004.16; P < 0.001) or pregnancy (class I & II MFI 11418, 17,837 vs class I and II MFI 8631.71, 7253.29; P < 0.001). CONCLUSIONS: Pregnancy and blood transfused had high allo-immunization rate for class I HLA antigens. While re-transplantation patients had high allo-immunization rate for both the HLA classes (HLA- class I and HLA- class II). Re-transplantation group showed significantly stronger antibody strength, followed by pregnancy and then transfusion.

The different predictive effects of the intensity and proportion of CD20 expression on the prognosis of B-lineage acute lymphocyte leukemia.

The prognostic effects of the CD20 positivity have been studied extensively in B-lineage acute lymphocyte leukemia (B-ALL) patients, but the results remain controversial. The aim of this study is to investigate the different predictive effects of the intensity and proportion of CD20 expression on the prognosis for B-ALL patients by retrospective analysis. The mean fluorescence intensity (MFI) and percentage of CD20 on B-ALL cells from 206 patients with B-ALL were dynamically measured by flow cytometry, and their optimal cut-off values were determined using the receiver operating characteristic curve. Changes in MFI and percentage of CD20 at various time points and their relationship with prognosis were analyzed. We found that a low baseline CD20 MFI or high CD20 proportion was significantly associated with shorter 5-year overall survival and progression-free survival, and the combination of these two factors could more accurately predict worse survival for B-ALL patients. Furthermore, low CD20 MFI or a high CD20 proportion had different predictive effects for ALL patients with different clinical characteristics and could serve as an independent risk factor for adverse prognosis. There were significant decreases in both the intensity and proportion of CD20 after recurrence in the absence of rituximab treatment, particularly with CD20 intensity. Notably, the decrease of CD20 intensity after recurrence indicated a more shortened survival time. Finally, we conclude that a low intensity or high proportion of CD20 expression may be used as an indicator for inferior prognosis for B-ALL patients. CD20 intensity is more likely to be a more universal biomarker for worse prognosis.

Significance of Luminex-crossmatch assay and its mean fluorescence intensity - a retrospective observation in 380 renal transplant cases.

<b>Introduction:</b> Cell-based complement-dependent cytotoxicity crossmatch (CDC-XM) and solid phase assays were introduced for assessing HLA antibodies. However, the complexity of data from cell-based and solid phase assays have led to potential confusion about how to use the results for clinical decision making. </br></br> <b> Aim:</b> Aim of this study was to compare results of cell-based assay and solid phase assay, to evaluate the usefulness of L-XM for pretransplant detection of HLA class I and II donor-specific IgG antibodies, correlate the mean fluorescence intensity (MFI) values of class I and class II L-XM assay and with CDC-XM and L-PRA (panel reactive antibodies) results. </br></br> <b> Methods:</b> In this retrospective study, 380 prospective renal transplant recipients were tested for the presence of HLA antibodies by CDC-XM, IgG-L-XM, IgG-L-PRA & L-SAB screening with their corresponding donor. </br></br> <b>Results:</b> Fifty-one recipients (13.42%) had a positive CDC-XM. L-XM was positive in 125 recipients (32.89%); class I-L-XM was positive in 46 (36.80%) cases, and class II-L-XM was positive in 58 (46.4%) cases and 21 (16.8%) samples were positive for class I and class II. High background was present in 22 (5.87%) samples, the results of which were confirmed by retesting or by correlation with L-PRA and L-SAB assays. </br></br> <b>Conclusion:</b> The introduction of more sensitive approaches for the detection of anti-HLA-IgG-antibodies, such as L-XM and L-PRA assay, has allowed the identification of anti-HLA-antibodies in recipient serum which is not usually identified by CDC-XM alone. However, L-XM has some limitations; they can be overcome if we combine this assay with L-PRA.

Natural exosome-like nanovesicles from edible tea flowers suppress metastatic breast cancer via ROS generation and microbiota modulation.

Although several artificial nanotherapeutics have been approved for practical treatment of metastatic breast cancer, their inefficient therapeutic outcomes, serious adverse effects, and high cost of mass production remain crucial challenges. Herein, we developed an alternative strategy to specifically trigger apoptosis of breast tumors and inhibit their lung metastasis by using natural nanovehicles from tea flowers (TFENs). These nanovehicles had desirable particle sizes (131 nm), exosome-like morphology, and negative zeta potentials. Furthermore, TFENs were found to contain large amounts of polyphenols, flavonoids, functional proteins, and lipids. Cell experiments revealed that TFENs showed strong cytotoxicities against cancer cells due to the stimulation of reactive oxygen species (ROS) amplification. The increased intracellular ROS amounts could not only trigger mitochondrial damage, but also arrest cell cycle, resulting in the in vitro anti-proliferation, anti-migration, and anti-invasion activities against breast cancer cells. Further mice investigations demonstrated that TFENs after intravenous (i.v.) injection or oral administration could accumulate in breast tumors and lung metastatic sites, inhibit the growth and metastasis of breast cancer, and modulate gut microbiota. This study brings new insights to the green production of natural exosome-like nanoplatform for the inhibition of breast cancer and its lung metastasis via i.v. and oral routes.

TGF-ß1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-ß1) and whether EMT could be induced through TGF-ß1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-ß1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-ß1. Analysis of EMT markers showed that mRNA levels changed with TGF-ß1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-ß1 treatment. Invasion assays showed that TGF-ß1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-ß1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.