MicroRNAs as Potential Graft Rejection or Tolerance Biomarkers and Their Dilemma in Clinical Routines Behaving like Devilish, Angelic, or Frightening Elements.

Allograft rejection is a widespread complication in allograft recipients with chronic kidney disease. Undertreatment of subclinical and clinical rejection and later post-transplant problems are caused by an imperfect understanding of the mechanisms at play and a lack of adequate diagnostic tools. Many different biomarkers have been analyzed and proposed to detect and monitor these crucial events in transplant outcomes. In this sense, microRNAs may help diagnose rejection or tolerance and indicate appropriate treatment, especially in patients with chronic allograft rejection. As key epigenetic regulators of physiological homeostasis, microRNAs have therapeutic potential and may indicate allograft tolerance or rejection. However, more evidence and clinical validation are indispensable before microRNAs are ready for clinical prime time.

Measurable residual disease study through three different methods can anticipate relapse and guide early interventions in childhood acute lymphoblastic leukemia.

INTRODUCTION: Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Measurable residual disease (MRD, previously named minimal residual disease) study can guide therapy adjustments or preemptive interventions that might avoid hematological relapse. METHODS: Clinical decision making and patient outcome were evaluated in 80 real-life childhood ALL patients, according to the results observed in 544 bone marrow samples analyzed with three MRD methods: multiparametric flow cytometry (MFC), fluorescent in-situ hybridization (FISH) on B or T-purified lymphocytes and patient-specific nested reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Estimated 5 year overall survival and event-free survival were 94% and 84.1%, respectively. A total of 12 relapses in 7 patients were associated with positive MRD detection with at least one of the three methods: MFC (p < 0.00001), FISH (p < 0.00001) and RT-PCR (p = 0.013). MRD assessment allowed the anticipation of relapse and adapted early interventions with different approaches including chemotherapy intensification, blinatumomab, HSCT and targeted therapy to halt relapse in five patients, although two of them relapsed afterwards. CONCLUSION: MFC, FISH and RT-PCR are complementary methods for MRD monitoring in pediatric ALL. Although, our data clearly show that MDR positive detection is associated with relapse, continuation of standard treatment, intensification or other early interventions were able to halt relapse in patients with different risks and genetic background. More sensitive and specific methods are warranted to enhance this approach. However, whether early treatment of MRD can improve overall survival in patients with childhood ALL needs to be evaluated in adequately controlled clinical trials.

The relapsed acute lymphoblastic leukemia network (ReALLNet): a multidisciplinary project from the spanish society of pediatric hematology and oncology (SEHOP).

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with survival rates exceeding 85%. However, 15% of patients will relapse; consequently, their survival rates decrease to below 50%. Therefore, several research and innovation studies are focusing on pediatric relapsed or refractory ALL (R/R ALL). Driven by this context and following the European strategic plan to implement precision medicine equitably, the Relapsed ALL Network (ReALLNet) was launched under the umbrella of SEHOP in 2021, aiming to connect bedside patient care with expert groups in R/R ALL in an interdisciplinary and multicentric network. To achieve this objective, a board consisting of experts in diagnosis, management, preclinical research, and clinical trials has been established. The requirements of treatment centers have been evaluated, and the available oncogenomic and functional study resources have been assessed and organized. A shipping platform has been developed to process samples requiring study derivation, and an integrated diagnostic committee has been established to report results. These biological data, as well as patient outcomes, are collected in a national registry. Additionally, samples from all patients are stored in a biobank. This comprehensive repository of data and samples is expected to foster an environment where preclinical researchers and data scientists can seek to meet the complex needs of this challenging population. This proof of concept aims to demonstrate that a network-based organization, such as that embodied by ReALLNet, provides the ideal niche for the equitable and efficient implementation of "what's next" in the management of children with R/R ALL.

Genotypic Frequencies of Mutations Associated with Alpha-1 Antitrypsin Deficiency in Unrelated Bone Marrow Donors from the Murcia Region Donor Registry in the Southeast of Spain.

Alpha-1 antitrypsin (AAT1) deficiency (AAT1D) is an inherited disease with an increased risk of chronic obstructive pulmonary disease (COPD), liver disease, and skin and blood vessel problems. AAT1D is caused by mutations in the SERPINE1 gene (Serine Protease Inhibitor, group A, member 1). Numerous variants of this gene, the Pi system, have been identified. The most frequent allelic variants are Pi*M, Pi*S, and Pi*Z. The development of COPD requires both a genetic predisposition and the contribution of an environmental factor, smoking being the most important. Studies on this deficiency worldwide are very scarce, and it is currently considered a rare disease because it is underdiagnosed. The aim of this study was to analyze the genotypic frequencies of mutations associated with AAT1 deficiency in unrelated bone marrow donors from the donor registry of the Region of Murcia in southeastern Spain due to the high risk of presenting with different pathologies and underdiagnosis in the population. A total of 112 DNA-healthy voluntary unrelated bone marrow donors from different parts of the Region of Murcia were analyzed retrospectively. AAT1 deficiency patient testing involved an automated biochemical screening routine. The three main variants, Pi*M, Pi*Z, and Pi*S, were analyzed in the SERPINE1 gene. Our results showed a frequency of 3.12% of the Pi*Z (K342) mutation in over 224 alleles tested in the healthy population. The frequency of Pi*S (V264) was 11.1%. The frequency of the haplotype with the most dangerous mutation, EK342 EE264, was 4.46%, and the frequency of EK342 EV264 was 1.78% in the healthy population. Frequencies of other EE342 EV264-mutated haplotypes accounted for 18.7%. As for the EE342 VV264 haplotype, 0.89% of the total healthy population presented heterozygous for the EV264 mutation and one individual presented homozygous for the VV264 mutation. In conclusion, the frequencies of Pi mutations in the healthy population of the Region of Murcia were not remarkably different from the few studies reported in Spain. The genotype and haplotype frequencies followed the usual pattern. Health authorities should be aware of this high prevalence of the Pi*S allelic variant and pathological genotypes such as Pi*MZ and Pi*SZ in the healthy population if they consider screening the smoking population.

Higher Expression of Activated CD8+ T Lymphocytes (CD8+CD25+, CD8+CD69+ and CD8+CD95+) Mediate Early Post-Transplant Acute Tubular Injury in Kidney Recipients.

BACKGROUND: Acute kidney injury (AKI) is a leading cause of early post-transplant kidney damage. Furthermore, acute tubular necrosis (ATN) is appointed as the most prevalent form of AKI, a frequent multifactorial process associated with high morbidity and mortality, yet giving rise to delayed graft function (DGF) and, ultimately, allograft dysfunction. Common factors such as prolonged cold ischemia time, advanced donor age, cadaveric versus living donor, donor history of hypertension, as well as donation after cardiac death have all been deemed risk factors for ATN. With the increasing number of older cadaveric and cardiac donors in the donation process, ATN could have a detrimental impact on patient welfare. Therefore understanding the underlying process would benefit the transplant outcome. We aimed to prospectively monitor several T cell subsets in a cohort of kidney transplant recipients (KTrs) to investigate whether there is an adaptive immune-mediated involvement in the ATN process. METHODS: Peripheral blood was collected from 31 KTrs at different time points within the first-year post-transplantation for in vitro stimulation with Concanavalin-A (Con-A) in a humidified 5% CO2 incubator at 37 °C for 72 hours. Upon cell stimulation, flow cytometry was applied to quantify the surface expression through the median fluorescence intensity (MFI) of CD4+CD25+, CD8+CD25+, CD4+CD38+, CD8+CD38+, CD4+CD154+, CD8+CD154+, CD4+CD69+, CD8+CD69+, CD4+CD95+, and CD8+CD95+ T cells. Statistical analysis was carried out with SPSS Statistics IBM v.25 (IBM Corp, Armonk, NY, USA). MFIs values were compared using a univariate analysis by a nonparametric U-Mann Whitney test. ROC analysis was applied to define cut-off values most capable of stratifying patients at high risk of ATN. Spearman's rank-order coefficient test was applied to correlate biomarkers with allograft function. Multivariate regression independently validated CD8+ T lymphocytes as surrogate biomarkers of ATN. A p-value < 0.05 was considered statistically significant. RESULTS: KTrs who developed ATN upon transplantation had significantly higher expression of CD25, CD69, and CD95 on CD8+ and lower expression of CD95 on CD4+ T lymphocytes than patients with stable graft function. ROC curve analysis showed that MFIs ≥1015.20 for CD8+CD25+, ≥2489.05 for CD8+CD69+, ≥4257.28 for CD8+CD95+, and ≤1581.98 for CD4+CD95+ were capable of stratifying KTrs at high risk of ATN. Furthermore, patients with an MFI below any cut-off were significantly less likely to develop ATN than those with other values. The allograft function was correlated with the CD4+CD95+/CD8+CD95+ ratio in KTrs who developed ATN. The multivariate analysis confirmed that, within the first-month post-transplant, MFI values of CD8+CD25+, CD4+CD95+, and CD8+CD95+ T lymphocytes, along with donor age, serum creatinine, and GFR were independent risk factors to ATN. Moreover, we were also able to corroborate previous immune factors of importance in immune-mediated response to the allograft, such as the patient's maximum panel reactive antibody (PRA) or the maintenance immunosuppression therapy. CONCLUSIONS: Our results demonstrate evidence for the implication of CD8+ T lymphocytes in the development of ATN early in the post-transplant phase. Post-transplant monitoring of activated CD8+ T lymphocytes may help identify which patients require further clinical intervention to prevent graft damage.

Early Cytomegalovirus Reactivation in Renal Recipients Is Associated with High Levels of B Cell Maturation Antigen Transcript Expression Prior to Transplantation.

Cytomegalovirus (CMV) infection is the most frequent infection episode in kidney transplant (KT) recipients. Reactivation usually occurs in the first three months after transplantation and is associated with higher cellular and/or antibody-mediated rejection rates and poorer graft performance. CMV induces the expression of BAFF (B-cell-activating factor, a cytokine involved in the homeostasis of B cells), which communicates signals for survival and growth to B cells and virus-specific plasma cells via the R-BAFF (BAFF receptor), TACI (the calcium modulator, the cyclophilin ligand interactor), and BCMA (B cell maturation antigen) receptors. These molecules of the BAFF system have also been suggested as biomarkers for the development of alloantibodies and graft dysfunction. This prospective study included 30 CMV-IgG seropositive KT recipients. The expression levels of the genes BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA) in peripheral blood leukocytes (PBL) pre-KT were determined using qPCR. qPCR was also used to monitor CMV reactivation in the first three months following KT. The remainder of the KT recipients were classified as CMV- reactivation, and those with more than 500 copies/mL in at least one sample were classified as CMV+ reactivation. There were no discernible variations in the BAFF-R and TACI transcript expression levels. In the CMV+ group, we examined the relationship between the transcript levels and peak viremia. Peak viremia levels and BCMA transcript levels showed a strong correlation. BAFF-R and TACI expressions showed no measurable differences. In patients with early CMV reactivation, high BCMA receptor expression was associated with increased plasmablast, lymphocyte B cell class-switched levels (LBCS), and viral load. Our findings demonstrate that pre-KT BCMA transcript levels increased in KT recipients with early CMV reactivation. These transcript levels positively correlate with peak viremia and weakly with plasmablast and LBCS levels in PBLs.

Role of vaccination and anti-SARS-CoV-2 antibodies in the clinical outcome of hospitalized COVID-19 patients.

Background: Although vaccination has considerably reduced the risk of hospitalization and death from COVID19, the impact of vaccination and anti-SARS-CoV-2 antibody status on the outcome of patients who required hospitalization has been poorly investigated. Material and methods: A prospective observational study in 232 patients hospitalized for COVID19 was carried out from October 2021 to January 2022 to evaluate the role on patient outcome of their vaccination and anti-SARS-CoV-2 antibody status and titer, comorbidities, analytical determinations, clinical presentation at admission, treatments and requirements for respiratory support. Cox regression and survival analyzes were performed. The SPSS and "R" programs were used. Results: Patients with complete vaccination schedule had higher S-protein antibody titers (log10 3.73 [2.83-4.6] UI/ml vs 1.6 [2.99-2.61] UI/ml; p < 0.001), lower probability of radiographic worsening (21.6% vs. 35.4%; p = 0.005), less likely required high doses of dexamethasone (28.4% vs. 45.4%; p = 0.012), high-flow oxygen (20.6% vs. 35.4%; p = 0.02), ventilation (13.7% vs, 33.8%; p = 0.001) and intensive care admissions (10.8% vs. 32.6%; p < 0.001). Remdesivir (HR = 0.38; p < 0.001) and complete vaccination schedule (HR = 0.34; p = 0.008) were protective factors. No differences in antibody status were detected between groups (HR = 0.58; p = 0.219). Conclusions: SARS-CoV-2 vaccination was associated with higher S-protein antibody titers and lower probability of radiological progression, immunomodulators requirement and respiratory support or death. However, vaccination but not antibody titters protected from adverse events pointing a role of immune-protective mechanisms in addition to humoral response.

Antecedentes: Aunque la vacunación ha reducido considerablemente el riesgo de hospitalización y muerte por COVID-19, se ha investigado poco el impacto de la vacunación y el estado de los anticuerpos anti-SARS-CoV-2 en la evolución de los pacientes que requieren hospitalización. Material y métodos: Se realizó un estudio observacional prospectivo en 232 pacientes hospitalizados por COVID-19 desde octubre del 2021 hasta enero del 2022 para evaluar el impacto en la evolución clínica del estado vacunal, el título de anticuerpos anti-SARS-CoV-2, la presencia de comorbilidades, analítica, la clínica al ingreso, tratamientos y soporte respiratorio. Se realizaron análisis de supervivencia y regresión de Cox. Se utilizaron los programas SPSS y «R¼. Resultados: Los pacientes con esquema de vacunación completo presentaron títulos de anticuerpos contra la proteína S más elevados (log10 3,73 [2,83-4,6] UI/mL vs. 1,6 [2,99-2,61] UI/mL; p < 0,001), menor probabilidad de empeoramiento radiográfico (21,6 vs. 35,4%; p = 0,005), requirieron con menor probabilidad dosis elevadas de dexametasona (28,4 vs. 45,4%; p = 0,012), oxígeno de alto flujo (20,6 vs. 35,4%; p = 0,02), ventilación (13,7 vs. 33,8%; p = 0,001) e ingresos en cuidados intensivos (10,8 vs. 32,6%; p < 0,001). El remdesivir (HR = 0,38; p < 0,001) y el esquema completo de vacunación (HR = 0,34; p = 0,008) fueron factores protectores de mala evolución. No se detectaron diferencias en el estado de los anticuerpos entre los grupos (HR = 0,58; p = 0,219). Conclusiones: La vacunación contra el SARS-CoV-2 se asoció con mayores títulos de anticuerpos contra la proteína S y menor probabilidad de progresión radiológica, requerimiento de inmunomoduladores y soporte respiratorio o muerte. Sin embargo, la vacunación, pero no los títulos de anticuerpos, protegió de los eventos adversos, lo que indica un papel de los mecanismos de protección inmunológica además de la respuesta humoral.

All That Glitters in cfDNA Analysis Is Not Gold or Its Utility Is Completely Established Due to Graft Damage: A Critical Review in the Field of Transplantation.

In kidney transplantation, a biopsy is currently the gold standard for monitoring the transplanted organ. However, this is far from an ideal screening method given its invasive nature and the discomfort it can cause the patient. Large-scale studies in renal transplantation show that approximately 1% of biopsies generate major complications, with a risk of macroscopic hematuria greater than 3.5%. It would not be until 2011 that a method to detect donor-derived cell-free DNA (dd-cfDNA) employing digital PCR was devised based on analyzing the differences in SNPs between the donor and recipient. In addition, since the initial validation studies were carried out at the specific moments in which rejection was suspected, there is still not a good understanding of how dd-cfDNA levels naturally evolve post-transplant. In addition, various factors, both in the recipient and the donor, can influence dd-cfDNA levels and cause increases in the levels of dd-cfDNA themselves without suspicion of rejection. All that glitters in this technology is not gold; therefore, in this article, we discuss the current state of clinical studies, the benefits, and disadvantages.

Role of vaccination and anti-SARS-CoV-2 antibodies in the clinical outcome of hospitalized COVID-19 patients / Papel de la vacunación y los anticuerpos anti-SARS-CoV-2 en el desenlace clínico de pacientes hospitalizados por COVID-19

Background Although vaccination has considerably reduced the risk of hospitalization and death from COVID19, the impact of vaccination and anti-SARS-CoV-2 antibody status on the outcome of patients who required hospitalization has been poorly investigated. Material and methods A prospective observational study in 232 patients hospitalized for COVID19 was carried out from October 2021 to January 2022 to evaluate the role on patient outcome of their vaccination and anti-SARS-CoV-2 antibody status and titer, comorbidities, analytical determinations, clinical presentation at admission, treatments and requirements for respiratory support. Cox regression and survival analyzes were performed. The SPSS and “R” programs were used. Results Patients with complete vaccination schedule had higher S-protein antibody titers (log10 3.73 [2.83–4.6]UI/ml vs 1.6 [2.99–2.61]UI/ml; p<0.001), lower probability of radiographic worsening (21.6% vs. 35.4%; p=0.005), less likely required high doses of dexamethasone (28.4% vs. 45.4%; p=0.012), high-flow oxygen (20.6% vs. 35.4%; p=0.02), ventilation (13.7% vs, 33.8%; p=0.001) and intensive care admissions (10.8% vs. 32.6%; p<0.001). Remdesivir (HR=0.38; p<0.001) and complete vaccination schedule (HR=0.34; p=0.008) were protective factors. No differences in antibody status were detected between groups (HR=0.58; p=0.219). Conclusions SARS-CoV-2 vaccination was associated with higher S-protein antibody titers and lower probability of radiological progression, immunomodulators requirement and respiratory support or death. However, vaccination but not antibody titters protected from adverse events pointing a role of immune-protective mechanisms in addition to humoral response (AU)

Antecedentes Aunque la vacunación ha reducido considerablemente el riesgo de hospitalización y muerte por COVID-19, se ha investigado poco el impacto de la vacunación y el estado de los anticuerpos anti-SARS-CoV-2 en la evolución de los pacientes que requieren hospitalización. Material y métodos Se realizó un estudio observacional prospectivo en 232 pacientes hospitalizados por COVID-19 desde octubre del 2021 hasta enero del 2022 para evaluar el impacto en la evolución clínica del estado vacunal, el título de anticuerpos anti-SARS-CoV-2, la presencia de comorbilidades, analítica, la clínica al ingreso, tratamientos y soporte respiratorio. Se realizaron análisis de supervivencia y regresión de Cox. Se utilizaron los programas SPSS y «R». Resultados Los pacientes con esquema de vacunación completo presentaron títulos de anticuerpos contra la proteína S más elevados (log10 3,73 [2,83-4,6] UI/mL vs. 1,6 [2,99-2,61] UI/mL; p < 0,001), menor probabilidad de empeoramiento radiográfico (21,6 vs. 35,4%; p = 0,005), requirieron con menor probabilidad dosis elevadas de dexametasona (28,4 vs. 45,4%; p = 0,012), oxígeno de alto flujo (20,6 vs. 35,4%; p = 0,02), ventilación (13,7 vs. 33,8%; p = 0,001) e ingresos en cuidados intensivos (10,8 vs. 32,6%; p < 0,001). El remdesivir (HR = 0,38; p < 0,001) y el esquema completo de vacunación (HR = 0,34; p = 0,008) fueron factores protectores de mala evolución. No se detectaron diferencias en el estado de los anticuerpos entre los grupos (HR = 0,58; p = 0,219). Conclusiones La vacunación contra el SARS-CoV-2 se asoció con mayores títulos de anticuerpos contra la proteína S y menor probabilidad de progresión radiológica, requerimiento de inmunomoduladores y soporte respiratorio o muerte. Sin embargo, la vacunación, pero no los títulos de anticuerpos, protegió de los eventos adversos, lo que indica un papel de los mecanismos de protección inmunológica además de la respuesta humoral (AU)

Remdesivir plus dexamethasone is associated to improvement in the clinical outcome of COVID-19 hospitalized patients regardless of their vaccination status.

INTRODUCTION: Remdesivir seems to reduce the risk of hospitalization and improve clinical outcome in hospitalized patients with COVID-19. OBJECTIVES: To compare the clinical outcome of COVID-19 hospitalized patients treated with remdesivir plus dexamethasone versus dexamethasone alone, according to their vaccination status. MATERIAL AND METHODS: A retrospective observational study was carried out in 165 patients hospitalized for COVID-19 from October 2021 to January 2022. Multivariate logistic regression, Kaplan-Meier and the log-rank tests were used to evaluate the event (need for ventilation or death). RESULTS: Patients treated with remdesivir plus dexamethasone (n=87) compared with dexamethasone alone (n=78) showed similar age (60±16, 47-70 vs. 62±37, 51-74 years) and number of comorbidities: 1 (0-2) versus 1.5 (1-3). Among 73 fully vaccinated patients, 42 (47.1%) were in remdesivir plus dexamethasone and 31 (41%) in dexamethasone alone. Patients treated with remdesivir plus dexamethasone needed intensive care less frequently (17.2% vs. 31%; p=0.002), high-flow oxygen (25.3% vs. 50.0%; p=0.002) and non-invasive mechanical ventilation (16.1% vs. 47.4%; p<0.001). Furthermore, they had less complications during hospitalization (31.0% vs. 52.6%; p=0.008), need of antibiotics (32.2% vs. 59%; p=0.001) and radiologic worsening (21.8% vs. 44.9%; p=0.005). Treatment with remdesivir plus dexamethasone (aHR, 0.26; 95% CI: 0.14-0.48; p<0.001) and vaccination (aHR 0.39; 95% CI: 0.21-0.74) were independent factors associated with lower progression to mechanical ventilation or death. CONCLUSIONS: Remdesivir in combination with dexamethasone and vaccination independently and synergistically protects hospitalized COVID-19 patients requiring oxygen therapy from progression to severe disease or dead.

Role of vaccination and anti-SARS-CoV-2 antibodies in the clinical outcome of hospitalized COVID-19 patients.

BACKGROUND: Although vaccination has considerably reduced the risk of hospitalization and death from COVID19, the impact of vaccination and anti-SARS-CoV-2 antibody status on the outcome of patients who required hospitalization has been poorly investigated. MATERIAL AND METHODS: A prospective observational study in 232 patients hospitalized for COVID19 was carried out from October 2021 to January 2022 to evaluate the role on patient outcome of their vaccination and anti-SARS-CoV-2 antibody status and titer, comorbidities, analytical determinations, clinical presentation at admission, treatments and requirements for respiratory support. Cox regression and survival analyzes were performed. The SPSS and "R" programs were used. RESULTS: Patients with complete vaccination schedule had higher S-protein antibody titers (log10 3.73 [2.83-4.6]UI/ml vs 1.6 [2.99-2.61]UI/ml; p<0.001), lower probability of radiographic worsening (21.6% vs. 35.4%; p=0.005), less likely required high doses of dexamethasone (28.4% vs. 45.4%; p=0.012), high-flow oxygen (20.6% vs. 35.4%; p=0.02), ventilation (13.7% vs, 33.8%; p=0.001) and intensive care admissions (10.8% vs. 32.6%; p<0.001). Remdesivir (HR=0.38; p<0.001) and complete vaccination schedule (HR=0.34; p=0.008) were protective factors. No differences in antibody status were detected between groups (HR=0.58; p=0.219). CONCLUSIONS: SARS-CoV-2 vaccination was associated with higher S-protein antibody titers and lower probability of radiological progression, immunomodulators requirement and respiratory support or death. However, vaccination but not antibody titters protected from adverse events pointing a role of immune-protective mechanisms in addition to humoral response.

Monitoring of Serological, Cellular and Genomic Biomarkers in Transplantation, Computational Prediction Models and Role of Cell-Free DNA in Transplant Outcome.

The process and evolution of an organ transplant procedure has evolved in terms of the prevention of immunological rejection with the improvement in the determination of immune response genes. These techniques include considering more important genes, more polymorphism detection, more refinement of the response motifs, as well as the analysis of epitopes and eplets, its capacity to fix complement, the PIRCHE algorithm and post-transplant monitoring with promising new biomarkers that surpass the classic serum markers such as creatine and other similar parameters of renal function. Among these new biomarkers, we analyze new serological, urine, cellular, genomic and transcriptomic biomarkers and computational prediction, with particular attention to the analysis of donor free circulating DNA as an optimal marker of kidney damage.

Clinical Significance of the Pre-Transplant CXCR3 and CCR6 Expression on T Cells In Kidney Graft Recipients.

BACKGROUND: T cells play a fundamental role in the processes that mediate graft rejection, tolerance, and defense against infections. The CXCR3 and CCR6 receptors, highly expressed in Th1 (type 1 T helper cells)/Tc1 (T cytotoxic cells, type 1), Th1-Tc1, and Th17-Tc17 lymphocytes, respectively, participate in cell migration toward inflamed tissues. The altered expression level of CXCR3 and CCR6 has been associated with different clinical events after renal transplantation, such as acute rejection (AR) and chronic graft dysfunction, but data are still limited. In this study, we evaluated the expression of the receptor CXCR3 and CCR6 in peripheral blood T lymphocytes from kidney transplant recipients (KTR) and their association with viral infections, AR, and allograft function. METHODS: Through flow cytometry, the peripheral blood expression of CXCR3 and CCR6 in T cells was evaluated in a pretransplant collection of KTR. The levels of these T subpopulations and their association with the incidence of AR, kidney graft function, viral infections, cytomegalovirus, and BK virus were studied. Adverse clinical events and graft function were monitored during the first year post transplant. RESULTS: KTRs with low pretransplantation levels of Th17 (CD4+CXCR3-CCR6+) (tertile 1, Th17<16.4%) had a higher risk of suffering AR during the first year post transplantation (P = .033). KTRs with viral infections or reactivations during the first 3 months post transplantation had significantly lower levels of Tc17 (CD8+CXCR3-CCR6+) and higher levels of Th1 (CD4+CXCR3+CCR6-). In patients with cytomegalovirus reactivations, the viral peak correlates negatively with the pretransplant levels of Th1 (r = -0.606, P = .037). CONCLUSIONS: Pretransplantation assessment of Th1-Th17 and Tc1-Tc17 levels may help predict post-transplant clinical events such as AR and reactivation of viral infections.

Measurable residual disease study through three different methods can anticipate relapse and guide pre-emptive therapy in childhood acute myeloid leukemia.

PURPOSE: Although outcomes of children with acute myeloid leukemia (AML) have improved over the last decades, around one-third of patients relapse. Measurable (or minimal) residual disease (MRD) monitoring may guide therapy adjustments or pre-emptive treatments before overt hematological relapse. METHODS: In this study, we review 297 bone marrow samples from 20 real-life pediatric AML patients using three MRD monitoring methods: multiparametric flow cytometry (MFC), fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). RESULTS: Patients showed a 3-year overall survival of 73% and a 3-year event-free survival of 68%. Global relapse rate was of 25%. All relapses were preceded by the reappearance of MRD detection by: (1) MFC (p = 0.001), (2) PCR and/or FISH in patients with an identifiable chromosomal translocation (p = 0.03) and/or (3) one log increase of Wilms tumor gene 1 (WT1) expression in two consecutive samples (p = 0.02). The median times from MRD detection to relapse were 26, 111, and 140 days for MFC, specific PCR and FISH, and a one log increment of WT1, respectively. CONCLUSIONS: MFC, FISH and PCR are complementary methods that can anticipate relapse of childhood AML by weeks to several months. However, in our series, pre-emptive therapies were not able to prevent disease progression. Therefore, more sensitive MRD monitoring methods that further anticipate relapse and more effective pre-emptive therapies are needed.

Killer Cell Immunoglobulin-like Receptors (KIR) and Human Leucocyte Antigen C (HLA-C) Increase the Risk of Long-Term Chronic Liver Graft Rejection.

Chronic liver rejection (CR) represents a complex clinical situation because many patients do not respond to increased immunosuppression. Killer cell immunoglobulin-like receptors/Class I Human Leukocyte Antigens (KIR/HLA-I) interactions allow for predicting Natural Killer (NK) cell alloreactivity and influence the acute rejection of liver allograft. However, its meaning in CR liver graft remains controversial. KIR and HLA genotypes were studied in 513 liver transplants using sequence-specific oligonucleotides (PCR-SSO) methods. KIRs, human leucocyte antigen C (HLA-C) genotypes, KIR gene mismatches, and the KIR/HLA-ligand were analyzed and compared in overall transplants with CR (n = 35) and no-chronic rejection (NCR = 478). Activating KIR (aKIR) genes in recipients (rKIR2DS2+ and rKIR2DS3+) increased CR compared with NCR groups (p = 0.013 and p = 0.038). The inhibitory KIR (iKIR) genes in recipients rKIR2DL2+ significantly increased the CR rate compared with their absence (9.1% vs. 3.7%, p = 0.020). KIR2DL3 significantly increases CR (13.1% vs. 5.2%; p = 0.008). There was no influence on NCR. CR was observed in HLA-I mismatches (MM). The absence of donor (d) HLA-C2 ligand (dC2-) ligand increases CR concerning their presence (13.1% vs. 5.6%; p = 0.018). A significant increase of CR was observed in rKIR2DL3+/dC1- (p = 0.015), rKIR2DS4/dC1- (p = 0.014) and rKIR2DL3+/rKIR2DS4+/dC1- (p = 0.006). Long-term patient survival was significantly lower in rKIR2DS1+rKIR2DS4+/dC1- at 5-10 years post-transplant. This study shows the influence of rKIR/dHLA-C combinations and aKIR gene-gene mismatches in increasing CR and KIR2DS1+/C1-ligands and the influence of KIR2DS4+/C1-ligands in long-term graft survival.

Monitoring of Soluble Forms of BAFF System (BAFF, APRIL, sR-BAFF, sTACI and sBCMA) in Kidney Transplantation.

BAFF system plays an essential role in B cells homeostasis and tolerance, although it has widely not been tested in transplantation with doubtful results. The main purpose was to study the BAFF soluble forms and their correlation with acute rejection (AR) and donor-specific antibodies production. Serum levels of BAFF, APRIL, and soluble forms of their receptors were analyzed in renal recipients with and without acute rejection (AR/NAR) appearance. All molecules were evaluated at pre- and post-transplantation. sTACI showed a significant correlation with BAFF and sR-BAFF levels, and sBCMA also showed a positive correlation with sAPRIL levels. A significant increase in sAPRIL levels in patients suffering AR was also found, and ROC curves analysis showed an AUC = 0.724, a concentration of 6.05 ng/ml (sensitivity: 66.7%; specificity: 73.3%), the best cutoff point for predicting AR. In the post-transplant dynamics of sAPRIL levels in the longitudinal cohort, we observed a significant decrease at 3 and 6 month post-transplantation compared to pretransplantation status. We also observed that recipients with high pre-transplant levels of sAPRIL generated antibodies earlier than those with lower sAPRIL levels, although their long-term post-transplantation was not different. Our results show that elevated serum levels of APRIL may be helpful as a biomarker for the diagnosis of AR, although the longitudinal study shows that it is not helpful as a prognostic biomarker.

Evaluating the Link between BAFF System Gene Expression and Acute Rejection Development in Kidney Transplantation.

B-cell activating factor (BAFF) system signaling is critical for B-cell homeostasis, effector functions, and tolerance maintenance in transplants, but it has not been studied in kidney transplant recipients (KTRs). The aim was to analyze the changes in BAFF system expression in KTRs with/without acute rejection (AR/NAR). The BAFF system expression was analyzed by qPCR in 40 KTRs. A meta-analysis of BAFF system expression and histological renal damage was identified by the Chronic Allograft Damage Index (CADI) and performed from the GEO database. Proliferation-inducing ligand (APRIL) expression increased at three- and six-months post-KT (p = 0.014 and p < 0.001). B-cell maturation antigen (BCMA) expression increased at six-months post-KT (p = 0.038). BAFF expression remained stable in NAR-KTRs, but was increased in CADI concerning the No-CADI group at one year (p = 0.008). BCMA expression increased in the CADI group at one- (p = 0.001) and six-years post-KT (p = 0.024). At three months, the transmembrane activator and calcium modulator interactor (TACI) gene significantly elevated KTRs with DSAs (donor-specific antibody; p = 0.034). KTRs with DSAs significantly increase the B-cell activating factor receptor (R-BAFF; p = 0.021) and TACI (p = 0.018) between pre- and three-month post-KT. Changes in the expression of the BAFF system increase during post-KTR in the development of AR and chronic allograft damage, and could be an important pathological tool to detect and prevent kidney graft outcomes.

Clonal heterogeneity and rates of specific chromosome gains are risk predictors in childhood high-hyperdiploid B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer. High hyperdiploidy (HHD) identifies the most frequent cytogenetic subgroup in childhood B-ALL. Although hyperdiploidy represents an important prognostic factor in childhood B-ALL, the specific chromosome gains with prognostic value in HHD-B-ALL remain controversial, and the current knowledge about the hierarchy of chromosome gains, clonal heterogeneity and chromosomal instability in HHD-B-ALL remains very limited. We applied automated sequential-iFISH coupled with single-cell computational modeling to identify the specific chromosomal gains of the eight typically gained chromosomes in a large cohort of 72 primary diagnostic (DX, n = 62) and matched relapse (REL, n = 10) samples from HHD-B-ALL patients with either favorable or unfavorable clinical outcome in order to characterize the clonal heterogeneity, specific chromosome gains and clonal evolution. Our data show a high degree of clonal heterogeneity and a hierarchical order of chromosome gains in DX samples of HHD-B-ALL. The rates of specific chromosome gains and clonal heterogeneity found in DX samples differ between HHD-B-ALL patients with favorable or unfavorable clinical outcome. In fact, our comprehensive analyses at DX using a computationally defined risk predictor revealed low levels of trisomies +18+10 and low levels of clonal heterogeneity as robust relapse risk factors in minimal residual disease (MRD)-negative childhood HHD-B-ALL patients: relapse-free survival beyond 5 years: 22.1% versus 87.9%, P < 0.0001 and 33.3% versus 80%, P < 0.0001, respectively. Moreover, longitudinal analysis of matched DX-REL HHD-B-ALL samples revealed distinct patterns of clonal evolution at relapse. Our study offers a reliable prognostic sub-stratification of pediatric MRD-negative HHD-B-ALL patients.

High BMP4 expression in low/intermediate risk BCP-ALL identifies children with poor outcomes.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) outcome has improved in the last decades, but leukemic relapses are still one of the main problems of this disease. Bone morphogenetic protein 4 (BMP4) was investigated as a new candidate biomarker with potential prognostic relevance, and its pathogenic role was assessed in the development of disease. A retrospective study was performed with 115 pediatric patients with BCP-ALL, and BMP4 expression was analyzed by quantitative reverse transcription polymerase chain reaction in leukemic blasts at the time of diagnosis. BMP4 mRNA expression levels in the third (upper) quartile were associated with a higher cumulative incidence of relapse as well as a worse 5-year event-free survival and central nervous system (CNS) involvement. Importantly, this association was also evident among children classified as having a nonhigh risk of relapse. A validation cohort of 236 patients with BCP-ALL supported these data. Furthermore, high BMP4 expression promoted engraftment and rapid disease progression in an NSG mouse xenograft model with CNS involvement. Pharmacological blockade of the canonical BMP signaling pathway significantly decreased CNS infiltration and consistently resulted in amelioration of clinical parameters, including neurological score. Mechanistically, BMP4 favored chemoresistance, enhanced adhesion and migration through brain vascular endothelial cells, and promoted a proinflammatory microenvironment and CNS angiogenesis. These data provide evidence that BMP4 expression levels in leukemic cells could be a useful biomarker to identify children with poor outcomes in the low-/intermediate-risk groups of BCP-ALL and that BMP4 could be a new therapeutic target to blockade leukemic CNS disease.

Moderate to Intense Physical Activity Is Associated With Improved Clinical, CD4/CD8 Ratio, and Immune Activation Status in HIV-Infected Patients on ART.

BACKGROUND: Physical activity has anti-inflammatory effects and reduces morbidity and mortality in the general population, but its role in the clinical, CD4/CD8 ratio, and immune activation status of HIV-infected patients has been poorly studied. METHODS: A cross-sectional study was carried out in a cohort of 155 HIV-infected patients on stable antiretroviral therapy (ART) to compare clinical, biochemical, CD4/CD8 ratio, and immune activation status according to their physical activity in the last 2 years (sedentary/low vs moderate/intense) assessed by the iPAQ. A binary logistic regression and mixed analysis of variance were performed to evaluate the impact of levels of physical activity on CD4/CD8 ratio. RESULTS: In our series, 77 (49.7%) out of 155 patients were sedentary, and 78 (50.3%) practiced moderate/intense physical activity. Moderate/intense physical activity was associated with better metabolic control (lower body mass index, P = .024; glucose, P = .024; and triglyceride, P = .002) and CDC HIV stage (P = .046), lower CD8+ (P =  .018), CD4+CD8+ (P = .026), CD4+CD86+ (P = .045), CD4+HLA-DR+ (P = .011), CD8+HLA-DR+ (P = .048) T lymphocytes and CD16+HLA-DR+ natural killer cells (P = .026), and higher CD3+CD4+ T lymphocytes (P = .016) and CD4/CD8 ratio (P = .001). Sedentary lifestyle (odds ratio [OR], 2.12; P = .042), CD4 nadir (OR, 1.005; P < .001), and CD8+CD38+ T cells (OR, 1.27; P = .006) were independently associated with low CD4/CD8 ratio (<0.8). Earlier and more intense CD4/CD8 ratio recovery was observed in patients with higher physical activity in the 2-year follow-up with a significant interaction between these variables: F(2, 124) = 3.31; P = .049; partial η2 = 0.042. CONCLUSIONS: Moderate to high physical activity is associated with beneficial health effects, improvement in metabolic profile, and reduction of chronic inflammation in patients with HIV. Although more studies and clinical trials are needed to confirm these findings, a healthy lifestyle including at least moderate physical activity should be recommended to HIV patients on stable ART.