Performance of Whole Blood Stimulation Assays for the Quantification of SARS-CoV-2 Specific T-Cell Response: A Cross-Sectional Study.

Since the identification of the new severe acute respiratory syndrome virus 2 (SARS-CoV-2), a huge effort in terms of diagnostic strategies has been deployed. To date, serological assays represent a valuable tool for the identification of recovered COVID-19 patients and for the monitoring of immune response elicited by vaccination. However, the role of T-cell response should be better clarified and simple and easy to perform assays should be routinely introduced. The main aim of this study was to compare a home-made assay for whole blood stimulation with a standardized ELISpot assay design in our laboratory for the assessment of spike-specific T-cell response in vaccinated subjects. Even if a good correlation between the assays was reported, a higher percentage of responder subjects was reported for immunocompromised subjects with ELISpot assay (56%) than home-made whole blood stimulation assay (33%). Additionally, three commercial assays were compared with our home-made assay, reporting a good agreement in terms of both positive and negative results.

Effect of a Third Dose of SARS-CoV-2 mRNA BNT162b2 Vaccine on Humoral and Cellular Responses and Serum Anti-HLA Antibodies in Kidney Transplant Recipients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has severely impacted on public health, mainly on immunosuppressed patients, including solid organ transplant recipients. Vaccination represents a valuable tool for the prevention of severe SARS-CoV-2 infection, and the immunogenicity of mRNA vaccines has been evaluated in transplanted patients. In this study, we investigated the role of a third dose of the BNT162b2 vaccine in a cohort of kidney transplant recipients, analyzing both humoral and cell-mediated responses. We observed an increased immune response after the third dose of the vaccine, especially in terms of Spike-specific T cell response. The level of seroconversion remained lower than 50% even after the administration of the third dose. Mycophenolate treatment, steroid administration and age seemed to be associated with a poor immune response. In our cohort, 11/45 patients experienced a SARS-CoV-2 infection after the third vaccine dose. HLA antibodies appearance was recorded in 7 out 45 (15.5%) patients, but none of the patients developed acute renal rejection. Further studies for the evaluation of long-term immune responses are still ongoing, and the impact of a fourth dose of the vaccine will be evaluated.

Three-D-printed simulator for kidney transplantation.

BACKGROUND: Three-Dimensional (3D) printing technology can be used to manufacture training platforms for surgeons. Kidney transplantation offers a suitable model, since it mostly entails vascular and ureteric anastomoses. METHODS: A new simulation platform for surgical training in kidney transplantation was realized and validated in this study. A combination of different 3-D printing technology was used to reproduce the key anatomy of lower abdomen, of pelvis, and of a kidney graft, including their mechanical properties. RESULTS: Thirty transplantations were performed by two junior trainees with no previous experience in the area. Analysis of the times required to perform the simulated transplantation showed that proficiency was reached after about ten cases, as indicated by a flattening of the respective curves that corresponded to a shortening of about 40% and 47%, respectively, of the total time initially needed to perform the whole simulated transplantation. Although an objective assessment of the technical quality of the anastomoses failed to show a significant improvement throughout the study, a growth in self-confidence with the procedure was reported by both trainees. CONCLUSION: The quality of the presented simulation platform aimed at reproducing in the highest possible way a realistic model of the operative setting and proved effective in providing an integrated training environment where technical skills are enhanced together with a team-training experience. As a result the trainees' self-confidence with the procedure resulted enforced. Three-D--printed models can also offer pre-operative patient-specific training when anatomical variants are anticipated by medical imaging. An analysis of the costs related to the use of this platform is also provided and discussed.

Psychiatric and psychological evaluation in living donor kidney transplantation: a single center experience.

Background: Living donor kidney transplantation (LDKT) is the treatment of choice for end stage renal disease. LDKT involves complex psychosocial issues, which remain partially unexplored. Methods: The study involved all potential living donors and recipient pairs consecutively referred for psychosocial evaluation from the nephrologist. Clinical and sociodemographic variables including prior psychiatric history, previous and current use of psychopharmacological therapy, motivation and information about the transplant procedure were collected. Study participants completed the Symptom Checklist-90-R (SCL-90-R) to assess psychopathological distress. Results: Fifty-three donor/recipient pairs underwent psychiatric and psychological evaluation. Seven subjects (13%) in the recipient group and 13 subjects (25%) in the donor group reported a history of psychological distress and/or psychiatric conditions. A psychiatric diagnosis was confirmed in 4 recipients (7.5% of the study sample, including autism spectrum disorder, histrionic personality disorder, and anxiety-depressive disorders) and 5 donors (9%, including narcissistic personality disorder in one case and anxiety-depressive disorders). SCL-90-R GSI mean scores were 0.3 ±0.3 and 0.2 ±0.2 for the recipient and donor groups, respectively. Overall, 8 couples (15%) suspended the living donation pathway before transplantation. Four couples were excluded for a new onset medical condition. The psychological and psychiatric evaluation excluded one candidate. One couple dropped out before completing the scheduled exams. One recipient refused to undergo crossover renal transplantation, while 1 donor candidate withdrew her consent for transplantation at the end of the evaluation process. Conclusions: Limited but significant psychopathological distress in donors and recipients supports the usefulness of psychiatric and psychological competencies within the transplant team.

Kidney Transplants From Donors on Extracorporeal Membrane Oxygenation Prior to Death Are Associated With Better Long-Term Renal Function Compared to Donors After Circulatory Death.

Donation after circulatory death (DCD) allows expansion of the donor pool. We report on 11 years of Italian experience by comparing the outcome of grafts from DCD and extracorporeal membrane oxygenation (ECMO) prior to death donation (EPD), a new donor category. We studied 58 kidney recipients from DCD or EPD and collected donor/recipient clinical characteristics. Primary non function (PNF) and delayed graft function (DGF) rates, dialysis need, hospitalization duration, and patient and graft survival rates were compared. The estimated glomerular filtration rate (eGFR) was measured throughout the follow-up. Better clinical outcomes were achieved with EPD than with DCD despite similar graft and patient survival rates The total warm ischemia time (WIT) was longer in the DCD group than in the EPD group. Pure WIT was the highest in the class II group. The DGF rate was higher in the DCD group than in the EPD group. PNF rate was similar in the groups. Dialysis need was the greatest and hospitalization the longest in the class II DCD group. eGFR was lower in the class II DCD group than in the EPD group. Our results indicate good clinical outcomes of kidney transplants from DCD despite the long "no-touch period" and show that ECMO in the procurement phase improves graft outcome, suggesting EPD as a source for pool expansion.

Calcineurin Inhibitor-Based Immunosuppression and COVID-19: Results from a Multidisciplinary Cohort of Patients in Northern Italy.

The role of immunosuppression in SARS-CoV-2-related disease (COVID-19) is a matter of debate. We here describe the course and the outcome of COVID-19 in a cohort of patients undergoing treatment with calcineurin inhibitors. In this monocentric cohort study, data were collected from the COVID-19 outbreak in Italy up to April 28th 2020. Patients were followed at our hospital for solid organ transplantation or systemic rheumatic disorders (RMDs) and were on calcineurin inhibitor (CNI)-based therapy. Selected patients were referred from the North of Italy. The aim of our study was to evaluate the clinical course of COVID-19 in this setting. We evaluated 385 consecutive patients (220 males, 57%; median age 61 years, IQR 48-69); 331 (86%) received solid organ transplantation and 54 (14%) had a RMD. CNIs were the only immunosuppressant administered in 47 patients (12%). We identified 14 (4%) COVID-19 patients, all transplanted, mainly presenting with fever (86%) and diarrhea (71%). Twelve patients were hospitalized and two of them died, both with severe comorbidities. No patients developed acute respiratory distress syndrome or infectious complications. The surviving 10 patients are now fully recovered. The clinical course of COVID-19 patients on CNIs is generally mild, and the risk of superinfection seems low.

Myostatin in the Arterial Wall of Patients with End-Stage Renal Disease.

AIM: Myostatin (Mstn) has been described as a trigger for the progression of atherosclerosis. In this study, we evaluated the role of Mstn in arterial remodeling in patients with end-stage renal disease (ESRD). METHODS: Vascular specimens were collected from 16 ESRD patients (56.4±7.9 years) undergoing renal transplant (recipients) and 15 deceased kidney non-uremic donors (55.4±12.1 years). We studied gene and protein expression of Mstn, ubiquitin ligases, Atrogin-1, and muscle ring finger protein-1 (MuRF-1), inflammatory marker CCL2, cytoskeleton components, and Klotho by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Moreover, we assessed vascular calcification and collagen deposition. Finally, we studied the effects of recombinant Mstn on rat vascular smooth muscle cells (VSMCs, A7r5) and evaluated the effects of uremic serum (US) on primary human VSMCs. RESULTS: Myostatin mRNA was upregulated in the arterial vascular wall of recipients compared with donors (~15- folds, p＜0.05). This response was accompanied by the upregulation of gene expression of Atrogin-1 and MuRF-1 (＋2.5- and ＋10-fold) and CCL2 (＋3-fold). Conversely, we found downregulation of protein expression of Smoothelin, α-smooth muscle actin (α-SMA), vimentin, and Klotho (-85%, -50%, -70%, and -80%, respectively; p＜0.05) and gene expression of vimentin and Klotho. Exposition of A7r5 to Mstn induced a time-dependent SMAD 2/SMAD 3 phosphorylation and expression of collagen-1 and transforming growth factor ß (TGFß) mRNA, while US induced overexpression of Mstn and Atrogin-1 and downregulation of Smoothelin and Klotho. CONCLUSIONS: Our data suggest that uremia might induce vascular Mstn gene expression together with a complex pathway of molecular and structural changes in the vascular wall. Myostatin, in turn, can translate the metabolic alterations of uremia into profibrotic and stiffness inducing signals.

Are there any relations among transplant centre volume, surgical technique and anatomy for donor graft selection? Ten-year multicentric Italian experience on mini-invasive living donor nephrectomy.

BACKGROUND: Selection of the right or left living donor kidney for transplantation is influenced by many variables. In the present multi centric study including 21 Italian transplant centres, we evaluated whether centre volume or surgical technique may influence the selection process. METHODS: Intra- and perioperative donor data, donor kidney function, and recipient and graft survival were collected among 693 mini-invasive living donor nephrectomies performed from 2002 to 2014. Centre volume (LOW, 1-50 cases; HIGH, >50 cases) and surgical technique (FULL-LAP, full laparoscopic and robotic; HA-LAP, hand-assisted laparoscopy; MINI-OPEN, mini-lumbotomy) were correlated with selection of right or left donor kidney and with donor and recipient outcome. RESULTS: HIGH-volume centres retrieved a higher rate of donor right kidneys (29.3% versus 17.6%, P < 0.01) with single artery (83.1% versus 76.4%, P < 0.05) compared with LOW-volume centres. Surgical technique correlated significantly with rate of donor right kidney and presence of multiple arteries: MINI-OPEN (53% and 13%) versus HA-LAP (29% and 22%) versus FULL-LAP (11% and 23%), P < 0.001 and P < 0.05, respectively. All donors had an uneventful outcome; donor bleeding was more frequent in LOW-volume centres (4% versus 0.9%, P < 0.05). CONCLUSIONS: Centre volume and surgical technique influenced donor kidney side selection. Donor nephrectomy in LOW-volume centres was associated with higher risk of donor bleeding.

Making a case for controlled organ donation after cardiac death: the story of Italy's first experience.

Donation after circulatory death (DCD) is a valuable option for the procurement of organs for transplantation. In Italy, organ procurement after controlled DCD is legally and ethically conceivable within the current legislative framework. However, although formal impediments do not exist, the health care team is faced with many obstacles that may hinder the implementation of such programs. We report the case of Italy's first controlled DCD, specifically discussing the role of the patient's family in the shared decision-making process. In our case, the death of the patient subsequent to the withdrawal of life-sustaining therapies was consistent with the patient's wishes, showing respect for his dignity and honoring his autonomy, as expressed to his family previously. By making donation possible, the medical team was able to fulfill the family's last request on behalf of the patient. This case should stimulate deliberation regarding the potential to shorten the 20-minute no-touch period currently in place in Italy. Such an action would not have injured this patient and would certainly have increased the quality of the procured organs.

Sirolimus vs cyclosporine after induction with basiliximab does not promote regulatory T cell expansion in de novo kidney transplantation: Results from a single-center randomized trial.

Regulatory T cells (Tregs), defined as CD4+CD25+highFoxP3+CD127- cells, could promote tolerance in renal transplantation (Tx). In an open-label, randomized, controlled trial 62 de-novo Tx recipients received induction with basiliximab and cyclosporine A (CsA) for the first month after Tx and then were assigned to treatment with sirolimus (SRL) or CsA and followed up for 2 years. The primary endpoint was to evaluate the effects of induction and maintenance treatments on circulating Tregs, while the secondary endpoint was the assessment of Treg renal infiltration and the relationship between Treg count and clinical outcomes. There were no significant differences in either circulating or tissue Treg number between the two groups. At 1 month post-Tx, all patients presented a profound Treg depletion, followed by a significant increase in Tregs that resulted stable during the follow-up. The same trend was also observed for non-activated Tregs (CD69-) and for other immunocompetent cells (CD4+ and CD8+ T cells, B cells and NK cells). Moreover, the Treg count did not correlate either with renal function or with acute rejection and graft loss. Initial immunosuppression is crucial to regulate circulating Tregs, regardless of subsequent immunosuppressive maintenance regimens. Strategies aiming to promote tolerance should consider the effects of different induction regimens.

[Non-heart-beating-donor transplant: the first experience in Italy]. / Trapianto di rene da donatore a cuore non battente: la prima esperienza in Italia.

A promising way to increase the number of kidneys for transplantation is to expand the donor pool by including non-heart-beating donors (NHBDs). The centers involved in NHBD transplantation programs have reported a 16-40% increase in kidney transplants. A key issue with NHBD is the significantly higher rate of delayed graft function (DGF) and primary non-function (PNF) compared with that associated with heart-beating donor (HBD) transplants. However, although transplants from NHBD are associated with a greater incidence of early adverse events, long-term graft survival appears to be similar to that observed after transplants from HBDs. In addition, the use of extracorporeal membrane oxygenation and mechanical perfusion, the careful selection of recipients and donors, and an adequate therapeutic strategy may at least partially reduce the risk of PNF and DGF and improve transplant outcome.

[Different approaches for medical or surgical management of Crohn's disease: the importance of the classification of Vienna]. / Modalità di approccio chirurgico nei pazienti con morbo di Crohn: inquadramento secondo la classificazione di Vienna.

The Vienna classification of Crohn's disease provides defined criteria for a phenotypic classification of the disease, considering that phenotypic parameters reflect the contribution of both genetic and environmental factors to the expression of disease. The classification includes mainly three criteria as part of the natural course of disease: age at the diagnosis, location and behaviour and it provides distinct definitions to categorize Crohn' patients into 24 subgroups. In this study we attempt to define the clinical importance of the Vienna classification and the possibility to optimize medical care of Crohn's disease according to patient subgroups. Early age at the diagnosis is associated with a greater prevalence of a family history, greater small bowel involvement, more complicated stricturing disease and a higher frequency of surgery. Surgery is based on exact knowledge of location: L1 patients might profit the most from resective surgery, L4 patients from strictureplasty. Stricturing behaviour (B2) might be the most appropriate subgroup for surgical treatment. Penetrating behaviour (B3) is confirmed as an important risk for early postoperative recurrence; patients in the penetrating behaviour group (i.e. fistulas) have a specific indication for immunosuppressive or anti-tumour necrosis factor-alpha therapy.