Preimplantation genetic diagnosis (PGD) for HLA typing: bases for setting up an open international collaboration when PGD is not available.

In severe forms of Diamond-Blackfan anemia, preimplantation genetic diagnosis (PGD) of histocompatibility leukocyte antigen-compatible embryos for enabling the next sibling in the family to be a stem-cell transplantation donor constitutes the sole lasting cure capable of terminating the enduring need for iterative transfusions. We report here an open collaboration between two renowned institutions to provide a family desiring this treatment even though they resided where the preimplantation genetic diagnosis procedure is banned.

Natural killer cell genes and functions after kidney transplantation

Natural Killer (NK) cells are of special interest in solid organ transplantation (SOT) because classical immunosuppressive drugs could enhance NK cells activity.We studied NK cells after kidney transplantation in three different situations. First, we analysed the peripheral repertoire reconstitution and function of NK cells after a polyclonal rabbit anti-thymocytes globulin (rATG) induction therapy, in 20 patients transplanted with living donor and with a low immunological risk. Second, we analysed the influence of KIR genes on the risk of CMV primo-infection or reactivation in 224 transplanted patients during the first year. Finally, we studied the risk of rejection and graft function during the first 5 years according to the KIR genes. Our study demonstrates that after an intial drop, NK cell reconstitution is fast with a ratio of CD56+/CD3− cells versus CD3+ cells that remains identical. The fraction of NK cells expressing the inhibitory receptor NKG2A significantly increases and the activating receptor NKG2D decreases after transplantation to retrieve the pretransplantation value after one year. The secretion of INF-f × and the cytotoxicity is maintained over time after transplantation. Then, we demonstrated that the presence of 2 KIR missing ligands and a large number of activating KIR gene protected against CMV primo-infection or reactivation during the first year post transplantation. Finally, the KIR genes and their HLA ligands do not influence the long term graft function after univariate and multivariate analysis. Our data suggest that despite the modification of the receptor repertoire, NK cell activity is preserved. NK cells are an important player of the immune response in the first year after transplantation mainly thanks to their anti-infectious activity.

Differential role of naïve and memory CD4 T-cell subsets in primary alloresponses.

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen-presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide-specific responses. In this study, we explored the contributions of naïve and memory CD4(+) T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4(+) T cells, both naturally occurring and induced by sensitization with multiple third-party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4(+)CD25(+) T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.

Hematopoietic stem cell transplantation in Switzerland: a comprehensive quality control report on centre effect.

Interest groups advocate centre-specific outcome data as a useful tool for patients in choosing a hospital for their treatment and for decision-making by politicians and the insurance industry. Haematopoietic stem cell transplantation (HSCT) requires significant infrastructure and represents a cost-intensive procedure. It therefore qualifies as a prime target for such a policy. We made use of the comprehensive database of the Swiss Blood Stem Cells Transplant Group (SBST) to evaluate potential use of mortality rates. Nine institutions reported a total of 4717 HSCT - 1427 allogeneic (30.3%), 3290 autologous (69.7%) - in 3808 patients between the years 1997 and 2008. Data were analysed for survival- and transplantation-related mortality (TRM) at day 100 and at 5 years. The data showed marked and significant differences between centres in unadjusted analyses. These differences were absent or marginal when the results were adjusted for disease, year of transplant and the EBMT risk score (a score incorporating patient age, disease stage, time interval between diagnosis and transplantation, and, for allogeneic transplants, donor type and donor-recipient gender combination) in a multivariable analysis. These data indicate comparable quality among centres in Switzerland. They show that comparison of crude centre-specific outcome data without adjustment for the patient mix may be misleading. Mandatory data collection and systematic review of all cases within a comprehensive quality management system might, in contrast, serve as a model to ascertain the quality of other cost-intensive therapies in Switzerland.

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide.

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.

Emotional experience in patients awaiting lung transplantation: A qualitative analysis

Emotional reactions in the course of transplantation are often explored through negative emotions (e.g. stress, guilt) or depression and its evaluation. However the emotional reactions in the course of transplantation describing positive and negative emotional experiences have not been comprehensively described. Qualitative semi-structured interviews were conducted shortly after registration on the waiting list with 15 lung patients waiting for an organ coming from a deceased donor. In a qualitative analysis, focussing on the emotional experience of transplantation, a very rich discourse was underlined. The described emotions in the interviews of the patients were related to specific situations, stakes and existential questions. All these emotions help to describe more precisely the very intimate experience of a difficult and stressful situation while awaiting transplantation. It also helps to better understand the impact of the paradoxical situation of transplantation when a person is waiting for an organ, which will improve quality of life and will allow to survive, but which also depends on the end of the life of a donor.

Programme latin de don d'organes (PLDO): une initiative efficace pour augmenter les dons d'organes en Suisse [Latin organ donation programme (LODP): an effective initiative to increase organ donation in Switzerland].

The 1st federal transplant law was enforced in July 2007 with the obligation to promote quality and efficiency in the procedures for organ and tissue donation for transplantation. The Latin organ donation programme (LODP) created in 2008 aims to develop organ donation in 17 public hospitals in 7 Latin cantons, covering 2.2 million people; 29% of the Swiss population. The implementation of various effective measures by the LODP enabled the increase in the number of donors by 70% between 2008 and 2010, with four organs procured per donor; greatly exceeding the European average of three. The results show that LODP has successfully professionalised the system and we can only hope that similar organisations will be put into place throughout Switzerland.

Strategies for preventing late-onset cytomegalovirus disease in organ transplant recipients.

Objective: The incidence of late-onset cytomegalovirus disease (i.e. disease appearing after discontinuation of antiviral prophylaxis) in solid-organ transplant recipients remains excessively high. This review will focus on describing the several strategies that could potentially reduce the incidence of late-onset cytomegalovirus disease. Methods: We reviewed the literature and presented our own clinical experience in the field. Results: The incidence of late-onset cytomegalovirus disease in recent trials can be as high as 36% in high-risk patients (donor positive/recipient negative for cytomegalovirus). The extension of antiviral prophylaxis to six months has recently proven in a prospective randomized controlled trial to be effective for reducing late-onset cytomegalovirus disease. The monitoring of cytomegalovirus viral load by PCR after the discontinuation of prophylaxis seems to be of moderate usefulness in low/intermediate-risk patients. The use of low-dose valganciclovir could reduce drug toxicity and costs while maintaining similar efficacy, but further studies are needed. A potentially interesting approach to predict the individual risk for development of cytomegalovirus disease appears to be the assessment of specific cell-mediated immune response. If cell-mediated immunity assays become widely available in transplant centers in the future, these assays may possibly be used to tailor the cytomegalovirus preventive strategy on an individual basis. Finally, recent prospective trials have evaluated novel cytomegalovirus vaccines that merit further evaluation in the transplant setting, although currently there is no cytomegalovirus vaccine that has been approved for routine clinical use. Conclusions: Several studies have recently evaluated novel strategies to reduce the incidence of late-onset cytomegalovirus disease. It is therefore expected that this improvement in preventive strategies will allow to further reduce the negative effects of cytomegalovirus disease after transplantation.

Pharmacokinetic and pharmacodynamic evaluation of valganciclovir in solid organ transplant patients

Goal: To validate oral vatgancictovir (VGC) in the prophylaxis of CMV infection in Lung (Lu) and Liver (L) recipients and in the treatment of CMV infection/disease in solid organ transplant recipients, using pharmacokinetic and pharmacodynamic studies in comparison with i/v gancicLovir (GCV). Methods: patients undergoing organ transpLantation donor or recipient CMV-seropositive receiving VGC prophylaxis for a period of 3 months (D+/R- Lung recipients, 6 months) were enroLLed. Heart (H), Lu, and L recipients received 900mg VGC q.d., adjusted to kidney (K) function. No K recipients received more than 450mg of VGC q.d. GCV trough (Ctrough) and peak (Cpeak = 3 hours after drug administration) LeveLs, and CMV DNA were measured at 7, 30, and 60 days post-transpLant (prophyLactic study). Patients who developed CMV infection/disease after stopping prophylaxis were treated with VGC (1800mg per day adjusted to K function and GCV blood LeveLs). GCV trough and peak LeveLs, and CMV DNA were measured weekly for the first 3 weeks and biweekly thereafter, until therapy cessation (therapeutic study). PLasma concentration of GCV is measured by HPLC. Results: In the first 8 prophyLaxed patients (6 K, and 1 L and 1 H transplant recipient) of 450mg VGC q.d., the average GCV concentration was 0.5±0.3 mg/t at trough, and 3.9±l.0mg/t 3 hours after administration. Inter-patient variability was substantiaL, especiaLLy for Ctrough (63% of total variance), which correlated with the patient's estimated gtomerutar filtration rate (r square = 42%). No CMV DNA was detected during VGC prophy- Laxis. Two patients (1 H and 1 L) were treated for Late CMV disease. Average GCV Cpeak were 8.9±2.3 mg/L and 4.6±0.5 rag/L, and GCV Ctrough were 2.0±0.9 mg/t and 1.6±0.2 mg/t respectively in each patient during induction phase. VGC treatment afforded a decrease in CMV DNA from 5.2 and 4.4 Log copies/10E6 cettutes at week 0, to 3.9 and 3.0 at week 1, and 3.3 and 2.1 at week 3, respectively.

No detectable indirect effects of late-onset Cytomegalovirus disease after valganciclovir (VGC) prophylaxis in kidney transplant recipients

Background: Cytomegalovirus (CMV) disease remains an important cause of morbidity after kidney transplantation and has been associated with acute rejection, graft loss and other indirect effects. A 3-month course of VGC prophylaxis reduces the incidence of CMV disease. However, little is known about the indirect effects of lateonset CMV disease after VGC prophylaxis. Objective: To evaluate the impact and indirect consequences of late-onset CMV disease after VGC prophylaxis in kidney transplant recipients. Methods: Retrospective analysis of 61 consecutive adult kidney transplant recipient with positive CMV serology (donor or recipient) who received VGC prophylaxis for 3 months and completed a follow-up of at least 2 years post-transplantation. Patients who developed CMV disease within 1 year after transplantation were compared to CMV disease-free patients for renal function (plasma creatinine values) at 1, 6, 12 and 24 months and for the incidence of graft loss, acute rejection, diabetes, cancer and opportunistic infections. Results: 8/61 (13%) patients developed CMV disease at a median of 131 days after transplantation (range: 98-220). The CMV incidence in D+/R- high risk patients was 6/18 (33%), while it was 2/43 (5%) in intermediate-risk patients (p < 0.01). All 8 patients were treated by oral valganciclovir (median 39 days; range: 19-119) with a complete resolution of CMV disease. As shown in the figure, there was no difference in creatinine values between the two groups at any time during follow-up. There was no graft loss, and the incidence of acute rejection, cancer and opportunistic infections did not differ between the two groups. The incidence of post-transplant diabetes was higher (38% vs 15%) in patients with CMV disease, but this difference was not significant (p = 0.4). Conclusions: An incidence of 13% of late-onset CMV disease was observed despite 3 months VGC prophylaxis. However, no indirect consequences were found. Moreover, therapy of CMV disease by oral VGC was effective and safe. Larger trials are needed to study whether late-onset CMV disease is associated with indirect consequences, as described with early-onset CMV.

Gene transfer of programmed death ligand-1.Ig prolongs cardiac allograft survival.

BACKGROUND: The CD28 homologue programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2 (which are homologous to B7), constitute an inhibitory pathway of T cell costimulation. The PD-1 pathway is of interest for immune-mediated diseases given that PD-1-deficient mice develop autoimmune diseases. We have evaluated the effect of local overexpression of a PD-L1.Ig fusion protein on cardiac allograft survival. METHODS: Adenovirus-mediated PD-L1.Ig gene transfer was performed in F344 rat donor hearts placed in the abdominal position in Lewis recipients. Inflammatory cell infiltrates in the grafts were assessed by immunohistochemistry. RESULTS: Allografts transduced with the PD-L1.Ig gene survived for longer periods of time compared with those receiving noncoding adenovirus or virus dilution buffer alone: median survival time (MST), 17 (range: 16-20) days vs. 11 (8-14) and 9 (8-13) days, respectively (P < 0.001). PD-L1.Ig gene transfer combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone: MST, 25 (15-42) vs. 15 (13-19) days (P < 0.05). PD-L1.Ig gene transfer was associated with decreased numbers of CD4 cells and monocytes/macrophages infiltrating the graft (P < 0.05). CONCLUSIONS: Localized PD-L1.Ig expression in donor hearts attenuates acute allograft rejection in a rat model. The effect is additive to that of a subtherapeutic regimen of CsA. These results suggest that targeting of PD-1 by gene therapy may inhibit acute cardiac allograft rejection in vivo.

LIGHT/HVEM/LTβR interaction as a target for the modulation of the allogeneic immune response in transplantation.

The exchange of information during interactions of T cells with dendritic cells, B cells or other T cells regulates the course of T, B and DC-cell activation and their differentiation into effector cells. The tumor necrosis factor superfamily member LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) is transiently expressed upon T cell activation and modulates CD8 T cell-mediated alloreactive responses upon herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR) engagement. LIGHT-deficient mice, or WT mice treated with LIGHT-targeting decoy receptors HVEM-Ig, LTβR-Ig or sDcR3-Ig, exhibit prolonged graft survival compared to untreated controls, suggesting that LIGHT modulates the course and severity of graft rejection. Therefore, targeting the interaction of LIGHT with HVEM and/or LTβR using recombinant soluble decoy receptors or monoclonal antibodies represent an innovative therapeutic strategy for the prevention and treatment of allograft rejection and for the promotion of donor-specific tolerance.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

The temporoparietal fascia flap: a versatile tool in head and neck reconstruction.

PURPOSE OF REVIEW: The article reviews recent significant advances and current applications of the temporoparietal fascia flap (TPFF) in head and neck surgery. RECENT FINDINGS: The recent literature describes a wide span of new applications of the TPFF in many areas. Significant developments and refinements in the reconstruction of orbitomaxillary composite defects and orbital exenteration cavities are reported. The TPFF combined with alloplastic framework is gaining in importance in external ear reconstruction. Innovative prefabricated skin or soft-tissue grafts based on the TPFF are used to restore facial contour or in the reconstruction of complex facial defects. The free TPFF finds a role in laryngotracheal reconstruction as a vascular carrier to support cartilage grafts. SUMMARY: Owing to its reliability and unequalled structural properties, the TPFF still plays a central role in facial reconstruction. Future investigation will likely incorporate the free TPFF as a vascular carrier of bioengineered tissues, such as cartilage and mucosa, for various head and neck indications.

Does Influenza Vaccination Induce De Novo HLA Alloantibody Formation in Transplant Recipients?

Introduction: Infl uenza vaccination is recommended for all solid organ transplant recipients. However, some centers are reluctant to give annual vaccination due to concerns about precipitating rejection. A proposed mechanism of this is vaccineinduced development of cellular and humoral responses to donor HLA antigens. We studied the induction of HLA Ab in a cohort of lung transplant recipients receiving infl uenza vaccination. Methods: Adult lung transplant recipients were immunized with 0.5 mL intramuscular seasonal infl uenza vaccine followed by 0.1 mL intradermal booster at 4 weeks as part of a previous study. Sera were collected pre-vaccination and at 4, 8 weeks post-vaccination. Post-vaccination sera were analyzed for HLA Ab using fl owPRA specifi c beads (One Lambda Inc). A positive result was defi ned as 5%. Positive samples were further analyzed for antibody specifi city by single antigen bead testing. Pre-vaccination sera were tested only only if post-vaccination sample screen was positive for HLA Ab. The presence of HLA Ab was correlated to vaccine seroresponse and rejection episodes. Results: Sixty patients were included with equal numbers of men and women. Mean age of patients was 47.3 years (range 20.7-72.4). Median time post-transplant was 1.3 years (range 85 days - 17 years). One patient was excluded due to an uninterpretable baseline screen result. 16/59 (27.1%) patients were positive for HLA Ab both in both pre- and post-vaccination samples. Of these, 12/16 (75%) had antibody against HLA Class I (majority A30,A31,B27,B44), 2/16 (12.5%) had antibody against HLA class II (majority DQ4, DQ7), and 2/16 (12.5%) had antibody against both Class I & II. There was no signifi cant increase in existing HLA Ab post-vaccination. Of the 16 patients, only one (6.3%) patient had de novo HLA Ab and this was determined to be non donor specifi c. Factors such as gender, time from transplant, immunosuppression, and acute rejection episodes did not correlate with presence of HLA Ab. HLA Ab was not associated with seroconversion to to vaccine antigens. Conclusions: Our data support that receiving the annual infl uenza vaccine does not lead to the generation of de novo donor specifi c antibodies in lung transplant recipients or upregulation of existing HLA Ab.