External ear canal mycobiome of some rabbit breeds.

The genus Malassezia is part of the normal skin mycobiota of a wide range of warm-blooded animals. In this genus, M. cuniculi is the only species described from rabbits. However, Malassezia species are rarely studied in lagomorphs. In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples. Although no growth was observed in the cultured plates, cytological examination revealed the presence of round cells similar to those of Malassezia yeasts. For metagenomics analysis, the D1/D2 domain of the large subunit of the ribosomal DNA (LSU rDNA) was PCR amplified and the resulting reads were mapped against a custom-made cured database of 26S fungal sequences. NGS analysis revealed that Basidiomycota was the most abundant phylum in all the samples followed by Ascomycota. Malassezia was the most common genus presenting the highest abundance in the external ear canal. Malassezia phylotype 131 and M. cuniculi were the main sequences detected in the external auditory canal of rabbits. The study included both lop-eared and erect-eared rabbits and no differences were observed in the results when comparing both groups. This is the first attempt to study the external ear canal mycobiome of rabbits of different breeds using NGS. LAY SUMMARY: In the present study, the presence of Malassezia was assessed in samples from the external ear canal of healthy rabbits of different breeds. Cytological and culture techniques, Sanger sequencing, and Next-generation sequencing (NGS) were used to describe the ear mycobiota in the samples.

Vertebral heart score to evaluate cardiac size in thoracic radiographs of 124 healthy rats (Rattus norvegicus).

Dilated cardiomyopathy is a relatively common disease in pet rats (Rattus norvegicus); however, there is a lack of radiographic references for the normal cardiac size in this species. The aim of this prospective, anatomical and reference interval study was to establish quantitative radiographic reference range measurements for the vertebral heart score (VHS) in rats. Right lateral (RL), ventrodorsal (VD), and dorsoventral (DV) radiographs of clinically healthy rats (n = 124) were evaluated. Measurements were performed by 2 expert readers who were unaware of signalment data. The mean values and references intervals of VHS were 7.7 and 7.0-8.5 for the RL, 7.5 and 6.6-8.6 for the VD, and 7.9 and 6.9-9.0 for the DV, with VHS values greater in males than in females. The measurements reported in this study can be used by the clinician as an objective tool to evaluate cardiac size in rats, in order to improve the diagnosis and treatment of cardiac diseases.

Successful use of nonantigen-specific immunoadsorption with antihuman Ig-columns in kidney graft antibody-mediated rejection.

INTRODUCTION: Nonantigen-specific immunoadsorption (IA) has proven to be effective in acute antibody-mediated rejection (aAMR). However, there is a lack of solid studies evaluating the safety and efficacy of IA with antihuman Ig-columns in aAMR. For chronic-active AMR (cAMR), no studies have evaluated the efficacy of nonantingen-specific IA with antihuman Ig-columns. The purpose of this study was to evaluate the role of nonantigen-specific IA with antihuman Ig-columns in the treatment of both aAMR and cAMR in kidney transplantation. MATERIAL AND METHODS: In retrospective and observational study, kidney graft and recipient survival rates were assessed after treatment of aAMR and cAMR with nonantigen-specific IA with Ig-Flex columns (Therasorb) between January 2012 and May 2018. Protocols included nonantigen-specific IA, rituximab, intravenous immunoglobulin, and rescue plasma exchange, if necessary. RESULTS: The study included 14 patients with AMR (acute in 9, chronic active in 5). For aAMR, mean follow-up was 13 ± 6 months, and patient and graft survival were, respectively, of 100% and 83%, with a mean increase in estimated glomerular filtration rate (eGFR) of 7.98 ± 12.96, 10.18 ± 16.71, and 11.43 ± 13.85 mL/min/1.72 m2 (P > .05) at 3, 12 months after treatment, and at the end of follow-up, respectively. For cAMR, mean follow-up was 14 ± 8 months, and patient and graft survival were, respectively, of 100% and 60%, with an average increase in eGFR of 4.30 ± 7.86, 5.64 ± 10.47, and 14.5 ± 7.86 mL/min/m2 (P > .05) at 3, 12 months after IA treatment, and at the end of the follow-up, respectively, although 40% did not respond and required chronic hemodialysis. CONCLUSION: Nonantigen-specific IA with Ig-Flex columns was safe and effective for aAMR treatment in kidney transplantation. In cAMR, IA with Ig-Flex columns was associated with a satisfactory kidney graft survival, suggesting that IA could potentially offer some benefits supporting its indication in cAMR.

Ultrasonographic appearance of the coelomic cavity organs in healthy veiled chameleons (Chamaeleo calyptratus) and panther chameleons (Furcifer pardalis).

Veiled chameleons (Chamaeleo calyptratus) and panther chameleons (Furcifer pardalis) are the most popular chameleons over the world, and consequently, two of the most frequent species presenting to veterinary practices. However, published studies on normal ultrasonographic anatomy for these lizards are currently lacking. The objectives of this prospective anatomic study were to develop an ultrasound protocol for evaluation of the coelomic cavity in these species and describe the normal ultrasonographic anatomy of the coelomic organs. Seventeen healthy veiled chameleons and 15 healthy panther chameleons were included. A linear 18 MHz transducer was used. Chameleons were sedated and restrained in right lateral recumbency by an assistant. Longitudinal and transverse images were acquired, and authors recorded qualitative and quantitative ultrasonographic characteristics of the coelomic structures. The kidneys, liver, caudal vena cava, hepatic veins, portal vein, gallbladder, wall of the stomach and intestine, gonads and, when distended, urinary bladder could be visualized during ultrasonography of the coelomic cavity in both species. The spleen, pancreas, and adrenal glands could not be identified. Findings from the current study supported the use of ultrasonography for veiled chameleons (Chamaeleo calyptratus) and panther chameleons (Furcifer pardalis) with suspected intracoelomic diseases and provided normal reference information for future studies of these chameleon species.

Ultrasonographic anatomy of the atlanto-occipital region and ultrasound-guided cerebrospinal fluid collection in rabbits (Oryctolagus cuniculus).

Cerebrospinal fluid analyses are important for diagnosis of neurologic problems in rabbits and for translational research projects using rabbits as models. Blind puncture of the cisterna magna is the current standard technique for sampling cerebrospinal fluid in this species. However, the complexity and small size of the cisterna magna and surrounding structures are limitations of this technique. Aims of this prospective, anatomic, pilot study were to (1) describe the normal anatomy of the atlanto-occipital region, (2) describe ultrasonographic anatomic landmarks, and (3) develop and evaluate a technique for ultrasound-guided puncture of the cisterna magna for cerebrospinal fluid sampling in rabbits. Thirty healthy rabbits were included and the study was conducted in three stages. Three rabbit cadavers were used for the first stage of the study. Then, the second stage was completed using 13 rabbit cadavers. Finally, the third stage was completed in 14 live rabbits. The ultrasound-guided puncture performed in 13 cadavers was successful at the first attempt in 10 cases, and at the second attempt in the remaining three cases. In the in vivo study, the ultrasound-guided puncture was successful in all 14 cases, without signs of complications. Findings supported the use of ultrasound-guided puncture of the cisterna magna as a safe technique that may be used routinely or when the sample of cerebrospinal fluid cannot be obtained with the blind technique in rabbits.

Posttransplant peripheral blood donor-specific interferon-γ enzyme-linked immune spot assay differentiates risk of subclinical rejection and de novo donor-specific alloantibodies in kidney transplant recipients.

Noninvasive diagnosis of kidney allograft inflammation in transplant recipients with stable graft function (subclinical rejection) could permit more effective therapy and prevent later development of de novo anti-donor HLA antibodies and/or graft dysfunction. Here we tested whether quantifying posttransplant donor-specific alloreactive T-cells by IFN-γ ELISPOT assay noninvasively detects subclinical T-cell mediated rejection and/or predicts development of anti-donor HLA antibodies. Using an initial cross-sectional cohort of 60 kidney transplant patients with six-month surveillance biopsies, we found that negative donor-specific IFN-γ ELISPOT assays accurately ruled out the presence of subclinical T-cell mediated rejection. These results were validated using a distinct prospective cohort of 101 patients where donor-specific IFN-γ ELISPOT results at both three- and six-months posttransplant significantly differentiated patients with subclinical T-cell mediated rejection at six months, independent of other clinical variables (odds ratio 0.072, 95% confidence interval 0.008-0.653). The posttransplant donor-specific IFN-γ ELISPOT results independently associated with subsequent development of significant anti-donor HLA antibodies (0.085, 0.008-0.862) and with significantly worse two-year function (estimated glomerular filtration rate) compared to patients with a negative test. Thus, posttransplant immune monitoring by donor-specific IFN-γ ELISPOT can assess risk for developing subclinical T-cell mediated rejection and anti-donor HLA antibodies, potentially limiting the need for surveillance biopsies. Our study provides a guide for individualizing immunosuppression to improve posttransplant outcomes.

Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation.

The accurate evaluation of donor-specific antibodies (DSAs) has allowed a precise identification of sensitized patients at risk of antibody-mediated rejection (ABMR). However, the scale of the humoral response is not always fully addressed, as it excludes the complete memory B-cell (mBC) pool such as that caused by antigen-specific mBC. Using a novel B-cell ELISpot assay approach, we assessed circulating mBC frequencies against class I and II HLA antigens in highly sensitized and nonsensitized patients in the waiting list for kidney transplantation. Also, kidney transplant patients undergoing ABMR were evaluated for the presence of donor-specific mBCs both at the time of rejection and before transplantation. For this purpose, 278 target HLA-sp antigens from 70 patients were studied and compared to circulating HLA-sp antibodies. Both class I and II HLA-sp mBC frequencies were identified in highly sensitized individuals but not in nonsensitized and healthy individuals, many years after first sensitization. Also, high donor-specific mBC responses were clearly found both during ABMR and before transplantation, regardless of circulating DSA. The higher the donor-specific mBC response, the more aggressive the allograft rejection. Thus, assessing donor-specific mBC frequencies may be relevant to better refine patient alloimmune-risk stratification, and provides new insight into the mechanisms of the adaptive humoral alloimmune response taking place in kidney transplantation.

Clinical response to pandemic H1N1 influenza virus from a fatal and mild case in ferrets.

BACKGROUND: The majority of pandemic 2009 H1N1 (A(H1N1)pdm09) influenza virus (IV) caused mild symptoms in most infected patients, however, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. The purpose of this work was to study in ferrets the dynamics of infection of two contemporary strains of human A(H1N1)pdm09 IV, one isolated from a patient showing mild disease and the other one from a fatal case. METHODS: Viral strains isolated from a patient showing mild disease-M (A/CastillaLaMancha/RR5661/2009) or from a fatal case-F (A/CastillaLaMancha/RR5911/2009), both without known comorbid conditions, were inoculated in two groups of ferrets and clinical and pathological conditions were analysed. RESULTS: Mild to severe clinical symptoms were observed in animals from both groups. A clinical score distribution was applied in which ferrets with mild clinical signs were distributed on a non-severe group (NS) and ferrets with severe clinical signs on a severe group (S), regardless of the virus used in the infection. Animals on S showed a significant decrease in body weight compared to animals on NS at 4 to 7 days post-infection (dpi). Clinical progress correlated with histopathological findings. Concentrations of haptoglobin (Hp) and serum amyloid A (SAA) increased on both groups after 2 dpi. Clinically severe infected ferrets showed a stronger antibody response and higher viral titres after infection (p = 0.001). CONCLUSIONS: The severity in the progress of infection was independent from the virus used for infection suggesting that the host immune response was determinant in the outcome of the infection. The diversity observed in ferrets mimicked the variability found in the human population.

Allogeneic adipose stem cell therapy in acute myocardial infarction.

BACKGROUND: Stem cell therapy offers a promising approach to reduce the long-term mortality rate associated with heart failure after acute myocardial infarction (AMI). To date, in vivo translational studies have not yet fully studied the immune response to allogeneic adipose tissue-derived mesenchymal stem cells (ATMSCs). We analysed the immune response and the histological and functional effects of allogeneic ATMSCs in a porcine model of reperfused AMI and determine the effect of administration timing. DESIGN: Pigs that survived AMI (24/26) received intracoronary administration of culture medium after reperfusion (n = 6), ATMSCs after reperfusion (n = 6), culture medium 7 days after AMI (n = 6) or ATMSCs 7 days after AMI (n = 6). At 3-week follow-up, cardiac function, alloantibodies and histological analysis were evaluated. RESULTS: Administration of ATMSCs after reperfusion and 7 days after AMI resulted in similar rates of cell engraftment; some of those cells expressed endothelial, smooth muscle and cardiomyogenic cell lineage markers. Delivery of ATMSCs after reperfusion compared with that performed at 7 days was more effective in increasing: vascular density (249 ± 64 vs. 161 ± 37 vessels/mm2; P < 0.01), T lymphocytes (1 ± 0.4 vs. 0.4 ± 0.3% of area CD3(+) ; P < 0.05) and expression of vascular endothelial growth factor (VEGF; 32 ± 7% vs. 20 ± 4% of area VEGF(+) ; P < 0.01). Allogeneic ATMSC-based therapy did not change ejection fraction but generated alloantibodies. CONCLUSIONS: The present study is the first to demonstrate that allogeneic ATMSCs elicit an immune response and, when administered immediately after reperfusion, are more effective in increasing VEGF expression and neovascularization.

Reference Range of Kaolin-Activated Thromboelastography (TEG) Values in Healthy Pet Rabbits (Oryctolagus cuniculus).

Thromboelastography (TEG) is a viscoelastic technique that allows the examination of both cellular and plasma protein clotting factors. Thromboelastography helps to investigate the underlying coagulopathy and to monitor therapeutic modalities. Although viscoelastic techniques have been used in human and veterinary medicine, reference ranges in pet rabbits are missing. The objective of this study is to establish the reference-range values of TEG parameters in healthy pet rabbits. 24 healthy pet rabbits of different breeds were included: 16 crossbreeds, four Californians, two lops, one lionhead, and one angora. Four rabbits were less than one year old and 20 were older than one year. Twelve rabbits were neutered females, 10 neutered males, and two were intact females. Health status was assessed through a physical examination, a complete blood work, and a coagulation profile. A TEG 5000 Thromboelastograph Hemostasis System was used with kaolin-activated citrated whole blood. All samples were analysed 30 min postextraction. The TEG reference ranges were reaction time (R) 1.4-6.9 min; clot formation time (K) 0.8-2.2 min; α angle 65.8-82.2 degrees; maximal amplitude (MA) 53.7-73.5 mm; measure of clot strength/firmness (G-value) 5796.6-13,885.9 dyn/cm2; and percentage of clot lysis in 30 min (LY30%) 0-41.5%. This study provides the reference ranges of TEG in pet rabbits.

Computed tomography of the coelomic cavity in healthy veiled chameleons (Chamaeleo calyptratus) and panther chameleons (Furcifer pardalis).

Background: Veiled chameleon (Chamaeleo calyptratus) and panther chameleon (Furcifer pardalis) are two of the most popular pet chameleons, and consequently, these species are frequently evaluated in veterinary practices. According to our review of the literature, normal computed tomography (CT) anatomy of these lizards has not been previously described. Aim: The purposes of this prospective study were to describe the normal CT anatomy of the coelomic organs in healthy patients and to provide normal reference values in these species. Methods: Seventeen clinically healthy veiled chameleons and 15 clinically healthy panther chameleons were included. All CT studies were performed with the chameleons under light anesthesia and positioned in sternal recumbency. Studies were performed with a 16-slice helical CT scanner with a slice thickness of 0.625 mm. The authors recorded qualitative and quantitative CT characteristics of the coelomic structures. Macroscopic cross-sectional anatomy was performed for comparison of the CT findings. Results: Heart, lungs, liver, including caudal vena cava, hepatic vessels, gallbladder, esophagus, stomach, intestines, gonads, fat bodies, kidneys, and urinary bladder could be visualized with CT. The spleen, pancreas, and adrenal glands could not be identified. Conclusion: This study provides a guide to the normal cross-sectional and computed tomographic anatomy of the coelomic cavity in veiled and panther chameleons. Our results could be used as a reference for future research studies or comparison of clinically ill patients.

Clinical, imaging, and pathologic features in cases of neurologic disease in 3 psittacine birds.

We used magnetic resonance imaging (MRI) to evaluate the CNS, and confirmed CNS lesions histologically, in 3 psittacine birds with neurologic signs. One bird was recumbent as a result of non-ambulatory paraparesis, and 2 birds were ataxic with impaired proprioception. In all 3 cases, imaging was performed, and infectious diseases were excluded in cases 1 and 2. In case 1, a large mass arose from the left lung; in case 2, a multinodular coelomic mass encompassed the left caudal pulmonary area to the left cranial renal pole; and in case 3, a diffuse hyperintensity affected the lumbar spinal cord. In the first 2 cases, masses invaded the vertebral canal, causing spinal cord compression. All 3 birds were euthanized given the poor prognosis, and postmortem examinations were performed. The final diagnoses were pulmonary adenocarcinoma in cases 1 and 2, and granulomatous and lymphocytic leptomeningitis caused by Mycobacterium genavense in case 3. MRI enabled visualization of the lesions in the affected area of the CNS, and MRI findings were confirmed by histopathology.

Health status of free-ranging pure and cross-mixed miniature swine population from Northeast Spain.

BACKGROUND: Miniature pigs have gained popularity as companion animals in the recent years in Spain. Due to the fact that their abandonment and crossing breeds with wild boars can cause severe problems, investigation about the health status is needed. OBJECTIVES: The aim of this study was to determine their health status according to the clinical findings during physical examination and the results of antibody serology tests against selected infectious diseases. METHODS: Two-hundred and eleven miniature pigs (Sus scrofa) were included in the study. Their origin, age, sex, housing conditions and diet were recorded. RESULTS: The housing of the animals ranged from wild animals to ones living in animal sanctuaries. The diet varied from a natural one in the wild to commercial and homemade food. Thirty animals out of two-hundred and eleven were hybrids between miniature pigs and wild boars according to morphological characteristics. Antibody serology techniques of Influenza A virus, Hepatitis E virus, brucellosis, tuberculosis, African swine fever, Classical swine fever and Aujeszky's disease was performed. The prevalence for Influenza A was 5.30%, for Hepatitis E was 5.35% and the rest tested negative. It is important to control and monitor these zoonotic infections to prevent Public Health problems. CONCLUSIONS: The results obtained from this investigation demonstrated that the animals' health status in this study is optimal and the diseases prevalence is similar or minor when compared to previous studies. This study confirms the hybridization of miniature pig and wild boar in Catalonia.

Undifferentiated Wing Sarcoma in a Peach-Faced Lovebird (Agapornis roseicollis).

A 10-year-old peach-faced lovebird (Agapornis roseicollis) was evaluated for an ulcerated and painful mass at the location of a fracture 2 years previously. Whole body radiographs showed a humeral fracture with a presumptive neoplastic proliferation in the distal diaphysis. Right wing amputation was elected but the animal died during recovery from surgery. Histopathological examination of the amputated wing revealed an infiltrative sarcomatous neoplastic proliferation. Immunohistochemistry (IHC) was carried out to characterize the tumour using antibodies against vimentin, desmin, smooth muscle actin (SMA), S-100, ionized calcium-binding adapter molecule-1 (IBA-1), CD18, cytokeratin and epithelial membrane antigen (EMA). The mesenchymal component of the mass was immunolabelled for vimentin and SMA and sparse epithelial cells were immunopositive for cytokeratin. Very few scattered cells were immunopositive for CD18 and IBA-1. The final diagnosis was consistent with an undifferentiated sarcoma with intralesional hyperplastic epithelium. According to the location, the history of a previous fracture and the histological pattern and IHC profile, the tumour was classified as an undifferentiated sarcoma with entrapped air sac epithelium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and humoral responses against different variants of concern in domestic pet animals and stray cats from North-Eastern Spain.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic in humans, is able to infect several domestic, captive and wildlife animal species. Since reverse zoonotic transmission to pets has been demonstrated, it is crucial to determine their role in the epidemiology of the disease to prevent further spillover events and major spread of SARS-CoV-2. In the present study, we determined the presence of virus and the seroprevalence to SARS-CoV-2, as well as the levels of neutralizing antibodies (nAbs) against several variants of concern (VOCs) in pets (cats, dogs and ferrets) and stray cats from North-Eastern of Spain. We confirmed that cats and dogs can be infected by different VOCs of SARS-CoV-2 and, together with ferrets, are able to develop nAbs against the ancestral (B.1), Alpha (B.1.1.7), Beta (B.1.315), Delta (B.1.617.2) and Omicron (BA.1) variants, with lower titres against the latest in dogs and cats, but not in ferrets. Although the prevalence of active SARS-CoV-2 infection measured as direct viral RNA detection was low (0.3%), presence of nAbs in pets living in COVID-19-positive households was relatively high (close to 25% in cats, 10% in dogs and 40% in ferrets). It is essential to continue monitoring SARS-CoV-2 infections in these animals due to their frequent contact with human populations, and we cannot discard the probability of a higher animal susceptibility to new potential SARS-CoV-2 VOCs.

Paradoxical Vestibular Syndrome Caused by a Presumptive Cerebellar Infarction in a Rabbit.

A 6-year-old, female spayed rabbit (Oryctolagus cuniculus) presented with right paradoxical vestibular signs. Magnetic resonance imaging was performed and findings were consistent with an ischemic infarct of the cerebellum. The patient improved gradually and was free of clinical signs at the time this article was written. To the authors' knowledge this is the first case report of a paradoxical vestibular syndrome in a rabbit secondary to a presumptive ischemic infarct. Strokes should be included in the differential diagnosis of central vestibular syndrome in rabbits.

A Europe wide acceptable mismatch program will enable transplantation of long waiting highly sensitised patients with a compatible donor.

Immunisation against Human Leucocyte Antigens (HLA) can be caused by pregnancy, blood transfusion, or organ transplants. The HLA antibody status of a given patient significantly influences their access and waiting time to transplant. For some highly sensitised patients (HSP) there is hardly any suitable donor available in the deceased donor pool of their allocation organisation and therefore they wait a very long time before being offered a kidney for transplant. Especially patients with rare HLA phenotypes in relation to the actual donor pool are waiting extremely long. As HLA phenotypes are different in the various European populations, we hypothesized that extension of the donor pool outside the respective allocation system will increase the chance of receiving a compatible transplant for this subgroup of highly sensitised patients. One of the objectives of the EUROSTAM project, (a Europe-wide Strategy to enhance Transplantation of highly sensitised patients on the basis of Acceptable HLA Mismatches) was to develop a tool to compare the chance of transplanting HSP in different European populations with donor organs from within and outside their own donor pool. Information on the HLA type and ABO blood group of the actual donor population, as well as the acceptable mismatches of long waiting HSP were obtained from the EUROSTAM partner organizations i.e. Eurotransplant (ET), UK National Health Service Blood and Transplant (NHSBT), Barcelona, Prague and Athens. Results from simulations using the newly developed tool shows that 195 (27%) of the 724 long waiting highly sensitised patients registered at each partner organisation have increased chances of transplant in a different European donor pool. This makes a strong case for sharing kidneys between European countries for selected difficult to transplant patients.

Detection of antibodies to denatured human leucocyte antigen molecules by single antigen Luminex.

The anti-HLA antibody detection has been improved in sensitivity and specificity with solid-phase antigen bead (SAB) assays based on Luminex. However, false positive results due to denatured HLA (dHLA) may arise after single antigen test. The aim of this study was to compare the performance of the two Luminex technology-based anti-HLA detection kits available in the market in showing undesired anti-HLA antibody results. A prospective cohort was assessed for anti-HLA antibodies with single antigen A manufacturer (AM) kit and a comparison cohort with single antigen B manufacturer (BM) kit. A total of 11 out of 90 patients in a prospective cohort presented monospecific HLA-I antibodies with AM, and 5 out of 11 confirmed monospecific reaction with BM. Despite the confirmation of monospecific reaction with both manufacturers, 80% were assigned as dHLA reaction by specific crossmatch. Further comparative cohorts detected four out of six monospecific reactions with BM that were confirmed as possible dHLA reactions. A positive SAB test should rule out a reaction against a dHLA molecule, thus avoidance of prolonged waitlist periods or misattribution of anti-HLA reactions after transplantation.

B Cell-Derived Extracellular Vesicles Reveal Residual B Cell Activity in Kidney Graft Recipients Undergoing Pre-Transplant Desensitization.

Background: Living-donor kidney transplant (LDKT) recipients undergoing desensitization for Human Leukocyte Antigen (HLA)-incompatibility have a high risk of developing antibody-mediated rejection (ABMR). The purpose of the study is to evaluate if residual B cell activity after desensitization could be estimated by the presence of circulating B cell-derived extracellular vesicles (BEVs). Methods: BEVs were isolated by Sepharose-based size exclusion chromatography and defined as CD19+ and HLA-II+ extracellular vesicles. We analyzed stored serum samples from positive crossmatch LDKT recipients before and after desensitization at first post-transplant biopsy and at 12-month protocol biopsy (n = 11). Control groups were formed by hypersensitized patients who were not submitted to desensitization (n = 10) and by low-risk recipients (n = 9). A prospective validation cohort of 11 patients also included the analysis of B cells subpopulations in recipients' blood and lymph nodes recovered upon graft implantation, along with BEVs analysis before and after desensitization. Results: We found out that CD19+ and HLA-II+BEVs dropped significantly after desensitization and relapse in patients who later developed ABMR was evident. We validated these findings in a proof-of-concept prospective cohort of 6 patients who received the same desensitization protocol and also in a control group of 5 LDKT recipients. In these patients, B cell subpopulations were also studied in recipients' blood and lymph nodes that were recovered before the graft implantation. We confirmed the significant drop in BEVs after desensitization and that this paralleled the reduction in CD19+cells in lymph nodes, while in peripheral blood B cells, this change was almost undetectable. Conclusions: BEVs reflected B cell residual activity after desensitization and this could be a valid surrogate of humoral alloreactivity in this setting.

[Immunologic study of the donor-receptor couple]. / Estudio inmunológico de la pareja donante-receptor.

The objective of the study is to evaluate the risk of graft failure. The presence of donor specific alloantibodies and the HLA incompatibilities between donor and receptor must be identified. There are several methods to identify alloandibodies that has different sensitivity and different Prognostic Value. Some define a high risk of hyperacute rejection, others an increase in the risk to loss the graft in defined subgroups. First steps of the pretransplant study identify: a) HLA typing of the receptor and available donors; b) alloantibodies by Complement Dependent Cytotoxicity against Panel (PRA-CDC) and screening of alloantibodies against HLA by Solid Phase; c) in sensitized receptors it can be useful to identify acceptable incompatibilities using Single Antigen Solid Phase technique and to evaluate the «Virtual Crossmatch». Pretransplant study (10 days): a) crossmatch by Citotoxicity (CM-CDC) between receptor and donor; b) crossmatch by Flow-Cytometry (FCCM) between receptor and donor specially indicated in the retransplant. Useful also to discard IgM auto-antibodies. Receptors desensitization: the necessity and success probability of desensitization should be evaluated before treatment. Post-Transplant Monitoring: identify alloantibodies for: a) the differential diagnostic of corticorresistant rejection episodes with humoral component, and b) as a marker of long term reduced graft survival probability in the long term. Final remarks: Evaluation should consider the allosensibilization history of the receptor. The cytotoxicity crossmatch indicates a high risk of hyperacute rejection and is considered a contraindication. The Flow Cytometry crossmatch indicates an increase in the probability to loss the graft in the first year that is low for first transplants (>10%) but higher for retransplantation (>30%). The virtual crossmatch by solid phase indicates an increase in the probability to have an antibody mediated rejection (from 5% to 55%) but did not contraindicate always the transplant.