Tomas Lorant

Standardized experimental brain death model for studies of intracranial dynamics, organ preservation, and organ transplantation in the pig

Objectives:Brain death impairs organ function and outcome after transplantation. There is a need for a brain death model to allow studies of organ viability and preservation. For neurointensive care research, it is also of interest to have a relevant brain death model for studies of intracranial dynamics and evaluation of cerebral monitoring devices. Therefore, the objective was to develop a standardized clinically relevant brain death model. Methods:Six pigs of both sexes (10–12 wks old; mean weight, 24.5 ± 1.4 kg) were included. Mean arterial blood pressure, heart rate, intracranial pressure, intracranial compliance, cerebral perfusion pressure, and brain tissue oxygenation (BtiPo2) were recorded during stepwise elevation of intracranial pressure by inflation of an epidural balloon catheter with saline (1 mL/20 mins). Brain death criteria were decided to be reached when cerebral perfusion pressure was <0 mm Hg for 60 mins and at least 10 mL saline was inflated epidurally. BtiPo2 and arterial injections of microspheres were used for confirmation of brain death. Results:A gradual volume-dependent elevation of intracranial pressure was observed. After 10 mL of balloon infusion, mean intracranial pressure was 89.8 ± 9.7 (sd) mm Hg. Intracranial compliance decreased from 0.137 ± 0.069 mL/mm Hg to 0.007 ± 0.001 mL/mm Hg. The mean arterial pressure decreased and the heart rate increased when the intracranial volume was increased to between 5 and 6 mL. All animals showed cerebral perfusion pressure ≤0 after 7 to 10 mL of infusion. In all animals, the criteria for brain death with negative cerebral perfusion pressure and BtiPo2 ∼0 mm Hg were achieved. Only a negligible amount of microspheres were found in the cerebrum, confirming brain death. The kidneys showed small foci of acute tubular necrosis. Conclusions:The standardized brain death model designed in pigs simulates the clinical development of brain death in humans with a classic pressure-volume response and systemic cardiovascular reactions. Brain death was convincingly confirmed.

IgG Endopeptidase in Highly Sensitized Patients Undergoing Transplantation

Background Donor‐specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor‐specific antibodies are limited and ineffective in the most highly HLA‐sensitized patients. The IgG‐degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab′)2 and Fc fragments inhibiting complement‐dependent cytotoxicity and antibody‐dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open‐label, phase 1–2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA‐incompatible donor. Methods We administered IdeS to 25 highly HLA‐sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA‐incompatible donor. Frequent monitoring for adverse events, outcomes, donor‐specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound. Results Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor‐specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody‐mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non‐HLA IgM and IgA antibodies, occurred. Conclusions IdeS reduced or eliminated donor‐specific antibodies and permitted HLA‐ incompatible transplantation in 24 of 25 patients. (Funded by Hansa Medical; ClinicalTrials.gov numbers, NCT02224820, NCT02426684, and NCT02475551.)

Sinus excision and primary closure versus laying open in pilonidal disease: a prospective randomized trial.

Background: Surgical excision is the standard treatment for chronic pilonidal disease, but all excisional techniques are associated with tissue loss, risk of wound break down, and chronic healing problems. OBJECTIVE: The aim of the study was to compare sinus excision and primary closure vs a laying open technique in a prospective randomized trial. DESIGN, PATIENTS, AND INTERVENTIONS: Eighty patients were randomly assigned to sinus excision and primary closure (n = 39) or laying open (n = 41). Follow-up was performed 1, 3, and 12 months after surgery. MAIN OUTCOME MEASURE: The main outcome measure was the healing rate after 1 year. RESULTS: The healing rate was significantly higher after excision and closure than after laying open at 1 month (20 of 39 vs 8 of 41; P = .005) and 3 months (36 of 38 vs 28 of 39; P = .013) after surgery. At follow-up 12 months after surgery no difference was seen in healing rate between the treatment arms (33 of 37 vs 37 of 38; P = .198). CONCLUSIONS: This prospective randomized trial shows that sinus excision and primary closure results in faster healing than laying open does, but there is no difference in healing rate after 1 year. The laying open procedure is minimally invasive with small risks for the patient, and it might therefore be considered more frequently as the first choice of treatment (www.clinicaltrials.gov. Unique identifier: NCT00997048).

Screening of mortality in transplant patients using an assay for immune function

BACKGROUND So far, the ImmuKnow Immune Cell Function Assay (Cylex, Inc., Columbia, MD, USA) has been used to assess risks of infection and rejection in transplant patients. We hypothesized that the ImmuKnow assay might be used for mortality screening in transplant patients overall. METHODS In the period of February 2007 to December 2009, at the Uppsala University Hospital, 362 patients who received either kidney, kidney+pancreas, kidney+islet cells, liver or liver+kidney allografts were randomly screened using the ImmuKnow assay. All causes of mortality were compared between two groups: patients with at least one ImmuKnow assay below 175ng/mL and patients with all ImmuKnow assays from 175ng/mL and above. Subsequently, the frequency of rejection within thirty days of the ImmuKnow assay was compared between these two groups. RESULTS The study included 1031 ImmuKnow assays obtained from the 362 patients. A total of 111 patients had at least one ImmuKnow below 175ng/mL and 251 patients had all their ImmuKnow assays from 175ng/mL and above. By January 31st 2010, 16 of 111 patients (14.4%) with at least one ImmuKnow assay below 175ng/mL were deceased, compared to 13 of 251 patients (5.2%) with all ImmuKnow assays from 175ng/mL and above (p=0.0053, Fishers exact test). There was no difference in the frequency of rejection between the two groups (19.8% versus 17.5%, p=0.66). CONCLUSIONS In addition to assessing relative risks of infection and rejection in transplant patients, the ImmuKnow assay may be used to identify patients with increased risk of short-term mortality. Transplant patients being highly overimmunosuppressed as assessed by the ImmuKnow assay do not seem to have a lower risk of short-term rejection.

Refined Microdialysis Method for Protein Biomarker Sampling in Acute Brain Injury in the Neurointensive Care Setting

There is growing interest in cerebral microdialysis (MD) for sampling of protein biomarkers in neurointensive care (NIC) patients. Published data point to inherent problems with this methodology including protein interaction and biofouling leading to unstable catheter performance. This study tested the in vivo performance of a refined MD method including catheter surface modification, for protein biomarker sampling in a clinically relevant porcine brain injury model. Seven pigs of both sexes (10-12 weeks old; 22.2-27.3 kg) were included. Mean arterial blood pressure, heart rate, intracranial pressure (ICP) and cerebral perfusion pressure was recorded during the stepwise elevation of intracranial pressure by inflation of an epidural balloon catheter with saline (1 mL/20 min) until brain death. One naïve MD catheter and one surface modified with Pluronic F-127 (10 mm membrane, 100 kDa molecular weight cutoff MD catheter) were inserted into the right frontal cortex and perfused with mock CSF with 3% Dextran 500 at a flow rate of 1.0 μL/min and 20 min sample collection. Naïve catheters showed unstable fluid recovery, sensitive to ICP changes, which was significantly stabilized by surface modification. Three of seven naïve catheters failed to deliver a stable fluid recovery. MD levels of glucose, lactate, pyruvate, glutamate, glycerol and urea measured enzymatically showed an expected gradual ischemic and cellular distress response to the intervention without differences between naïve and surface modified catheters. The 17 most common proteins quantified by iTRAQ and nanoflow LC-MS/MS were used as biomarker models. These proteins showed a significantly more homogeneous response to the ICP intervention in surface modified compared to naïve MD catheters with improved extraction efficiency for most of the proteins. The refined MD method appears to improve the accuracy and precision of protein biomarker sampling in the NIC setting.

Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials

Adoptive transfer of regulatory T cells (Tregs) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with Treg therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4+CD25highCD127low Tregs from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare Treg preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded Treg preparations have been characterized by their purity, cytokine production and in‐vitro suppressive ability. The results show that Treg preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the Treg preparations. In particular, FACS‐sorted Treg preparations expanded with mature DCs secrete more interleukin (IL)‐10 and granzyme B than FACS‐sorted Treg preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with Tregs from uraemic patients that may guide future efforts to produce clinical‐grade Tregs for use in kidney transplantation.

Modifying the vessel walls in porcine kidneys during machine perfusion.

BACKGROUND Endothelial glycocalyx regulates the endothelial function and plays an active role in maintaining vascular homeostasis. During ischema and reperfusion, the glycocalyx is rapidly shed into the blood stream. A Corline heparin conjugate (CHC; Corline systems AB, Uppsala, Sweden) consists of 70 heparin molecules that have the capacity to adhere strongly to biological tissues expressing heparin affinity. We hypothesized that CHC could be used to restore disrupted glycocalyx in vivo in kidneys from brain-dead pigs. MATERIALS AND METHODS Brain death was induced in male landrace pigs (n = 6) by inflating a balloon catheter in the epidural space until obtaining negative cerebral perfusion. The recovered kidneys (n = 5 + 5) were perfused by hypothermic machine perfusion using two Lifeport kidney transporters (Organ Recovery Systems, Chicago, IL). CHC (50 mg) (including 25 mg biotinylated CHC) or 50 mg unfractionated heparin (control) was added to the perfusion fluid in the respective machines. In one case, the kidneys were used only for dose escalation of CHC with the same procedure. RESULTS CHC was detected by immunofluorescence and confocal microscopy in the inner surface of the vessel walls. The binding of CHC in the kidney was confirmed indirectly by consumption of CHC from the perfusion fluid. CONCLUSIONS In this first attempt, we show that CHC maybe used to coat the vessel walls of perfused kidneys during hypothermic machine perfusion, an approach that could become useful in restoring endothelial glycocalyx of kidneys recovered from deceased donors to protect vascular endothelium and possibly ameliorate ischemia and reperfusion injuries.

Isolation of mouse‐to‐rat cardiac xenograft‐infiltrating cells by ex vivo propagation

Ex vivo propagation of graft infiltrating lymphocytes has become a useful method for the examination of the cellular response after allogeneic transplantation. The aim of the present study was to investigate if this method can be used also for isolation of xenograft infiltrating cells, and, if so, to further characterize these cells.

Initial Experience With Hypothermic Machine Perfusion of Kidneys From Deceased Donors in the Uppsala Region in Sweden

BACKGROUND Simple cold storage (CS) is the gold standard for organ preservation. Recently, evidence has been presented suggesting compared with CS hypothermic machine perfusion (HMP) improves the quality and outcome of kidneys for transplantation. Uppsala has used the LifePort Kidney Transporter to preserve deceased donor kidneys. We evaluated our first single-center 52 cases retrospectively. METHODS Deceased donor kidneys preserved with HMP between July 2010 and July 2012 (n = 52) were compared with a matched historical cohort of organs preserved by CS between January 2009 and July 2012 (n = 87). We evaluated delayed graft function (DGF), creatinine level at hospital discharge, length of hospital stay, incidence of acute rejection episodes during the first year after transplantation, and graft survival. RESULTS Both groups included approximately 69% expanded criteria donors (ECD). Median cold ischemia time (CIT) was 12.8 hours in the HMP group and 11.7 hours in the CS group. The incidence of DGF was 11.5% with HMP and 20.7% with CS. Compared with CS, HMP significantly reduced the occurrence of DGF from 21.4% to 0% using standard criteria kidneys (P = .046), whereas the use of HMP did not impact the occurrence of DGF with ECD kidneys. The creatinine level at hospital discharge was lower after HMP than after CS (P = .047). No difference in graft survival was observed between the groups. CONCLUSIONS Machine perfusion resulted in a lower occurrence of DGF using kidneys from standard criteria donors with a lower creatinine at hospital discharge among the cohort with reasonably low CIT. Using machine perfusion seems to be safe; no adverse surgical events occurred during the study period.

Efficacy and Safety of Continuous Local Infusion of Ropivacaine after Retroperitoneoscopic Live Donor Nephrectomy

Morphine‐based analgesia is effective but can compromise donor safety. We investigated whether continuous infusion of local anesthetics (CILA) can provide sufficient pain control and reduce morbidity related to opiate analgesics after hand‐assisted retroperitoneoscopic (HARS) live donor nephrectomy. Forty consecutive live kidney donors underwent HARS and were treated with the ON‐Q system providing CILA with 0.5% ropivacaine through two SilvaGard® catheters placed in the retroperitoneal cavity and the rectus sheath, respectively. The case control group consisted of 40 donors matched with regard to sex, age, BMI and surgical technique. All donors were maintained on standardized multimodal analgesia combining nurse‐controlled oxycodone treatment and acetaminophen. CILA donors had lower median cumulative consumption of morphine equivalents (CCME) (7 mg [0–56] vs. 42 mg [15–127]; p < 0.0000001), lower incidence of nausea (18 [45%] vs. 35 [87.5%] donors; p < 0.001), shorter time in postoperative care unit (160 vs. 242.5 min; p < 0.001) and shorter hospital stay (4 [4–7] vs. 6 [4–11] days; p < 0.001). In 32.5% of CILA donors the CCME was 0 mg (0% in matched control group, p < 0.001). CILA with 0.5% ropivacaine provides effective postoperative pain relief, reduces the need for opioid treatment and promotes postoperative recovery.