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1.  Rapid generation of clinical-grade antiviral T cells: selection of suitable T-cell donors and GMP-compliant manufacturing of antiviral T cells 
The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.
To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ+ T cells exceeded 0.03% of CD3+ lymphocytes as determined by IFN-γ cytokine secretion assay.
Starting with low concentration of IFN-γ+ T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ+CD3+ T cells (1.42 × 106, 0.05 × 106, and 1.15 × 106) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8+ than CD4+ memory T cells, both of which were effectively enriched to a total of 81.0% CD8+IFN-γ+ and 38.4% CD4+IFN-γ+ T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.
The generation of antiviral CD4+ and CD8+ T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0336-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4335407  PMID: 25510656
Adoptive immunotherapy; Antiviral T cells; alloCELL; GMP-compliant manufacturing; CliniMACS CCS; Stem cell transplantation; Adoptive T-cell transfer
2.  Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells 
Cell replacement therapy may be considered as an alternate approach to provide therapeutic dose of plasma factor VIII (FVIII) in patients with hemophilia A (HA). However, immune rejection limits the use of allogeneic cells in this mode of therapy. Here, we have examined the role of donor major histocompatibility complex (MHC)-stimulated host CD4+CD25+ regulatory T (Treg) cells in suppressing immune responses against allogeneic uncommitted (Lin−) bone marrow cells (BMCs) for correction of bleeding disorder in HA mice.
Allogeneic donor Lin− BMCs were co-transplanted with allo-antigen sensitized Treg cells in HA mice having acetaminophen-induced acute liver injury. Plasma FVIII activity was determined by in vitro functional assay, and correction of bleeding phenotype was assessed on the basis of capillary blood clotting time and tail-clip challenge. The immunosuppression potential of the sensitized Treg cells on CD4+ T cells was studied both in vitro and in vivo. Suppression of inflammatory reactions in the liver against the homed donor cells by sensitized Treg cells was analysed by histopathological scoring. Allo-specificity of sensitized Treg cells and long-term retention of immunosuppression were examined against a third-party donor and by secondary challenge of allogeneic donor cells, respectively. The engraftment and phenotype change of donor BMCs in the liver and their role in synthesis of FVIII and liver regeneration were also determined.
Co-transplantation of allogeneic Lin− BMCs with sensitized Treg cells led to systemic immune modulation and suppression of inflammatory reactions in the liver, allowing better engraftment of allogeneic cells in the liver. Allo-antigen priming led to allo-specific immune suppression even after 1 year of transplantation. Donor-derived endothelial cells expressed FVIII in HA mice, leading to the correction of bleeding phenotype. Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.
A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin− BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells. This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s13287-015-0119-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4513683  PMID: 26152192
3.  Proliferative alloresponse of T-cytotoxic cells identifies rejection-prone children with steroid-free liver transplantation 
Donor- and third-party-induced proliferation of T-helper (Th) and T-cytotoxic (Tc) cells, and their naïve and memory subsets was evaluated simultaneously in single blood samples from 77 children who received steroid-free liver transplantation (LTx) after induction with rabbit anti-human thymocyte globulin. Proliferation was measured by dilution of the intravital dye carboxy-flourescien-succinimidyl-ester (CFSE) in 3–4 day MLR co-culture. The ratio of donor: third-party-induced proliferated, (CFSElow) T-cells was reported as the immunoreactivity index (IR) for each subset. Rejectors were defined as those who experienced biopsy-proven acute cellular rejection within 60 days of the assay. IR>1 signified increased risk of rejection and IR<1 implied decreased risk.
Demographics for 32 Rejectors and 45 Non-Rejectors were similar. Proliferated CFSElow T-cells and subsets were significantly higher among Rejectors, compared with Non-Rejectors. In 33 of 77 randomly selected children, logistic regression, leave-one-out cross-validation and ROC analyses showed that the IR of Tc associated best with biopsy-proven rejection (sensitivity>75%, specificity>88%). Sensitivity/specificity were replicated in the remaining 44 children, comprising the validation cohort. IR of CFSElow Tc correlated significantly with IR of pro-inflammatory, allospecific CD154+Tc (r=0.664, p=0.0005), and inversely with IR of allospecific, anti-inflammatory, CTLA4+Tc (r=−0.630, p=0.007).
Proliferative alloresponses of T-cytotoxic cells can identify rejection-prone children receiving LTx. (200)
PMCID: PMC2997467  PMID: 19642137
4.  Metabolic effects of liver transplantation in cirrhotic patients. 
Journal of Clinical Investigation  1997;99(4):692-700.
To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.
PMCID: PMC507852  PMID: 9045872
5.  Allospecific CD154+ T-cells associate with rejection risk after pediatric liver transplantation 
Antigen-specific T-cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16-hour MLR of 58 identically-immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy-proven rejection within 60 days post-transplantation). Thirty one children were sampled once, cross-sectionally. Twenty seven children were sampled longitudinally, pre-LTx, and at 1–60 and 61–200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE-dye dilution (n=23), and CTLA4, a negative T-cell costimulator, which antagonizes CD154-mediated effects (n=31). In cross-sectional observations, logistic regression and leave-one-out cross-validation identified donor-specific, CD154+T-cytotoxic (Tc)-memory cells as best associated with rejection outcomes. In the longitudinal cohort, 1) the association between CD154+Tc-memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1–60 days, and 2) elevated pre-LTx CD154+Tc-memory cell responses were associated with significantly increased incidence (p=0.02) and hazard (HR=7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r=0.835, p=7.1e-07), and inversely with CTLA4 expression of allospecific CD154+Tc-memory cells (r=−0.706, p=3.0e-05). Allospecific CD154+T-helper-memory cells, not CD154+Tc-memory, were inhibited by increasing Tacrolimus concentrations (p=0.026). Collectively, allospecific CD154+T-cells provide an estimate of rejection risk in children with LTx.
PMCID: PMC2997472  PMID: 18976293
6.  Complement activation is not required for obliterative airway disease induced by antibodies to MHC Class I: Implications for Chronic Lung Rejection 
Role of non-complement activating antibodies (Abs) to mismatched donor HLA in pathogenesis of chronic lung rejection is not known. We utilized a murine model of obliterative airway disease (OAD) induced by Abs to MHC class I and serum from donor specific Abs (DSA) developed human lung transplantation (LTx) recipients (LTxR) to test the role of non-complement activating Abs in development of OAD and bronchiolitis obliterans syndrome (BOS).
Noncomplement activating anti-MHC (ncAbs) were administered intrabronchially in B.10 mice or in C3 knockout (C3KO) mice. Lungs were analyzed by histopathology. Lymphocytes secreting IL-17, IFN-γ or IL-10 to Collagen V (ColV) and K-alpha 1 tubulin (Kα1T) were enumerated by ELISpot. Serum Abs to ColV and Kα1T determined by ELISA. Cytokine and growth factor expression in lungs was determined by RTPCR. DSA from patients with BOS and control BOS-negative LTxR were analyzed by C1q assay.
Administration of ncAbs in B.10 mice or C3KO resulted in OAD lesions. There were significant increases in IL-17 and IFNγ secreting cells to ColV and Kα1T along with serum Abs to these antigens. There was also augmented expression of MCP-1, IL-6, IL-1β, VEGF, TGFβ, and FGF in ncAbs administered mice by day 3. Among LTxR with BOS only 1/5 had C1q binding DSA.
Complement activation by Abs to MHC class I is not required for development of OAD and human BOS. Therefore, anti-MHC binding to epithelial and endothelial cells can directly activate pro-fibrotic and pro-inflammatory cascades leading to immune response to self-antigens and chronic rejection.
PMCID: PMC3472127  PMID: 22980951
7.  Impact of Center on Graft Failure after Liver Transplantation 
The hospital where liver transplantation (LTx) is performed has a substantial impact on post-LTx outcome. Center-specific outcome data are closely monitored not only by the centers themselves but also by patients and government regulatory agencies. However, the true magnitude of this center effect, apart from the effects of the region and donor service area (DSA) as well as recipient and donor determinants of graft survival has not been examined.
We analyzed data submitted to the Organ Procurement and Transplantation Network (OPTN) on all adult (age≥18 years) primary LTx recipients (2005-2008).Using the mixed effects proportional hazard regression analysis, we modeled graft failure within 1 year after LTx based on center (de-identified), region, DSA, and donor and recipient characteristics.
There were 14,654 recipients transplanted at 115 unique centers. Rates of graft loss within a year varied from 5.9% in the lowest quartile of centers to 20.2% in the highest quartile. When gauged by the comparison between the 75th and 25th percentiles of the data, the magnitude of the effect of center on graft survival (1.49 fold change) was similar to that of the recipient MELD score (1.47) and DRI (1.45). The center effect was similar across quartiles of DRI and MELD scores, and not associated with the center’s annual volume of LTx. After stratification by region and DSA, the magnitude of center effect, though decreased, remained significant and substantial (1.30 fold interquartile difference).
Center of LTx was a significant predictor of graft failure, independent of region and DSA, as well as donor and recipient characteristics.
PMCID: PMC4130473  PMID: 23784730
Mixed Effects Model; donor factors; organ allocation; Donor Risk Index; Donor Service Area
8.  Extracorporeal Lung Support Technologies – Bridge to Recovery and Bridge to Lung Transplantation in Adult Patients 
Executive Summary
For cases of acute respiratory distress syndrome (ARDS) and progressive chronic respiratory failure, the first choice or treatment is mechanical ventilation. For decades, this method has been used to support critically ill patients in respiratory failure. Despite its life-saving potential, however, several experimental and clinical studies have suggested that ventilator-induced lung injury can adversely affect the lungs and patient outcomes. Current opinion is that by reducing the pressure and volume of gas delivered to the lungs during mechanical ventilation, the stress applied to the lungs is eased, enabling them to rest and recover. In addition, mechanical ventilation may fail to provide adequate gas exchange, thus patients may suffer from severe hypoxia and hypercapnea. For these reasons, extracorporeal lung support technologies may play an important role in the clinical management of patients with lung failure, allowing not only the transfer of oxygen and carbon dioxide (CO2) but also buying the lungs the time needed to rest and heal.
The objective of this analysis was to assess the effectiveness, safety, and cost-effectiveness of extracorporeal lung support technologies in the improvement of pulmonary gas exchange and the survival of adult patients with acute pulmonary failure and those with end-stage chronic progressive lung disease as a bridge to lung transplantation (LTx). The application of these technologies in primary graft dysfunction (PGD) after LTx is beyond the scope of this review and is not discussed.
Clinical Applications of Extracorporeal Lung Support
Extracorporeal lung support technologies [i.e., Interventional Lung Assist (ILA) and extracorporeal membrane oxygenation (ECMO)] have been advocated for use in the treatment of patients with respiratory failure. These techniques do not treat the underlying lung condition; rather, they improve gas exchange while enabling the implantation of a protective ventilation strategy to prevent further damage to the lung tissues imposed by the ventilator. As such, extracorporeal lung support technologies have been used in three major lung failure case types:
As a bridge to recovery in acute lung failure – for patients with injured or diseased lungs to give their lungs time to heal and regain normal physiologic function.
As a bridge to LTx – for patients with irreversible end stage lung disease requiring LTx.
As a bridge to recovery after LTx – used as lung support for patients with PGD or severe hypoxemia.
Ex-Vivo Lung Perfusion and Assessment
Recently, the evaluation and reconditioning of donor lungs ex-vivo has been introduced into clinical practice as a method of improving the rate of donor lung utilization. Generally, about 15% to 20% of donor lungs are suitable for LTx, but these figures may increase with the use of ex-vivo lung perfusion. The ex-vivo evaluation and reconditioning of donor lungs is currently performed at the Toronto General Hospital (TGH) and preliminary results have been encouraging (Personal communication, clinical expert, December 17, 2009). If its effectiveness is confirmed, the use of the technique could lead to further expansion of donor organ pools and improvements in post-LTx outcomes.
Extracorporeal Lung support Technologies
The ECMO system consists of a centrifugal pump, a membrane oxygenator, inlet and outlet cannulas, and tubing. The exchange of oxygen and CO2 then takes place in the oxygenator, which delivers the reoxygenated blood back into one of the patient’s veins or arteries. Additional ports may be added for haemodialysis or ultrafiltration.
Two different techniques may be used to introduce ECMO: venoarterial and venovenous. In the venoarterial technique, cannulation is through either the femoral artery and the femoral vein, or through the carotid artery and the internal jugular vein. In the venovenous technique cannulation is through both femoral veins or a femoral vein and internal jugular vein; one cannula acts as inflow or arterial line, and the other as an outflow or venous line. Venovenous ECMO will not provide adequate support if a patient has pulmonary hypertension or right heart failure. Problems associated with cannulation during the procedure include bleeding around the cannulation site and limb ischemia distal to the cannulation site.
Interventional Lung Assist (ILA) is used to remove excess CO2 from the blood of patients in respiratory failure. The system is characterized by a novel, low-resistance gas exchange device with a diffusion membrane composed of polymethylpentene (PMP) fibres. These fibres are woven into a complex configuration that maximizes the exchange of oxygen and CO2 by simple diffusion. The system is also designed to operate without the help of an external pump, though one can be added if higher blood flow is required. The device is then applied across an arteriovenous shunt between the femoral artery and femoral vein. Depending on the size of the arterial cannula used and the mean systemic arterial pressure, a blood flow of up to 2.5 L/min can be achieved (up to 5.5 L/min with an external pump). The cannulation is performed after intravenous administration of heparin.
Recently, the first commercially available extracorporeal membrane ventilator (NovaLung GmbH, Hechingen, Germany) was approved for clinical use by Health Canada for patients in respiratory failure. The system has been used in more than 2,000 patients with various indications in Europe, and was used for the first time in North America at the Toronto General Hospital in 2006.
Evidence-Based Analysis
The research questions addressed in this report are:
Does ILA/ECMO facilitate gas exchange in the lungs of patients with severe respiratory failure?
Does ILA/ECMO improve the survival rate of patients with respiratory failure caused by a range of underlying conditions including patients awaiting LTx?
What are the possible serious adverse events associated with ILA/ECMO therapy?
To address these questions, a systematic literature search was performed on September 28, 2009 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1, 2005 to September 28, 2008. Abstracts were reviewed by a single reviewer and, for those studies meeting the eligibility criteria, full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with an unknown eligibility were reviewed with a second clinical epidemiologist and then a group of epidemiologists until consensus was established.
Inclusion Criteria
Studies in which ILA/ECMO was used as a bridge to recovery or bridge to LTx
Studies containing information relevant to the effectiveness and safety of the procedure
Studies including at least five patients
Exclusion Criteria
Studies reporting the use of ILA/ECMO for inter-hospital transfers of critically ill patients
Studies reporting the use of ILA/ECMO in patients during or after LTx
Animal or laboratory studies
Case reports
Outcomes of Interest
Reduction in partial pressure of CO2
Correction of respiratory acidosis
Improvement in partial pressure of oxygen
Improvement in patient survival
Frequency and severity of adverse events
The search yielded 107 citations in Medline and 107 citations in EMBASE. After reviewing the information provided in the titles and abstracts, eight citations were found to meet the study inclusion criteria. One study was then excluded because of an overlap in the study population with a previous study. Reference checking did not produce any additional studies for inclusion. Seven case series studies, all conducted in Germany, were thus included in this review (see Table 1).
Also included is the recently published CESAR trial, a multicentre RCT in the UK in which ECMO was compared with conventional intensive care management. The results of the CESAR trial were published when this review was initiated. In the absence of any other recent RCT on ECMO, the results of this trial were considered for this assessment and no further searches were conducted. A literature search was then conducted for application of ECMO as bridge to LTx patients (January, 1, 2005 to current). A total of 127 citations on this topic were identified and reviewed but none were found to have examined the use of ECMO as bridge to LTx.
Quality of Evidence
To grade the quality of evidence, the grading system formulated by the GRADE working group and adopted by MAS was applied. The GRADE system classifies the quality of a body of evidence as high, moderate, low, or very low according to four key elements: study design, study quality, consistency across studies, and directness.
Trials on ILA
Of the seven studies identified, six involved patients with ARDS caused by a range of underlying conditions; the seventh included only patients awaiting LTx. All studies reported the rate of gas exchange and respiratory mechanics before ILA and for up to 7 days of ILA therapy. Four studies reported the means and standard deviations of blood gas transfer and arterial blood pH, which were used for meta-analysis.
Fischer et al. reported their first experience on the use of ILA as a bridge to LTx. In their study, 12 patients at high urgency status for LTx, who also had severe ventilation refractory hypercapnea and respiratory acidosis, were connected to ILA prior to LTx. Seven patients had a systemic infection or sepsis prior to ILA insertion. Six hours after initiation of ILA, the partial pressure of CO2 in arterial blood significantly decreased (P < .05) and arterial blood pH significantly improved (P < .05) and remained stable for one week (last time point reported). The partial pressure of oxygen in arterial blood improved from 71 mmHg to 83 mmHg 6 hours after insertion of ILA. The ratio of PaO2/FiO2 improved from 135 at baseline to 168 at 24 hours after insertion of ILA but returned to baseline values in the following week.
Trials on ECMO
The UK-based CESAR trial was conducted to assess the effectiveness and cost of ECMO therapy for severe, acute respiratory failure. The trial protocol were published in 2006 and details of the methods used for the economic evaluation were published in 2008. The study itself was a pragmatic trial (similar to a UK trial of neonatal ECMO), in which best standard practice was compared with an ECMO protocol. The trial involved 180 patients with acute but potentially reversible respiratory failure, with each also having a Murray score of ≥ 3.0 or uncompensated hypercapnea at a pH of < 7.2. Enrolled patients were randomized in a 1:1 ratio to receive either conventional ventilation treatment or ECMO while on ventilator. Conventional management included intermittent positive pressure ventilation, high frequency oscillatory ventilation, or both. As a pragmatic trial, a specific management protocol was not followed; rather the treatment centres were advised to follow a low volume low pressure ventilation strategy. A tidal volume of 4 to 8 mL/kg body weight and a plateau pressure of < 30 cm H2O were recommended.
Bridge to recovery
No RCTs or observational studies compared ILA to other treatment modalities.
Case series have shown that ILA therapy results in significant CO2 removal from arterial blood and correction of respiratory acidosis, as well as an improvement in oxygen transfer.
ILA therapy enabled a lowering of respiratory settings to protect the lungs without causing a negative impact on arterial blood CO2 and arterial blood pH.
The impact of ILA on patient long-term survival cannot be determined through the studies reviewed.
In-hospital mortality across studies ranged from 20% to 65%.
Ischemic complications were the most frequent adverse events following ILA therapy.
Leg amputation is a rare but possible outcome of ILA therapy, having occurred in about 0.9% of patients in these case series. New techniques involving the insertion of additional cannula into the femoral artery to perfuse the leg may lower this rate.
Bridge to LTx
The results of one case series (n=12) showed that ILA effectively removes CO2 from arterial blood and corrects respiratory acidosis in patients with ventilation refractory hypercapnea awaiting a LTx
Eight of the 12 patients (67%) awaiting a LTx were successfully transplanted and one-year survival for those transplanted was 80%
Since all studies are case series, the grade of the evidence for these observations is classified as “LOW”.
Bridge to recovery
Based on the results of a pragmatic trial and an intention to treat analysis, referral of patient to an ECMO based centre significantly improves patient survival without disability compared to conventional ventilation. The results of CESAR trial showed that:
For patients with information about disability, survival without severe disability was significantly higher in ECMO arm
Assuming that the three patients in the conventional ventilation arm who did not have information about severe disability were all disabled, the results were also significant.
Assuming that none of these patients were disabled, the results were at borderline significance
A greater, though not statistically significant, proportion of patients in ECMO arm survived.
The rate of serious adverse events was higher among patients in ECMO group
The grade of evidence for the above observations is classified as “HIGH”.
Bridge to LTx
No studies fitting the inclusion criteria were identified.
There is no accurate data on the use of ECMO in patients awaiting LTx.
Economic Analysis
The objective of the economic analysis was to determine the costs associated with extracorporeal lung support technologies for bridge to LTx in adults. A literature search was conducted for which the target population was adults eligible for extracorporeal lung support. The primary analytic perspective was that of the Ministry of Health and Long-Term Care (MOHLTC). Articles published in English and fitting the following inclusion criteria were reviewed:
Full economic evaluations including cost-effectiveness analyses (CEA), cost-utility analyses (CUA), cost-benefit analyses (CBA);
Economic evaluations reporting incremental cost-effectiveness ratios (ICER) i.e. cost per quality adjusted life year (QALY), life years gained (LYG), or cost per event avoided; and
Studies in patients eligible for lung support technologies for to lung transplantation.
The search yielded no articles reporting comparative economic analyses.
Resource Use and Costs
Costs associated with both ILA and ECMO (outlined in Table ES-1) were obtained from the University Health Network (UHN) case costing initiative (personal communication, UHN, January 2010). Consultation with a clinical expert in the field was also conducted to verify resource utilization. The consultant was situated at the UHN in Toronto. The UHN has one ECMO machine, which cost approximately $100,000. The system is 18 years old and is used an average of 3 to 4 times a year with 35 procedures being performed over the last 9 years. The disposable cost per patient associated with ECMO is, on average, $2,200. There is a maintenance cost associated with the machine (not reported by the UHN), which is currently absorbed by the hospital’s biomedical engineering department.
The average capital cost of an ILA device is $7,100 per device, per patient, while the average cost of the reusable pump $65,000. The UHN has performed 16 of these procedures over the last 2.5 years. Similarly, there is a maintenance cost not that was reported by UHN but is absorbed by the hospital’s biomedical engineering department.
Resources Associated with Extracorporeal Lung Support Technologies
Hospital costs associated with ILA were based on the average cost incurred by the hospital for 11 cases performed in the FY 07/08 (personal communication, UHN, January 2010). The resources incurred with this hospital procedure included:
Device and disposables
OR transplant
Surgical ICU
Laboratory work
Medical imaging
Clinical nutrition
Occupational therapy
Speech and language pathology
Social work
The average length of stay in hospital was 61 days for ILA (range: 5 to 164 days) and the average direct cost was $186,000 per case (range: $19,000 to $552,000). This procedure has a high staffing requirement to monitor patients in hospital, driving up the average cost per case.
PMCID: PMC3415698  PMID: 23074408
9.  Analysis of T cell responses in liver allograft recipients. Evidence for deletion of donor-specific cytotoxic T cells in the peripheral circulation. 
Journal of Clinical Investigation  1993;91(3):900-906.
Analysis of cell-mediated lympholysis in long-term liver allograft recipients indicated that there was a donor-specific unresponsiveness that could not be reversed by the addition of rIL-2 and/or mixed lymphocyte culture supernatant or by nonspecific stimulation of the cultures with PHA. Stimulation of recipient cells with semisyngeneic cells having both donor and third-party HLA antigens failed to reveal the presence of cytotoxic T cells (CTL) specific to the donor, whereas the CTL response to third-party antigens remained normal. Removal of B lymphocytes from the responding cell population did not influence the responses. Furthermore, limiting dilution analysis showed that the liver transplant recipients did not have detectable levels of CTL precursors (CTLp) reactive to the donor antigens, whereas their CTLp to third-party antigens remained normal. Donor-specific CTLp were present before and during the early post-transplant period; these cells were eliminated from the peripheral circulation by 10 mo after transplantation. Taken together, these results indicate that there is a deletion of CTLp specific to donor MHC antigens in the peripheral circulation of long-term liver allograft recipients that may account in part for the success of liver transplantation across MHC barriers.
PMCID: PMC288042  PMID: 8450068
10.  Murine Cytomegalovirus Immediate-Early 1 Gene Expression Correlates with Increased GVHD after Allogeneic Hematopoietic Cell Transplantation in Recipients Reactivating from Latent Infection 
PLoS ONE  2013;8(4):e61841.
The success of allogeneic (allo) hematopoietic cell transplantation (HCT) is limited by its treatment related complications, mostly graft versus host disease (GVHD) and fungal and viral infections. CMV reactivation after HCT has been associated with increased morbidity and mortality, and a causal relation between GVHD, immunosuppressive therapy and vice versa has been postulated. Using a low GVHD severity murine HCT model, we assessed the role of MCMV reactivation and GVHD development. BALB/c mice were infected with either murine CMV (MCMV) or mock and monitored for 25 weeks to establish latency, followed by sublethal irradiation conditioning and infusion of bone marrow plus splenocytes from either syngeneic (syn) BALB/c or allo B10.D2 donors. Engraftment of allo donor cells was confirmed by PCR for D2Mit265 gene product size. Day+100 mortality and overall GVHD severity in allo MCMV pre-infected recipients was higher than in allo mock controls. Pathologic changes of lung and liver GVHD in immediate-early gene 1 (IE1) positive recipients were significantly increased compared to mock controls, and were only slightly increased in IE1 negative. No significant gut injury was seen in any group. Aggravated lung injury in IE1 positive recipients correlated with higher BAL cell counts both for total cells and for CD4+ T cells when compared with mock controls, and also with protein expression of lung IFN-gamma and liver TNF. No evidence for CMV specific morphologic changes was seen on histopathology in any organ of IE1 positive recipients, suggesting that CMV reactivation is related to increased GVHD severity but does not require active CMV disease, strengthening the concept of a reciprocal relationship between CMV and GVHD.
PMCID: PMC3626592  PMID: 23596528
Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality post lung transplantation (LTx). We sought to determine the relationship between alloimmune responses and autoimmunity, and subsequently how autoimmunity leads to chronic rejection.
We analyzed the development of donor specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T and ColV specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELSIPOT.
In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor specific anti-HLA Abs, Abs to self-antigens, and BOS (p<0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor specific Abs (DSA) and Abs to self-antigens. 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS+ patients had higher frequency of T-cells secreting IL-17 (p<0.01) and IFNγ (p<0.05) with decreased IL-10 (p<0.05) compared to BOS- patients.
Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be important in preventing the development of chronic rejection.
PMCID: PMC3091959  PMID: 21414808
12.  Induction of Foxp3-Expressing Regulatory T-Cells by Donor Blood Transfusion Is Required for Tolerance to Rat Liver Allografts 
PLoS ONE  2009;4(11):e7840.
Donor-specific blood transfusion (DST) prior to solid organ transplantation has been shown to induce long-term allograft survival in the absence of immunosuppressive therapy. Although the mechanisms underlying DST-induced allograft tolerance are not well defined, there is evidence to suggest DST induces one or more populations of antigen-specific regulatory cells that suppress allograft rejection. However, neither the identity nor the regulatory properties of these tolerogenic lymphocytes have been reported. Therefore, the objective of this study was to define the kinetics, phenotype and suppressive function of the regulatory cells induced by DST alone or in combination with liver allograft transplantation (LTx).
Methodology/Principal Findings
Tolerance to Dark Agouti (DA; RT1a) rat liver allografts was induced by injection (iv) of 1 ml of heparinized DA blood to naïve Lewis (LEW; RT1l) rats once per week for 4 weeks prior to LTx. We found that preoperative DST alone generates CD4+ T-cells that when transferred into naïve LEW recipients are capable of suppressing DA liver allograft rejection and promoting long-term survival of the graft and recipient. However, these DST-generated T-cells did not express the regulatory T-cell (Treg) transcription factor Foxp3 nor did they suppress alloantigen (DA)-induced activation of LEW T-cells in vitro suggesting that these lymphocytes are not fully functional regulatory Tregs. We did observe that DST+LTx (but not DST alone) induced the time-dependent formation of CD4+Foxp3+ Tregs that potently suppressed alloantigen-induced activation of naïve LEW T-cells in vitro and liver allograft rejection in vivo. Finally, we present data demonstrating that virtually all of the Foxp3-expressing Tregs reside within the CD4+CD45RC− population whereas in which approximately 50% of these Tregs express CD25.
We conclude that preoperative DST, in the absence of liver allograft transplantation, induces the formation of CD4+ T-cells that are not themselves Tregs but give rise directly or indirectly to fully functional CD4+CD45RC−Foxp3+Tregs when transferred into MHC mismatched recipients prior to LTx. These Tregs possess potent suppressive activity and are capable of suppressing acute liver allograft rejection. Understanding the mechanisms by which preoperative DST induces the generation of tolerogenic Tregs in the presence of alloantigens may lead to the development of novel antigen-specific immunological therapies for the treatment of solid organ rejection.
PMCID: PMC2776304  PMID: 19956764
13.  Cryptic B cell response to renal transplantation 
Renal transplantation reliably evokes allo-specific B cell and T cell responses in mice. Yet, human recipients of kidney transplants with normal function usually exhibit little or no antibody specific for the transplant donor during the early weeks and months after transplantation. Indeed, the absence of anti-donor antibodies is taken to reflect effective immunosuppressive therapy and to predict a favorable outcome. Whether the absence of donor-specific antibodies reflects absence of a B-cell response to the donor, tolerance to the donor or immunity masked by binding of donor-specific antibodies to the graft is not known. To distinguish between these possibilities, we devised a novel ELISPOT, using cultured donor, recipient and third-party fibroblasts as targets. We enumerated donor-specific antibody-secreting cells in the blood of nine renal allograft recipients with normal kidney function before and after transplantation. Although none of the nine subjects had detectable donor-specific antibodies before or after transplantation, all exhibited increases in the frequency of donor-specific antibody-secreting cells eight weeks after transplantation. The responses were directed against the donor HLA-class I antigens. The increase in frequency of donor-specific antibody-secreting cells after renal transplantation indicates that B cells respond specifically to the transplant donor more often than previously thought.
PMCID: PMC3764435  PMID: 23750851
accommodation; rejection; tolerance; renal transplant
14.  Correlation of Chimerism with Acute Graft-versus-Host Disease in Rats following Liver Transplantation 
The accurate diagnosis of acute graft-versus-host disease following liver transplantation (LTx-aGVHD) has been hampered. Chimerism appears in the majority of recipients after LT and its significance in the diagnosis of LTx-aGVHD has not been clearly established. To demonstrate the significance of chimerism on the diagnosis of LTx-aGVHD, we compared the change of chimerism in syngeneic LT recipients, semiallogeneic LT recipients, and LTx-aGVHD induced recipients. Chimerism in PBMCs following sex-mismatched LT was identified by real-time PCR based on a rat Y-chromosome-specific primer. All recipients in semiallogeneic group grew in a normal pattern. However, when 4 × 108 donor splenocytes were transferred simultaneously during LT, the morbidity of lethal aGVHD was 100%. The chimerism appeared slightly higher in the semiallogeneic group than in the syngeneic LT group, but the difference was not significant. However, when the recipients developed lethal aGVHD after LT, chimerism in the PBMCs increased progressively, and even at an early time, a significant increase in chimerism was observed. In conclusion, high level chimerism correlated well with LTx-aGVHD, and detection of chimerism soon after transplantation may be of value in the diagnosis of LTx-aGVHD prior to the onset of symptoms.
PMCID: PMC3170856  PMID: 21994878
Transplantation  1994;58(1):1-8.
Spontaneous orthotopic liver allograft acceptance associated with microchimerism in mice induces tolerance to subsequent skin or heart transplants from the donor but not third-party animals. Despite in vivo hyporesponsiveness, in vitro MLC and CTL assays showed continuing antidonor reactivity. Cells isolated from recipients’ spleens and grafted livers, when tested in MLC and CTL assays, were antidonor reactive out to 3 months to the same degree as splenocytes obtained from either naive or presensitized (with skin or heart) mice. Nevertheless, passive transfer of splenocytes or liver lymphocytes from liver tolerant mice, but not naive or sensitized donor strain mice, were able to prolong skin graft survival significantly in naive irradiated recipients. By using a strain combination in which the donor but not the recipient expressed the stimulatory endogenous super-Ag (Mlsf), it was possible to determine whether super-Ag-reactive T cells bearing Vβ5 and Vβ11 were deleted or anergic. Phenotypic analysis of cells isolated from recipients’ spleens and grafted livers (up to 90 days after transplant), when compared with naive animals, showed no significant difference in Vβ5 and Vβ11 TCR expression. Additionally, when these isolated spleen cells were tested for antibody-mediated stimulation, both anti-Vβ5 and Vβ11 TCR mAb led to marked proliferation of cells obtained from naive and liver-transplanted recipients, but as expected, proliferation was very low in cells from naive donors. These results suggest that liver transplantation induces donor-specific tolerance in vivo, which may not be reflected in in vitro proliferative and cytotoxicity assays (split tolerance). Furthermore, this tolerance does not seem to be induced by clonal deletion or anergy of minor-lymphocyte-stimulating-antigen-reactive T cells in the recipients.
PMCID: PMC3208349  PMID: 8036695
16.  Role of defensins in the pathogenesis of chronic lung allograft rejection 
Human immunology  2013;75(4):370-377.
Chronic rejection predominantly manifested as bronchiolitis obliterans syndrome (BOS), still remains a major problem affecting long-term outcomes in human lung transplantation (LTx). Donor specific antibodies (DSA) and infiltration of neutrophils in the graft have been associated with the development of BOS. This study determines the role of defensins, produced by neutrophils, and its interaction with α-1-antitrypsin (AAT) towards induction of airway inflammation and fibrosis which are characteristic hallmarks of BOS. Bronchoalveolar lavage (BAL) and serum from LTx recipients, BOS+ (n=28), BOS-(n=26) and normal healthy controls (n=24) were analyzed. Our results show that BOS+ LTx recipients had higher α-defensins (HNP1-3) and β-defensin2 HBD2 concentration in BAL and serum compared to BOS-DSA-recipients and normal controls (p=0.03). BOS+ patients had significantly lower serum AAT along with higher circulating concentration of HNP-AAT complexes in BAL (p=0.05). Stimulation of primary small airway epithelial cells (SAECs) with HNPs induced expression of HBD2, adhesion molecules (ICAM and VCAM), cytokines (IL-6, IL-1β, IL-13, IL-8 and MCP-1) and growth-factor (VEGF and EGF). In contrast, anti-inflammatory cytokine, IL-10 expression decreased 2 fold (p=0.002). HNPs mediated SAEC activation was completely abrogated by AAT. In conclusion, our results demonstrates that neutrophil secretory product, α-defensins, stimulate β-defensin production by SAECs causing upregulation of pro-inflammatory and pro-fibrotic signaling molecules. Hence, chronic stimulation of airway epithelial cells by defensins can lead to inflammation and fibrosis the central events in the development of BOS following LTx.
PMCID: PMC4011133  PMID: 24380698
HNP- Human Neutrophil Peptide; BOS-Bronchiolitis Obliterans Syndrome; BAL-Bronchoalveolar Lavage; HBD2-Human Beta Defensin 2; SAEC- Small Airway Epithelial Cells
17.  Development of Antibodies to HLA Precedes Development of Antibodies to MICA and Are Significantly Associated With Development of Chronic Rejection Following Human Lung Transplantation 
Human immunology  2010;71(6):560-565.
The development of antibodies (Abs) to major histocompatibility (MHC) class I related chain A (MICA) and human leukocyte antigen (HLA) and their role in the immunopathogenesis of chronic rejection (bronchiolitis obliterans syndrome (BOS)) following human lung transplantation (LTx) was analyzed. Sera from 80 LTx recipients were analyzed for anti-MICA and anti-HLA Abs using Luminex and flow PRA (panel reactive assay). Development of Abs either to MICA alone or MICA and HLA together significantly correlated (P<0.01) with development of BOS. Kinetic analysis in the post-LTx period revealed that development of anti-HLA Abs (7.6±4.7 months) preceded the development of anti-MICA Abs (10.0±3.5 months). Abs to MICA alleles (*001 and *009) developed approximately 6 months following LTx and peak titers were present at the time of clinical diagnosis of BOS (16.3±2.7 months). The development of Abs to both MICA and HLA was strongly associated with the development of BOS thereby suggesting a synergistic effect. Furthermore, immune response to mismatched HLA can lead to development of Abs to other MHC related antigens expressed on the airway epithelial cells. Cumulatively, these immune responses contribute to the pathogenesis of chronic rejection following human LTx.
PMCID: PMC2874120  PMID: 20211214
MICA; HLA; antibodies; lung transplantation; BOS; Chronic rejection
18.  High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures 
Analytical and Bioanalytical Chemistry  2015;407(10):2847-2858.
Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3–9.9 % coefficient of variation), and accurate (0.1–12.7 %error; n ≥ 25). Diagnostic sensitivity was 100 % (n = 27 animal/clinical cases). Diagnostic specificity was 100 % (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.
Graphical AbstractMethod schematic for analysis of anthrax lethal toxin activity by ID-MALDI-TOF MS
PMCID: PMC4369318  PMID: 25673244
Anthrax; MALDI-TOF MS; Quantification; Lethal toxin; Lethal factor; Protective antigen; Diagnostic; Bacillus anthracis
19.  Visualization of immune response kinetics in full allogeneic chimeras 
Background: Donor Lymphocyte Infusion (DLI) is a well-recognized tool for augmentation of the anti-leukemia effect after mismatched bone marrow transplantation. Experimental results show, however, that DLI efficacy is strongly dependent on the number of donor hematopoietic cells persisting in recipient after transplantation. It is strong in mixed chimeras and relatively weak in full chimeras (FC) that replace host antigen-presenting cells by donor antigen-presenting cells. In this study we applied a new in vivo cytotoxicity monitoring method for evaluation of the changes in FC anti-host immunity after co-transplantation of donor and host hematopoietic cells together. Method: Full hematopoietic chimeras and naïve control mice were transplanted with a mixture of equivalent numbers of donor and recipient or donor and third party splenocytes labeled by a cell-permeable fluorescent dye CFDA-SE. The animals were sacrificed at various time points, and their splenocyte suspensions were prepared, depleted of red blood cells, stained with allophycocyanin-labeled anti-H2b antibodies, and analyzed using fluorescence-activated cell sorting. The immune response was assessed according to the percentage of single positive CFDA-SE+/ H2b- cells of all CFDA-SE+ cells. Results: FC grafted with splenocytes from similar FC mixed with splenocytes from naïve host-type or third-party-type mice rejected host cells within 14 days, and third-party cells within 7 days. NK cell depletion in vivo had no influence on host cell rejection kinetics. Co-infusion of host-type splenocytes with splenocytes obtained from naïve donor-type mice resulted in significant acceleration of host cell rejection (10 days). Naïve mice rejected the same amount of allogeneic lymphocytes within 3 days. Conclusions: Proposed method provides a simple and sensitive tool to evaluate in vivo post-transplant cytotoxicity in different experimental settings. The method demonstrates that FC is specifically deficient in their ability to reject host lymphocytes even when antigen-presenting host cells are provided. DLI improve anti-host immune response in FC but can not restore it to the level observed in naïve donor-type mice.
PMCID: PMC3301426  PMID: 22432073
transplantation; chimerism; immune response; leukemia; donor lymphocyte infusion
20.  MHC Haplotype Matching for Unrelated Hematopoietic Cell Transplantation 
PLoS Medicine  2007;4(1):e8.
Current criteria for the selection of unrelated donors for hematopoietic cell transplantation (HCT) include matching for the alleles of each human leukocyte antigen (HLA) locus within the major histocompatibility complex (MHC). Graft-versus-host disease (GVHD), however, remains a significant and potentially life-threatening complication even after HLA-identical unrelated HCT. The MHC harbors more than 400 genes, but the total number of transplantation antigens is unknown. Genes that influence transplantation outcome could be identified by using linkage disequilibrium (LD)-mapping approaches, if the extended MHC haplotypes of the unrelated donor and recipient could be defined.
Methods and Findings
We isolated DNA strands extending across 2 million base pairs of the MHC to determine the physical linkage of HLA-A, -B, and -DRB1 alleles in 246 HCT recipients and their HLA-A, -B, -C, -DRB1, -DQB1 allele-matched unrelated donors. MHC haplotype mismatching was associated with a statistically significantly increased risk of severe acute GVHD (odds ratio 4.51; 95% confidence interval [CI], 2.34–8.70, p < 0.0001) and with lower risk of disease recurrence (hazard ratio 0.45; 95% CI, 0.22–0.92, p = 0.03).
The MHC harbors genes that encode unidentified transplantation antigens. The three-locus HLA-A, -B, -DRB1 haplotype serves as a proxy for GVHD risk among HLA-identical transplant recipients. The phasing method provides an approach for mapping novel MHC-linked transplantation determinants and a means to decrease GVHD-related morbidity after HCT from unrelated donors.
A novel method of MHC haplotype matching provides a means to decrease graft-versus-host disease-related morbidity after transplantation from unrelated donors.
Editors' Summary
Graft rejection and graft-versus-host disease (GVHD) are feared complications of hematopoietic cell transplantation (HCT). GVHD can affect all parts of the body, and, if severe (grade III to IV out of a scale of IV), can lead to the death of the transplant recipient. GVHD or rejection of the graft occurs when there are differences in specific proteins involved in the immune response (known as HLA antigens) between donor and recipient that stimulate the immune reaction. GVHD and graft rejection occur most often in people who receive transplants from unrelated donors because, although when donors are matched to recipients matching is done for the most important HLA antigens known to be involved, it has not technically been possible to match for all possible antigens. However, the human genome is organized into segments or blocks of closely linked genetic variants that are inherited as “haplotypes” on the same DNA strand of a chromosome. Most of the genes that code for HLA antigens are physically located together in one part of the human genome, known as the MHC region. Currently three HLA markers from this region (HLA-A, -B, -DRB1) are matched when matching donors and recipients. If it were possible to better map the structure of this region, it would be possible to better match recipients and donors (especially unrelated donors) for the unidentified transplantation antigens and reduce the chance of recipients getting GVHD or rejecting their grafts.
Why Was This Study Done?
Current strategies to define MHC haplotype blocks look at, on average, a length of only 18,000 base pairs and hence cannot define extended MHC haplotypes. Previously, this group of researchers developed a method of defining the HLA-A, B, DR haplotypes in recipients and their HLA-matched unrelated transplant donors using high-quality DNA containing 2 million base pairs across the MHC region. They wanted see if using this technique might provide a way to better assess the risk recipients have of developing GVHD or of having recurrent disease.
What Did the Researchers Do and Find?
They studied 246 HCT recipients and their donors who had been matched for HLA-A, -B, -C, -DRB1, -DQB1 by current techniques. The recipients were having HCT for a variety of hematological cancers: acute lymphoid leukemia, acute myeloid leukemia, chronic myeloid leukemia, or myelodysplastic syndrome. They found that, using the new technique, 22% of the donor–recipient pairs were haplotype-mismatched. Taking various other factors into account, including age, and patient and donor gender, MHC haplotype mismatching was associated with an approximately four times greater risk of severe acute GVHD but with a lower risk of disease recurrence. The lower risk of recurrence is believed to be because transplanted cells do not only replace abnormal cancerous cells but also react against them and therefore decrease the chance of the cancer recurring; mismatched cells are known to be more stimulated to react against the cancerous cells.
What Do These Findings Mean?
The results here suggest that this new haplotype matching method can provide a way to assess the risk of GVHD after HCT from unrelated donors, and in future could be considered as a technique to match donors and recipients.
Additional Information.
Please access these Web sites via the online version of this summary at
• Medline Plus has a page of information on stem cell transplantation, including HCT
• The Anthony Nolan Trust holds one of the largest databases of unrelated donors in the world
• The National Cancer Institute has a page of questions and answers on HCT
• The Center for International Blood & Marrow Transplant Research describes outcomes research in transplantation
• The National Marrow Donor Program describes how HLA-typed unrelated donors are identified
• The World Marrow Donor Association is involved in facilitating stem cell donation across international boundaries
PMCID: PMC1796628  PMID: 17378697
21.  In Situ-Targeting of Dendritic Cells with Donor-Derived Apoptotic Cells Restrains Indirect Allorecognition and Ameliorates Allograft Vasculopathy 
PLoS ONE  2009;4(3):e4940.
Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11chi CD8α+ and CD8− DCs, but not by CD11cint plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-γ-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV.
PMCID: PMC2660580  PMID: 19333400
22.  An Essential Role for IFN-γ in Regulation of Alloreactive CD8 T Cells Following Allogeneic Hematopoietic Cell Transplantation 
We have previously shown that CD8 T cells from IFN-γ gene knockout (GKO) donors induces more severe lethal graft-vs.-host disease (GVHD) compared to CD8 T cells from wild type (WT) donors in fully MHC-mismatched strain combinations. In this study, we investigated the mechanisms by which IFN-γ inhibits GVHD in a parent→F1 (B6→;B6D2F1) allogeneic hematopoietic cell transplantation (allo-HCT) model. IFN-γ was strongly protective against GVHD in this parent→;F1 haplotype-mismatched allo-HCT model. Irradiated B6D2F1 mice that received GKO B6 CD4-depleted splenocytes develop lethal GVHD with severe lung and liver injury, whereas those receiving a similar cell population from WT B6 donors survived long-term. Donor CD8 cells showed rapid activation, accelerated cell division and reduced/delayed activation-induced cell death in allogeneic recipients in which donor cells were incapable of producing IFN-γ. Consequently, the numbers of activated/effector (i.e., CD25+, CD62L− and CD44high) donor CD8 T cells in the recipients of GKO allo-HCT significantly exceeded those in mice receiving WT allo-HCT. These data show that IFN-γ negatively regulates the CD8 T cell response by inhibiting cell division and promoting cell death, and suggest that blockade of IFN-γ could augment the severity of GVHD in allo-HCT recipients.
PMCID: PMC1893089  PMID: 17222752
23.  CD39 deficiency in murine liver allografts promotes inflammatory injury and immune-mediated rejection 
Transplant immunology  2015;32(2):76-83.
Adenosine triphosphate (ATP), an essential metabolic energy source, is released following cell apoptosis or necrosis. It acts as a damage-associated molecule pattern to stimulate innate immune cells. The ectonucleotidase CD39 regulates immune activation by hydrolysis of extracellular ATP. We have shown previously that CD39 expression by donor livers helps protect syngeneic grafts with extended (24 hr) cold preservation time from ischemia reperfusion injury. Given its immune regulatory properties, we hypothesized that CD39 expression in donor livers might modulate transplant tolerance that occurs following mouse allogeneic liver transplantation (LTx). Livers from C57BL/6 (B6) wild-type (WT) or CD39 KO mice were transplanted into normal C3H recipients with minimal (approximately 1 hr) cold ischemia. Serum alanine aminotransferase levels at day 4 post LTx were significantly higher in animals given CD39KO compared with WT livers. Moreover, IFN-γ production by liver-infiltrating CD8+ T cells at day 4 was significantly higher in CD39KO than in WT grafts. Furthermore, splenic T cells from CD39KO liver recipients exhibited greater proliferative responses to donor alloantigens than those from mice given WT grafts. By contrast, there was a concomitant significant reduction in the frequency of regulatory T cells (Treg) in CD39KO than in WT livers. Whereas WT liver allografts survived > 100 days, no CD39KO grafts survived beyond 40 days (median survival time [MST]: WT: >100 days vs CD39KO: 8 days; p<0.01). In addition, soluble CD39 administration significantly prolonged CD39KO liver allograft survival (MST: 27.5 days). These novel data suggest that CD39 expression in liver allografts modulates tissue injury, inflammation, anti-donor effector T cell responses and Treg infiltration and can suppress transplant rejection.
PMCID: PMC4368493  PMID: 25661084
adenosine triphosphate; CD39; liver allograft; T cells
24.  HLA variants related to primary sclerosing cholangitis influence rejection after liver transplantation 
AIM: To investigate influence of human leukocyte antigen (HLA) and killer immunoglobuline-like receptor (KIR) genotypes on risks of acute rejection (AR) after liver transplantation (LTX).
METHODS: In this retrospective study we included 143 adult donor-recipient pairs with a minimum of 6 mo follow-up after LTX for whom DNA was available from both donor and recipients. Clinical data, all early complications including episodes and severity of AR and graft/patient survival were registered. The diagnosis of AR was based on clinical, biochemical and histological criteria. All suspected episodes of AR were biopsy confirmed. Key classical HLA loci (HLA-A, HLA-B, HLA-C and HLA-DRB1) were genotyped using Sanger sequencing. 16 KIR genes were genotyped using a novel real time PCR approach which allows for determination of the diploid copy number of each KIR gene. Immunohistochemical staining for T (CD3), B (CD20) and natural killer (NK) cells (CD56 and CD57) were performed on liver biopsies from 3 different patient groups [primary sclerosing cholangitis (PSC), primary biliary cirrhosis and non-autoimmune liver disease], 10 in each group, with similar grade of AR.
RESULTS: Fourty-four (31%) patients were transplanted on the basis of PSC, 40% of them had AR vs 24% in the non-PSC group (P = 0.04). No significant impact of donor-recipient matching for HLA and KIR genotypes was detected. In the overall recipient population an increased risk of AR was detected for HLA-B*08 (P = 0.002, OR = 2.5; 95%CI: 1.4-4.6), HLA-C*07 (P = 0.001, OR = 2.4; 95%CI: 1.4-4.0) and HLA-DRB1*03 (P = 0.03, OR = 1.9; 95%CI: 1.0-3.3) and a decreased risk for HLA-DRB1*04 (P = 0.001, OR = 0.2; 95%CI: 0.1-0.5). For HLA-B*08, HLA-C*07 and DRB1*04 the associations remained evident in a subgroup analysis of non-PSC recipients (P = 0.04, P = 0.003 and P = 0.02, respectively). In PSC recipients corresponding P values were 0.002, 0.17 and 0.01 for HLA-B*08, HLA-C*07 and DRB1*04, respectively. A dosage effect of AR prevalence according to the PSC associated HLA alleles was also notable in the total recipient population. For HLA-B*08 the frequency of AR was 56% in HLA-B*08 homozygous recipients, 39% in heterozygous recipients and 21% in recipients lacking HLA-B*08 (P = 0.02). The same was observed for the HLA-C*07 allele with AR in 57%, 27% and 18% in recipients being homozygous, heterozygous and lacking HLA-C*07 respectively (P = 0.003). Immunohistochemical analysis showed similar infiltration of T, B and NK cells in biopsies with AR in all three groups.
CONCLUSION: We found significant associations between the PSC-associated HLA-B*08, HLA-C*07, HLA-DRB1*03 and HLA-DRB1*04 alleles and risk of AR in liver transplant recipients.
PMCID: PMC3983454  PMID: 24744588
Liver transplantation; Primary sclerosing cholangitis; Acute rejection; Human leukocyte antigen; Killer immunoglobulin-like receptor
25.  The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner 
PLoS ONE  2013;8(7):e69483.
The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)2C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.
PMCID: PMC3709891  PMID: 23874964

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