Chapter 2: Source pigs

Abstract: An islet xenotransplantation product includes live cells from a non‐human source, in this study, a pig source. A live product cannot be subjected to conventional disinfection, and therefore the source pig must be depleted of infectious agents that can transmit to the recipient and cause disease. Among other requirements, regulatory guidances specify that donor animals fulfill the designated pathogen‐free (DPF) status. Donor pigs fulfilling DPF status are generally bred and maintained in biosecure facilities, where they are shielded from the outside by filtered air, disinfected water, and irradiated food that is certified free of any mammalian protein. All materials are autoclaved upon entry, and personnel enter by shower‐in/shower‐out, and are wearing special clothes. The operation of such facilities is in compliance with Current Good Manufacturing Practices. To ensure DPF status, pigs are brought into such facilities via Cesarean section, and the source pigs are kept as a closed herd. The DPF status cannot be realized for endogenous viruses, such as porcine endogenous retrovirus. Therefore, regulatory authorities require patient monitoring after xenotransplantation. Considering the infectious pathogen status and necessary regulatory compliance, it is recommended that organ procurement be conducted at the animal facility and that cell manufacturing facilities be located nearby. To enable assessment of as‐yet unknown pathogens long after xenotransplantation, regulatory guidances mandate archiving donor materials for at least 50 yr. As this is essentially a public health issue, governmental institutions are urged to be responsible for the archive.

Table of Contents

• ? Introduction
• ? Designated pathogen‐free status
• ? Biosecure barrier facility
• ? Organ retrieval and islet manufacturing
• ? Alternatives

     

Preventing adverse drug events in hospital practice: an overview

Adverse drug events (ADEs) are a considerable cause of morbidity and mortality in hospital practice. The precise frequency is unknown, but studies give an incidence number ranging from 2 until 52 ADEs per 100 patients. There are many different methods for definition, causality assessment, severity classification and detection which make it difficult to compare the different studies. A substantial part (in some studies up to 70%) of ADEs can be prevented and it is important to, besides their detection, focus on the prevention of these ADEs. In this literature review we give an overview of methods for preventing ADEs. There are many different tools with different impact on a particular part of the distribution system which has the potential to prevent ADEs. A multifaceted approach is needed. Two interesting strategies of prevention, pharmacist participation on ward rounds and computerised physician order entry with clinical decision support systems (CDSS), are highlighted. Moreover, two promising CDSS are discussed in more detail, namely computer-based monitoring systems and information systems which link laboratory and pharmacy data.     

Value of platelet pharmacogenetics in common clinical practice of patients with ST-segment elevation myocardial infarction

Background

Antiplatelet drug resistance is a well-known problem, causing recurrent cardiovascular events. Multiple genetic polymorphisms have been related to antiplatelet resistance by several large trials, however data from common clinical practice is limited. We examined the influence of previously described polymorphisms, related to aspirin and clopidogrel resistance, on treatment outcome in a real life unselected population of patients presenting with ST-segment elevation myocardial infarction (STEMI) treated with percutaneous coronary intervention.

Methods and results

This cohort study consisted of 1327 patients with STEMI. Patients were treated according to a standardized guideline-based protocol. Nine polymorphisms, COX1 (− 842A > G), P2Y₁ (893C > T), GPIa (807C > T), GPIIIa (PlA1/A2), CYP2C19 (*2, *3 and *17), ABCB1 (3435T > C) and PON1 (576A > G), were genotyped. During 1 year of follow up the primary endpoint, a composite of cardiac death or recurrent myocardial infarction, was reached in 86 patients. The COX1 and CYP2C19*2 polymorphisms were associated with the primary endpoint, HR 2.55 (95% CI 1.48–4.40), P = 0.001 and HR 2.03 (1.34–3.09) P = 0.001, respectively. The combined analysis demonstrated a 2.5-fold increased risk for individuals with ≥ 2 risk alleles, P = 6.9 × 10^–9. The association of COX1 was driven by mortality related events whereas that of CYP2C19*2 was mainly attributed to myocardial infarction and stent thrombosis.

Conclusion

In this unselected, real life population of STEMI patient on dual-antiplatelet therapy, the polymorphisms COX1 –842A > G and CYP2C19*2 were determinants of thrombotic complications during follow-up. We show that in a clinical setting, testing for these polymorphisms could be of value in the identification of STEMI patients at risk for recurrent cardiovascular events.     

Relationship between genetic variants in the adenosine pathway and outcome of methotrexate treatment in patients with recent-onset rheumatoid arthritis

OBJECTIVE: Among patients with rheumatoid arthritis (RA), there is a high degree of interindividual variability in the degree of response to methotrexate (MTX) treatment. This study was undertaken to explore polymorphisms in genes contributing to antiinflammatory adenosine release as novel predictors of MTX treatment outcome. METHODS: In 205 patients with newly diagnosed RA, 5 polymorphisms in 5 genes coding for enzymes related to the release of adenosine were analyzed. All patients received standardized MTX treatment (up to 25 mg per week orally), combined with folic acid. MTX efficacy was evaluated by the Disease Activity Score (DAS) and compared among genotypes. The association between MTX-related adverse events and genotype was also assessed. The following polymorphisms were determined: AMPD1 34C>T, ATIC 347C>G, ITPA 94C>A, MTR 2756A>G, and MTRR 66A>G. When significant differences were found by chi-square analysis, odds ratios (ORs) and 95% confidence intervals were calculated. RESULTS: Patients carrying the AMPD1 34T allele, ATIC 347CC, or ITPA 94CC were more likely to have a good clinical response, as defined by a DAS of < or =2.4 (OR [95% confidence interval] 2.1 [1.0-4.5], 2.5 [1.3-4.7], and 2.7 [1.1-8.1], respectively). The likelihood of a good clinical response was increased if patients possessed all 3 favorable genotypes (OR 27.8 [95% confidence interval 3.2-250]). Regarding toxicity, only ATIC G allele carriers experienced a greater frequency of adverse events (OR 2.0 [95% confidence interval 1.1-3.7]). CONCLUSION: Polymorphisms in the AMPD1, ATIC, and ITPA genes are associated with good clinical response to MTX treatment. These findings indicate that genotyping may help in the identification of patients who will benefit most from MTX treatment and may assist clinicians in making treatment decisions regarding patients with recent-onset RA.     

The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane

Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4-rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4-rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.     

Species incompatibilities in the pig-to-macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation

Graham ML, Bellin MD, Papas KK, Hering BJ, Schuurman H‐J. Species incompatibilities in the pig‐to‐macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation. Xenotransplantation 2011; 18: 328–342. © 2011 John Wiley & Sons A/S. Abstract: Background: Porcine islet transplantation into diabetic non‐human primates is considered most relevant in translational research supporting a clinical application. Most studies have focused on immunosuppressive protocols, while metabolic aspects have mainly been utilized in graft monitoring. We evaluated data from our group regarding human and non‐human primate (NHP) allotransplantation and pig‐to‐NHP xenotransplantation to identify incompatibilities in metabolic factors and their consequences for transplant outcomes. Methods: Basic gluco‐metabolic parameters (fasting blood glucose, C‐peptide, and response to stimulation with arginine or glucose) were derived from literature (humans), 72 macaques, and 47 adult Landrace pigs. Islet preparations from 15 human deceased donors, 61 macaques, and 23 adult pigs were compared with respect to yield, fractional viability assessed by oxygen consumption normalized for DNA, and in vitro glucose‐induced insulin release. Metabolic parameters at day 75 after a single islet transplantation in the liver were compared for 19 patients and 9 macaques receiving an allotransplant and 11 macaques receiving a porcine xenotransplant: recipients received chronic immunosuppression. Results: Pigs differ from NHPs and humans by a much lower C‐peptide level (0.42 vs. 1.3 to 2.0 ng/ml, respectively) and a 2‐ to 7‐fold lower C‐peptide response to arginine stimulation. In contrast, NHPs have the highest metabolic demand as evidenced by a high C‐peptide and high C‐peptide response to arginine stimulation; values are about twice higher than in humans. For manufactured islet preparations, these differences are reflected by glucose‐stimulated insulin release (the stimulation index for pigs is 1.5, for humans 3.8, and for macaques 7.7), but not by fractional viability, which was in the same range. The day 75 outcome after transplantation assessed by C‐peptide was similar for allotransplanted humans and NHPs (80 to 90% good graft function) and lower in xenografted NHPs (36% good graft function); gluco‐metabolic parameters were in accordance with graft function, albeit different between species because normoglycemia under exogenous insulin is maintained more aggressively in patients than in NHPs. In xenografted NHPs, the shift in glycemic control with respect to normal values, combined with low values of circulating porcine C‐peptide, resembled more the normal condition in a pig than that in a macaque. Conclusions: The substantially lower glucose‐induced insulin response in adult porcine islet preparations as opposed to islets manufactured from humans or macaques combined with the much higher need for insulin in macaques than in humans creates an imbalance between the metabolic demand and the engrafted islet mass in the pig‐to‐NHP xenograft recipient. Engrafted islet mass is affected by dose, suggesting that a much higher dose level of islets is necessary in the xenogeneic setting than in human or NHP allotransplantation, and pig islets need to be given at a higher dose in macaques than the anticipated effective dose in humans. To cope with differences in metabolic demand and presumably also metabolic dynamics, a liberal regime in supportive exogenous insulin might be essential to achieve long‐term survival. These intrinsic characteristics of the NHP model deserve consideration to optimally design experimental studies with the perspective of translational value of results.