Comparative analysis of swine leukocyte antigen gene diversity in Göttingen Minipigs.

Worldwide, pigs represent economically important farm animals, also representing a preferred preclinical large animal model for biomedical studies. The need for swine leukocyte antigen (SLA) typing is increasing with the expanded use of pigs in translational research, infection studies, and for veterinary vaccine design. Göttingen Minipigs (GMP) attract increasing attention as valuable model for pharmacological studies and transplantation research. This study represents a first-time assessment of the SLA gene diversity in Göttingen Minipigs in combination with a comparative metadata analysis with commercial pig lines. As Göttingen Minipigs could harbor private as well as potential novel SLA allele combinations, future research projects would benefit from the characterization of their SLA background. In 209 Göttingen Minipigs, SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes were characterized by PCR-based low-resolution (Lr) haplotyping. Criteria and nomenclature used for SLA haplotyping were proposed by the ISAG/IUIS-VIC SLA Nomenclature Committee. Haplotypes were assigned based on the comparison with already known breed or farm-specific allele group combinations. In total, 14 SLA class I and five SLA class II haplotypes were identified in the studied cohort, to manifest in 26 SLA class I but only seven SLA class II genotypes. The most common SLA class I haplotypes Lr-24.0 (SLA-1*15XX or Blank-SLA-3*04:04-SLA-2*06:01~02) and Lr-GMP-3.0 (SLA-1*16:02-SLA-3*03:04-SLA-2*17:01) occurred at frequencies of 23.44 and 18.66%, respectively. For SLA class II, the most prevalent haplotypes Lr-0.21 (DRB1*01XX-DQB1*05XX-DQA*04XX) and Lr-0.03 (DRB1*03:02-DQB1*03:01-DQA*01XX) occurred at frequencies of 38.28 and 30.38%. The comparative metadata analysis revealed that Göttingen Minipigs only share six SLA class I and two SLA class II haplotypes with commercial pig lines. More importantly, despite the limited number of SLA class I haplotypes, the high genotype diversity being observed necessitates pre-experimental SLA background assessment of Göttingen Minipigs in regenerative medicine, allo-transplantation, and xenograft research.

L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. METHODS: In several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

l-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. l-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. l-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating l-citrulline. METHODS: In several repeat-dose toxicity studies, plasma l-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma l-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline l-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma l-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of l-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute l-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in l-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute l-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in l-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of l-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of l-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of l-citrulline as a small intestine biomarker.

Biomechanical evaluation of the docking nail concept in periprosthetic fracture fixation around a stemmed total knee arthroplasty.

Intramedullary femoral nails provide an ideal mechanical axis for periprosthetic fracture fixation. Slotted nails allow a connection to a total knee arthroplasty (TKA) stem. This study aims to compare implant and construct stiffness, interfragmentary movement and cycles to failure between an antegrade slotted femoral nail construct docked to a TKA stem and a distal femoral locking plate in a human periprosthetic femoral fracture model. In eight pairs of fresh-frozen human femora with stalked TKA, a 10 mm transverse osteotomy gap was set simulating a Rorabeck type II, Su type I fracture. The femora were pairwise instrumented with either an antegrade slotted nail coupled to the prosthesis stem, or a locking plate. Cyclic testing with a progressively increasing physiologic loading profile was performed at 2 Hz until catastrophic construct failure. Relative movement at the osteotomy site was monitored by means of optical motion tracking. In addition, four-point bending implant stiffness, torsional implant stiffness and frictional fit of the stem-nail connection were investigated via separate non-destructive tests. Intramedullary nails exhibited significantly higher four-point bending and significantly lower torsional implant stiffness than plates, P < 0.01. Increasing difference between nail and stem diameters decreased frictional fit at the stem-nail junction. Nail constructs provided significantly higher initial axial bending stiffness and cycles to failure (200 ± 83 N/mm; 16'871 ± 5'227) compared to plate constructs (93 ± 35 N/mm; 7'562 ± 1'064), P = 0.01. Relative axial translation at osteotomy level after 2'500 cycles was significantly smaller for nail fixation (0.14 ± 0.11 mm) compared with plate fixation (0.99 ± 0.20 mm), P < 0.01. From a biomechanical perspective, the docking nail concept offers higher initial and secondary stability under dynamic axial loading versus plating in TKA periprosthetic fracture fixation.

Biomechanical evaluation of retrograde docking nailing to a total hip arthroplasty stem in a periprosthetic femur fracture model.

INTRODUCTION: Slotted nails allow a connection to a total hip arthroplasty (THA) stem and act as intramedullary load carrier. This study compares construct stiffness, cycles to failure and failure load between a retrograde slotted femur nail construct docked to a THA stem and a lateral locking plate in a human periprosthetic femur fracture model. MATERIALS AND METHODS: In seven pairs of fresh-frozen human anatomic femora with cemented THA, a transverse osteotomy was set simulating a Vancouver type B1 fracture. The femora were instrumented pairwise with either a retrograde slotted nail coupled to the prosthesis stem, or a locking plate plus a locking attachment plate. Four-point mediolateral bending, torsional and axial bending construct stiffness was investigated via non-destructive tests. Cyclic testing under progressively increasing physiologic loading was performed at 2 Hz until catastrophic construct failure. RESULTS: Mediolateral bending stiffness did not differ significantly between the two groups (P=0.17) but exhibited a biphasic profile with significantly increased stiffness in both groups (P<0.01). Nail constructs provided a significantly lower torsional stiffness (0.49 ± 0.66 Nm/°) than plate constructs (1.70 ± 0.86 Nm/°), P=0.03. Axial bending stiffness did not differ significantly between the groups (Nail: 605 ± 511 N/mm; Plate: 381 ± 428 N/mm), P=0.61. Cycles to failure and failure load were significantly higher for the plate constructs (25'700 ± 8'341; 3'070 ± 1334 N) compared with the nail constructs (20'729 ± 7'949; 2'573 ± 1295 N), P=0.04. CONCLUSION: The docking nail construct provides an intramedullary fixation with connection to the prosthesis stem; however, it is biomechanically weaker in stable fractures compared to the plate construct.