Production and characterization of anti-porcine CXCL10 monoclonal antibodies.

Research on C-X-C motif chemokine ligand 10 (CXCL10) has been widely reported for humans and select animal species, yet immune reagents are limited for pig chemokines. Our goal is to provide veterinary immunologists and the biomedical community with new commercial immune reagents and standardized assays. Recombinant porcine CXCL10 (rPoCXCL10) protein was produced by yeast expression and used to generate a panel of α CXCL10 monoclonal antibodies (mAbs). All mAbs were assessed for cross-inhibition and reactivity to orthologous yeast expressed CXCL10 proteins. Characterization of a panel of nine α PoCXCL10 mAbs identified six distinct antigenic determinants. A sensitive quantitative sandwich ELISA was developed with anti-PoCXCL10-1.6 and -1.9 mAb; reactivity was verified with both rPoCXCL10 and native PoCXCL10, detected in supernatants of peripheral blood mononuclear cells stimulated with rPoIFNγ or PMA/Ionomycin. Immunostaining of in vitro rPoIFNγ stimulated pig spleen and blood cells verified CXCL10 + cells as CD3-CD4-CD172+, with occasional CD3-CD4 + CD172 + subsets. Comparison studies determined that α PoCXCL10-1.4 mAb was the ideal mAb clone for intracellular staining, whereas with α PoCXCL10-1.1 and -1.2 mAbs were best for immunohistochemistry analyses. These techniques and tools will be useful for evaluating swine immune development, responses to infectious diseases and vaccines, as well as for improving utility of pigs as an important biomedical model.

Rescue therapy for symptomatic spinal cord ischemia after thoracic endovascular aortic repair.

OBJECTIVE: Spinal cord ischemia (SCI) after thoracic endovascular aortic repair (TEVAR) can cause permanent neurologic deficits and poor long-term survival. Targeted treatment of new SCI symptoms after TEVAR (rescue therapy [RT]) might improve/resolve neurologic symptoms but few data characterize the association of specific interventions with SCI outcomes. We evaluated the effectiveness of post-TEVAR RT at our tertiary aortic center. METHODS: Our institutional TEVAR database was reviewed for SCI incidence and details of RT. This included cerebrospinal fluid drainage (CSFD), medical therapy, and optimization of spinal cord oxygen delivery. SCI outcomes were categorized at discharge as paralysis/paraparesis and temporary/permanent. RESULTS: Nine hundred forty-three TEVAR procedures were performed in 869 patients from 2011 to 2020. Post-TEVAR SCI occurred in 7.8% (n = 74) with permanent paraplegia in 1.5%. Older patient age, chronic obstructive pulmonary disease, and previous abdominal aortic surgery were predictive of SCI. Half (n = 37) of SCI episodes resulted in only temporary paralysis/paraparesis. Rescue postoperative cerebrospinal fluid drains were implanted in 3.7% (n = 35) of procedures and was predicted by higher American Society of Anesthesiologists class, lower serum hemoglobin level, elevated international normalized ratio, bilateral iliac artery occlusion, nonelective procedures, and penetrating atherosclerotic ulcer/intramural hematoma indication. The most commonly used RTs were emergent placement of or increased drainage from an existing cerebrospinal fluid drain (87.8%), induced/permissive hypertension (77.0%), corticosteroid bolus (36.5%), and naloxone infusion (33.8%). Neurologic improvement occurred in 68.9% (n = 51/74). New/increased drainage was associated with improved SCI outcome. CONCLUSIONS: Permanent paraplegia from post-TEVAR SCI is rare (1.5%). Older patients with comorbidities carry greater post-TEVAR SCI risk. SCI symptoms improved/resolved with CSFD and multimodal RT in 68.9% of patients, but no intervention was independently associated with improvement. TEVAR centers should have robust protocols for timely and safe CSFD placement to augment RT strategies for SCI.

Safety of cerebrospinal fluid drainage for spinal cord ischemia prevention in thoracic endovascular aortic repair.

Objective: Spinal cord ischemia (SCI) after thoracic endovascular aortic repair (TEVAR) is associated with permanent neurologic deficit and decreased survival. Prophylactic cerebrospinal fluid (CSF) drainage (CSFD) in TEVAR is controversial. We evaluated the usage of CSFD in TEVAR at our tertiary aortic center. Methods: Our institutional TEVAR database was reviewed to determine the frequency of CSFD usage/complications. Complications were categorized as mild (headache/CSF leak not requiring intervention, urinary retention), moderate (headache/CSF leak requiring intervention, drain malfunction requiring replacement), or severe (intrathecal hemorrhage, CSFD-attributable neurologic deficit). The relationships between CSFD complications and patient/procedural characteristics, CSFD placement timing, and survival were analyzed. Results: Nine hundred thirty-six TEVAR procedures were performed in 869 patients from 2011 to 2020. Three hundred ninety CSFD drains were placed in 373 (41.7%) TEVAR patients. Most CSFD drains (89.5%) were pre-TEVAR. Most post-TEVAR drains were placed for new SCI symptoms (n = 21). Twenty-five patients (6.4%) suffered 32 CSFD complications. Most (n = 17) were mild in severity. Severe CSFD complications occurred in 5/432 (1.1% CSF drains) patients. No patient/procedural characteristics were predictive of CSFD complications. Post implant CSFD placement for new SCI symptoms conferred an increased risk of CSFD complication (odds ratio, 6.9; 95% CI, 2.42-19.6; P < .01). The long-term survival of the CSFD complication cohort did not differ from the overall population. Conclusions: Post-TEVAR CSFD placement for new SCI symptoms was associated with substantially greater risk of CSFD complications. Avoidance of post-implant therapeutic drain placement might be the key to prevention of CSFD complications, favoring a strategy of selective pre-implant drain placement in patients at higher risk for SCI.

Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma.

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Importance of the pig as a human biomedical model.

Pigs have substantial potential as biomedical models for studying human developmental processes, congenital diseases, and pathogen response mechanisms in addition to utility as xenotransplant organ donors and tools for vaccine and drug design. The similarity of pigs to humans in anatomical size and structure, physiology, immunology, and genome enhances their potential as models for humans. Hence, it is imperative that research is relevant and reproducible in animal models that more closely resemble humans, such as the pig. This review summarizes the current status of pigs as an investigative model for humans and highlights their future applications.

Gene expression in tonsils in swine following infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. RESULTS: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. CONCLUSIONS: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.

Differential responses in placenta and fetal thymus at 12 days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection.

BACKGROUND: A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC). RESULTS: The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways. CONCLUSION: Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.

The effects of two analgesic regimes on behavior after abdominal surgery in Steller sea lions.

This study examined the effects of two non-steroidal anti-inflammatory drug (NSAID) treatment protocols on the behavioral responses of juvenile Steller sea lions after abdominal surgery. Sea lions were randomly assigned to one of two treatments designed to control post-operative pain. The flunixin group (n=6) received flunixin meglumine (1mg/kg) administered as a single intramuscular (IM) injection before extubation from surgery. The carprofen group (n=5) received carprofen (4.4 mg/kg) as an IM injection before extubation, then orally at 24, 48 and 72 h after surgery. Seven behaviors related to post-operative pain were monitored by observers, blinded to treatment, for a total of 10 days (3 days pre-, day of surgery, and 6 days post-surgery). All seven behaviors changed after surgery regardless of NSAID treatment, two of which returned to baseline within 6 days of surgery. Only one behavior was mildly affected by analgesic treatment: sea lions in the carprofen group tended to spend less time lying down in Days 1-3 following surgery (i.e., the days which they received oral carprofen). These results suggested that neither treatment, at the dose administered, was effective in controlling pain in the days following this surgery.