Species-specific Immunoassays Research Articles

Exocrine pancreatic insufficiency in dogs and cats.

Exocrine pancreatic insufficiency (EPI) is a malabsorptive syndrome caused by insufficient secretion of digestive enzymes from pancreatic acini. The most common causes of EPI in dogs and cats are pancreatic acinar atrophy and chronic pancreatitis. EPI is diagnosed by measurement of species-specific immunoassays for serum trypsin-like immunoreactivity, the concentration of which directly reflects the mass of functioning pancreatic acinar tissue. EPI is treated by pancreatic enzyme replacement therapy, nutritional management (low-residue diets with moderate fat content), and supplementation of cobalamin. Some dogs and cats have persistent clinical signs despite these treatments. Growing evidence suggests that these clinical signs may be due to enteric microbiota dysbiosis or the presence of concurrent diseases such as chronic enteropathies. Management of these abnormalities may improve outcome in dogs and cats with EPI. The long-term prognosis for dogs and cats with EPI is generally good if high-quality medical therapy is provided. Future studies are needed to further understand the causes of persistent dysbiosis in animals with EPI following initiation of pancreatic enzyme replacement therapy and assess the efficacy of treatments to ameliorate these abnormalities.

Clinical utilization of species-specific immunoassays for identification of Staphylococcus aureus and Streptococcus agalactiae in orthopedic infections.

Staphylococcus aureus and Streptococcus agalactiae (Group B streptococcus, GBS) are common causes of deep musculoskeletal infections (MSKI) and result in significant patient morbidity and cost to the healthcare system. One of the major challenges with MSKI is the lack of faithful diagnostics to correctly identify the primary pathogen, as standard culture-based assays are prone to false positives in the case of polymicrobial infections, and false negatives due to limitations in sample acquisition and antibiotic use before presentation. To improve upon our current diagnostic methods for MSKI, we developed a multiplex immunoassay for antigen-specific IgGs in serum (Luminex), and medium enriched for newly synthesized antibodies (MENSA) for anti-S. aureus and GBS generated from cultured peripheral blood mononuclear cells (PBMCs) of orthopedic infection patients undergoing surgical treatment. Samples were obtained from 110 MSKI patients: 80 diabetic foot ulcer, 21 periprosthetic joint infection, 5 septic arthritis, 2 spine, 1 hand, and 1 fracture-related infection (FRI). Anti-S. aureus and anti-GBS antibody titers were compared to culture results to assess their concordance in identifying the pathogens. Immunoassay, particularly MENSA, showed high diagnostic potential for monomicrobial S. aureus and GBS orthopedic infections (AUC > 0.95). MENSA also demonstrated diagnostic potential for GBS polymicrobial orthopedic infection and for GBS DFU (AUC > 0.83 for both). Serum showed high diagnostic potential for S. aureus PJI (AUC > 0.95). Taken together, these findings support the development of species-specific immunoassays for the identification of causal pathogens in active MSKI, especially in conjunction with standard culture.

Species-Specific Immunoassay Aids Identification of Pathogen and Tracks Infectivity in Foot Infection.

Conventional bacterial cultures frequently fail to identify the dominant pathogen in polymicrobial foot infections, in which Staphylococcus aureus is the most common infecting pathogen. Previous work has shown that species-specific immunoassays may be able to identify the main pathogen in musculoskeletal infections. We sought to investigate the clinical applicability of a S. aureus immunoassay to accurately identify the infecting pathogen and monitor its infectivity longitudinally in foot infection. We hypothesized that this species-specific immunoassay could aid in the diagnosis of S. aureus and track the therapeutic response in foot infections. From July 2015 to July 2019, 83 infected foot ulcer patients undergoing surgical intervention (debridement or amputation) were recruited and blood was drawn at 0, 4, 8, and 12 weeks. Whole blood was analyzed for S. aureus-specific serum antibodies (mix of historic and new antibodies) and plasmablasts were isolated and cultured to quantify titers of newly synthesized antibodies (NSAs). Anti-S. aureus antibody titers were compared with culture results to assess their concordance in identifying S. aureus as the pathogen. The NSA titer changes at follow-ups were compared with wound healing status to evaluate concordance between evolving host immune response and clinically resolving or relapsing infection. Analysis of serum for anti-S. aureus antibodies showed significantly increased titers of 3 different anti-S. aureus antibodies, IsdH (P = .037), ClfB (P = .025), and SCIN (P = .005), in S. aureus culture-positive patients compared with culture-negative patients. Comparative analysis of combining antigens for S. aureus infection diagnosis increased the concordance further. During follow-up, changes of NSA titers against a single or combination of S. aureus antigens significantly correlated with clinically resolving or recurring infection represented by wound healing status. In the management of foot infection, the use of S. aureus-specific immunoassay may aid in diagnosis of the dominant pathogen and monitoring of the host immune response against a specific pathogen in response to treatment. Importantly, this immunoassay could detect recurrent foot infection, which may guide a surgeon's decision to intervene. Level II, prospective comparative study.

Abstract A26: A self-sustained gradient of CCL8 drives breast cancer metastasis

Abstract The establishment of chemoattractive gradients between the neoplastic epithelium, the stroma and the peripheral tissues represents an attractive, yet elusive, mechanism that may contribute to metastasis. Here we provide evidence for the maintenance of a gradient of increasing CCL8 concentration between the epithelium, the stroma and the periphery that is instrumental for breast cancer cells dissemination promoting the directional movement of cancer cells from the site of primary tumor towards the periphery. Experiments in breast cancer cells cultured in vitro and in vivo studies in tumor-bearing mice showed that CCL8 production is enhanced in stromal fibroblasts at the tumor margins and in tissues at which breast cancer cells tend to metastasize such as the lungs and the brain. This stimulation of CCL8 expression in the stroma was driven by signals elicited by the neoplastic epithelium and could be recapitulated by administration of breast cancer cells conditioned media in tumor free mice. Experiments involving transwell-based migration assays established that CCL8-mediated chemoattraction was produced in breast cancer cells while genetic and antibody-based manipulation of CCL8 activity in mice influenced the histology of the tumors and promoted all major steps of the metastatic process such as invasion to adjacent stroma, intravasation and ultimately extravasation and seeding. Specifically, tumors growing in CCL8 deficient animals had well defined borders and high cellularity as opposed to the ill-defined margins of the tumors of wild type animals. In tumor-bearing mice acute i.v. administration of CCL8 increased the number of circulating tumor cells while antibody-mediated inhibition of CCL8 activity reduced the number of cancer cells invading towards adjacent fibroblasts that had been enclosed in matrigel and implanted proximally to the tumors. Only fibroblasts inoculated in the vicinity of breast tumors exhibited elevated CCL8 expression but not the fibroblasts growing in tumor-free mice, which is consistent with the active role of the epithelium in triggering CCL8 expression in the stroma. The stromal origin of CCL8 is also consistent with the strong correlation observed between circulating levels of CCL8 and tumor size, in EO771 mouse breast cancers at which CCL8 expression is minimal and in human MDA-MB-231 breast cancers at which species specific immunoassays permit the distinction between CCL8 originating from the neoplastic epithelium or the host. Analysis of publicly available data suggested that CCL8 is overexpressed in breast tumors as compared to normal breast tissue while high CCL8 expression in clinical breast cancers is associated with poor prognosis. These findings exemplify how self-sustained CCL8-like gradients of chemoattractive factors, between the epithelium, the stroma and the periphery can be induced by breast cancer cells to drive the metastatic process and suggest that interference with their operation may provide means for breast cancer management. Citation Format: Elena Farmaki, Ioulia Chatzistamou, Vimala Kaza, Hippokratis Kiaris. A self-sustained gradient of CCL8 drives breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A26.

Species Specific Immunoassays to Measure Blood Platelet and Coagulation Activation in the Rat

The purpose of this study was to develop specific and sensitive immunoassays to detect early indices of hypercoagulability in the rat. Rat platelet factor 4 (rPF4) and rat fibrinopeptide A (rFPA) assays, tools for the detection of activation of platelets and coagulation respectively, were designed using antibodies raised against purified rPF4 and against synthetic rFPA. The relevance of these new assays and of the commercially available ELISA kit for thrombin-antithrombin III (TAT) complexes was demonstrated in a rat model of a prethrombotic state induced by intravenous infusion of varying doses of thrombo-plastin (90 to 2400 microliters/kg/h). In this model, the immunoassays allowed simultaneous detection of low levels of rFPA and rPF4 which were correlated with fibrinogen and platelet consumption and TAT generation and further proved to be of higher sensitivity than the classical methods of platelet count or measurement of fibrinogen levels. Plasma concentrations of rFPA, rPF4 and TAT were dependent on infusion time and thromboplastin dose, while hirudin (1 mg/kg) prevented their appearance. Thus the new specific immunoassays for rPF4 and rFPA and the commercial human TAT assay represent useful tools for pathophysiological studies or the screening of antithrombotic drugs in rats.