Genetic testing to resolve the source of haemolytic antibody in solid organ transplantation.

BACKGROUND: Antibody-mediated haemolysis due to passenger lymphocyte syndrome arising in the setting of solid organ transplant can be devastating. Some degree of passenger lymphocyte syndrome is said to occur in up to 10% of ABO mismatched renal transplants, 40% of ABO mismatched liver transplants, and 70% of ABO mismatched heart-lung transplants; a reflection of the number of memory B cells transplanted with the organ. Passenger lymphocyte syndrome is less common with minor red cell antigens but can still be severe. MATERIALS AND METHODS: We review a series of patients who developed passenger lymphocyte syndrome after solid organ transplantation. Conventional serological testing was performed using tube and solid-phase testing. Molecular testing was performed using a gene-chip array. RESULTS: In patients receiving a minor antigen mismatched organ transplant and multiple allogenic red cell transfusions, serological methods proved insufficient to resolve the source of minor blood group antibodies that arose in the aftermath of the transplant. Genetic testing was able to clearly resolve donor and recipient types. DISCUSSION: Passenger lymphocyte syndrome after mismatched organ transplantation is not rare, but the syndrome associated with non-ABO antibodies occurs in a much smaller subset of these cases. The mixtures of organ donor, recipient, and other transfused red blood cells profoundly limit the usefulness of serological testing. Genetic assignment of minor blood types to donor and recipient can guide therapy and inform prognosis.

Polyclonal human antibodies against glycans bearing red meat-derived non-human sialic acid N-glycolylneuraminic acid are stable, reproducible, complex and vary between individuals: Total antibody levels are associated with colorectal cancer risk.

BACKGROUND: N-glycolylneuraminic acid (Neu5Gc) is a non-human red-meat-derived sialic acid immunogenic to humans. Neu5Gc can be metabolically incorporated into glycan chains on human endothelial and epithelial surfaces. This represents the first example of a "xeno-autoantigen", against which circulating human "xeno-autoantibodies" can react. The resulting inflammation ("xenosialitis") has been demonstrated in human-like Neu5Gc-deficient mice and contributed to carcinoma progression via antibody-mediated inflammation. Anti-Neu5Gc antibodies have potential as biomarkers for diseases associated with red meat consumption such as carcinomas, atherosclerosis, and type 2 diabetes. METHODS: ELISA assays measured antibodies against Neu5Gc or Neu5Gc-glycans in plasma or serum samples from the Nurses' Health Studies, the Health Professionals Follow-up Study, and the European Prospective Investigation into Cancer and Nutrition, including inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over 1-3 years in archived samples. We also assessed associations between antibody levels and coronary artery disease risk (CAD) or red meat intake. A glycan microarray was used to detected antibodies against multiple Neu5Gc-glycan epitopes. A nested case-control study design assessed the association between total anti-Neu5Gc antibodies detected in the glycan array assay and the risk of colorectal cancer (CRC). RESULTS: ELISA assays showed a wide range of anti-Neu5Gc responses and good inter-assay reproducibility, stability with delayed sample processing, and within-person reproducibility over time, but these antibody levels did not correlate with CAD risk or red meat intake. Antibodies against Neu5Gc alone or against individual Neu5Gc-bearing epitopes were also not associated with colorectal cancer (CRC) risk. However, a sialoglycan microarray study demonstrated positive association with CRC risk when the total antibody responses against all Neu5Gc-glycans were combined. Individuals in the top quartile of total anti-Neu5Gc IgG antibody concentrations had nearly three times the risk compared to those in the bottom quartile (Multivariate Odds Ratio comparing top to bottom quartile: 2.98, 95% CI: 0.80, 11.1; P for trend = 0.02). CONCLUSIONS: Further work harnessing the utility of these anti-Neu5Gc antibodies as biomarkers in red meat-associated diseases must consider diversity in individual antibody profiles against different Neu5Gc-bearing glycans. Traditional ELISA assays for antibodies directed against Neu5Gc alone, or against specific Neu5Gc-glycans may not be adequate to define risk associations. Our finding of a positive association of total anti-Neu5Gc antibodies with CRC risk also warrants confirmation in larger prospective studies.

Performance characteristics of the Beckman Coulter UniCel DxI 800 TSH (3rd IS) assay.

INTRODUCTION: Beckman Coulter recently reformulated their commercial TSH assay with primary calibration to the World Health Organization 3rd TSH international standard. An extensive evaluation of the performance characteristics for this assay was completed. METHODS: Intra-day and inter-day precision was evaluated using 3 concentrations of commercial quality control material. Linearity, reportable range, stability, sensitivity and susceptibility to common inferences were determined using pooled patient specimens. Inter-assay variability was assessed across 5 different platforms (n=47 patient specimens). RESULTS: Intra-day and inter-day CVs were <10% at all concentrations evaluated. The LOQ, LOD and LOB were 0.0047µIU/ml (10% CV), 0.0012µIU/ml and 0.0005µIU/ml, respectively. Variable bias was observed for the TSH3 assay when evaluated against the previous generation assay and other platforms, but overall TSH3 gave comparable results. CONCLUSIONS: The TSH3 assay for UniCel DxI 800, is precise, highly sensitive and comparable to the previous generation assay. The assay is acceptable for clinical testing.

Siglec receptors impact mammalian lifespan by modulating oxidative stress.

Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan.

A red meat-derived glycan promotes inflammation and cancer progression.

A well known, epidemiologically reproducible risk factor for human carcinomas is the long-term consumption of "red meat" of mammalian origin. Although multiple theories have attempted to explain this human-specific association, none have been conclusively proven. We used an improved method to survey common foods for free and glycosidically bound forms of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc), showing that it is highly and selectively enriched in red meat. The bound form of Neu5Gc is bioavailable, undergoing metabolic incorporation into human tissues, despite being a foreign antigen. Interactions of this antigen with circulating anti-Neu5Gc antibodies could potentially incite inflammation. Indeed, when human-like Neu5Gc-deficient mice were fed bioavailable Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Such mice are already prone to develop occasional tumors of the liver, an organ that can incorporate dietary Neu5Gc. Neu5Gc-deficient mice immunized against Neu5Gc and fed bioavailable Neu5Gc developed a much higher incidence of hepatocellular carcinomas, with evidence of Neu5Gc accumulation. Taken together, our data provide an unusual mechanistic explanation for the epidemiological association between red meat consumption and carcinoma risk. This mechanism might also contribute to other chronic inflammatory processes epidemiologically associated with red meat consumption.

Involvement of a non-human sialic Acid in human cancer.

Sialic acids are common monosaccharides that are widely expressed as outer terminal units on all vertebrate cell surfaces, and play fundamental roles in cell-cell and cell-microenvironment interactions. The predominant sialic acids on most mammalian cells are N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Neu5Gc is notable for its deficiency in humans due to a species-specific and species-universal inactivating deletion in the CMAH gene encoding the hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc. However, Neu5Gc is metabolically incorporated into human tissues from dietary sources (particularly red meat), and detected at even higher levels in some human cancers. Early life exposure to Neu5Gc-containing foods in the presence of certain commensal bacteria that incorporate dietary Neu5Gc into lipooligosaccharides can lead to generation of antibodies that are also cross-reactive against Neu5Gc-containing glycans in human tissues ("xeno-autoantigens"). Such anti-Neu5Gc "xeno-autoantibodies" are found in all humans, although ranging widely in levels among individuals, and displaying diverse and variable specificities for the underlying glycan. Experimental evidence in a human-like Neu5Gc-deficient Cmah(-) (/) (-) mouse model shows that inflammation due to "xenosialitis" caused by this antigen-antibody interaction can promote tumor progression, suggesting a likely mechanism for the well-known epidemiological link between red meat consumption and carcinoma risk. In this review, we discuss the history of this field, mechanisms of Neu5Gc incorporation into tissues, the origin and specificities of human anti-Neu5Gc antibodies, their use as possible cancer biomarkers, implications of xenosialitis in cancer initiation and progression, and current and future approaches toward immunotherapy that could take advantage of this unusual human-specific phenomenon.