large-scale-sars-cov-2-testing-utilizing-saliva-transposition-sample

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

Michigan State University

The human lymphocyte antigen (HLA) typing community was one of the early groups to adopt molecular testing. This action was borne out of the need to identify the many alleles of the highly polymorphic HLA system. Early paradigms used restriction fragment length polymorphism regimes, but the polymerase chain reaction method of amplification quickly replaced that less-than-discriminating choice. Methods currently in use for HLA typing, with commercial kits available, are sequence-specific oligonucleotide probe (both dot blot and the reverse blot dot), sequence-specific primer amplification, restriction fragment length polymorphism of amplified products, double-stranded sequence conformation polymorphism (with and without reference strand), sequence-based typing, and microarray technologies. More than 1250 alleles are recognized by the World Health Organization and meet their criteria for assignment. These alleles can be identified by molecular methods and represent alleles present at class I and class II loci of the HLA complex. On occasion, ambiguous results still persist, even with the best molecular typing methods. Therefore, it is clear to the HLA typing community that a combination of the above methods may be needed to allow true discrimination of the possible alleles an individual carries in their genetic makeup. It is also clear that a typing laboratory may need to resort to nonmolecular serology to understand the significance and impact of the type generated by the HLA molecular typing laboratory.

Human lymphocyte antigen molecular typing: how to identify the 1250+ alleles out there.

BACKGROUND: Reactions are commonly associated with the chemotherapy of onchocerciasis. However unmanageable reactions are uncommon when ivermectin (Mectizan(R)) is used for the treatment of this infection, and this drug has proved to be a great improvement over previously used agents. Serious adverse events (SAE) nevertheless have occurred, and there is considerable concern about the negative effect such events may have on mass drug administration programs.This paper reviews the basic pathogenic mechanisms that can be involved in the destruction of microfilaria by chemotherapeutic agents. A central challenge to filarial chemotherapy is the need to remove parasites from biologically sensitive tissues, a more difficult medical challenge than eliminating nematodes from the gastrointestinal tract.Explanations for the etiology of the serious adverse reactions occurring with ivermectin treatment in specific geographic areas where there is coincident heavy Loa loa infections are hampered by a lack of specific pathological case material. Ways to investigate these possibilities are reviewed. Possible pathogenic mechanisms include embolic vascular pathology accompanied by local inflammation, blood brain barrier mdr1 abnormalities, and genetic predisposition to excessive inflammatory responses. CONCLUSION: It is important to keep ivermectin, and all its associated adverse clinical events, in perspective with the many other chemotherapeutic agents in general use - many of which produce serious adverse events even more frequently than does ivermectin. Currently available evidence indicates that the pathogenesis of the Loa-associated adverse reactions are probably related to inflammatory responses to microfilariae in specific tissues. However, the possibility of genetic predispositions to pathology should also be considered.

Possible pathogenic pathways in the adverse clinical events seen following ivermectin administration to onchocerciasis patients.

Class I major histocompatibility complex (MHC) presents intracellular-derived peptides on the majority of cells within the human body. Intracellular proteins are degraded into peptides of 8-11 amino acids, allowing them to fit into the groove of an empty MHC class I molecule. Detection of MHC-associated peptides can be challenging with the major difficulty being the ability to obtain peptides in adequate concentration. Published protocols require a large sample size that is unrealistic for a clinically available sample. Based on calculations, it should be possible to characterize MHC-associated peptides from cells obtained from 30 ml of whole blood. A citric acid wash of whole platelets was implemented to release the peptides with sample cleanup by reversed-phase high-performance liquid chromatography on a peptide trap. Peptides were analyzed by liquid chromatography tandem mass spectrometry. Four peptides were identified from an individual's platelets. The binding motifs of the peptides were consistent with the published MHC binding motif of the individual. Since red blood cells do not express MHC, they were used as a negative control. Using citric acid wash of whole cells and a peptide trap, the more abundant MHC-associated peptides can be identified. This report demonstrates the identification of peptides from a sample volume compatible with reasonable clinical availability.

Characterization of major histocompatibility complex-associated peptides from a small volume of whole blood.

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.

MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura.

Alterations in the protein tyrosine phosphatase N22 (PTPN22) gene affect the threshold for lymphocyte activation. The PTPN22 1858T polymorphism leads to uninhibited T-cell receptor cascade propagation. An elevated PTPN22 1858C/T genotype frequency has been correlated with several autoimmune disorders which have T-cell and humoral components. However, a recent Tunisian report demonstrated no association between PTPN22 1858T and patients with Pemphigus vulgaris (PV), an autoantibody-associated blistering disorder. Because PTPN22 1858T allele frequency is known to vary across ethnic populations, we conducted a case-control study investigating the relationship between PTPN22 1858T and PV in North American patients of either Ashkenazi Jewish or Caucasian (non-Ashkenazi) decent. Participant genotype was determined in 102 PV patients and 102 healthy controls by restriction fragment length polymorphism-polymerase chain reaction genotyping. Relationships were calculated using Fisher's exact tests and chi-squared tests. We report that the PTPN22 1858C/T genotype is not significantly associated with PV in either Caucasians (P = 0.83) or Ashkenazi Jews (P = 0.60). Further stratification of the patient population by gender, age of disease onset, HLA-type, family history of autoimmune disease, history of anti-desmoglein (anti-Dsg) 3 or anti-Dsg1 antibody response, history of lesion morphology, and disease duration did not uncover significant associations between the PTPN22 1858T allele and PV subgroups. Our data indicate that the PTPN22 1858T mutation is not associated with PV in the North American population. We do observe an elevation of PTPN22 1858C/T genotype frequency in male PV patients. Further investigation will be required to determine if this trend reaches significance in larger studies.

PTPN22 1858T is not a risk factor for North American pemphigus vulgaris.

In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs).

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1.

Emanuel Hackel: 1925-2012.

Alopecia areata (AA) is a genetically determined autoimmune hair loss disorder. A polymorphism in protein tyrosine phosphatase N22 (PTPN22), which normally suppresses T-cell proliferation, has been associated with human autoimmune disease, including AA in European populations. PTPN22 genotype frequency in known to vary geographically. Accordingly, we conducted a case-control study of the PTPN22 1858C/1858T (C1858T) genotype frequency in North American Caucasians and non-Caucasians. Allele status was determined in 365 AA patients, 196 healthy related control subjects (RC) and 77 unrelated healthy control subjects (UrC). We found that AA patients are more likely to carry the PTPN22 C1858T genotype than UrCs (p = 0.075), and this association reached significance in patients with the most severe disease presentation (Alopecia universalis vs. UrC, p = 0.024). PTPN22 C1858T genotype frequency in RC did not differ from AA patients (p = 0.657), but was significantly increased in comparison with UrC (p = 0.050). PTPN22 1858C/T genotype frequency increased in related control subjects most closely associated with patients (one family members of AA patients vs. UrC subjects, p = 0.040). Our data suggests that AA patients (particularly those that are severely affected) and closely related control subjects may belong to a shared inheritance group with increased disease risk, distinct from secondary and tertiary relatives and unrelated individuals. These findings have implications for the study of candidate genes and susceptibility to AA that may influence future clinical monitoring of unaffected, but closely related family members of patients.

PTPN22 profile indicates a novel risk group in Alopecia areata.

Pemphigus vulgaris (PV) belongs to an autoimmune disease cluster that includes autoimmune thyroid disease (AITD), suggesting common mechanisms driving autoimmune susceptibility. Our group has shown that PV patients exhibit significant reactivity to AITD-related anti-thyroid peroxidase (anti-TPO), and anti-TPO antibodies affect signaling pathways in keratinocytes similar to anti-desmoglein (Dsg) 3 antibodies. To further assess the relevance of anti-TPO reactivity in PV, we analyzed anti-TPO levels in 280 PV and 167 healthy control serum samples across a comprehensive set of variable and static parameters of disease activity and etiopathogenesis. PV patients have significantly higher activity rates (A.R.s) for anti-TPO than healthy controls, but levels do not differ between phases of clinical activity and remission. Patients that carry both the PV-associated human leukocyte antigen (HLA) alleles DRB1*0402 and DQB1*0503, or DQB1*0503 alone show a low prevalence of anti-TPO (A.R. 9.5 and 4.8%, respectively), while patients that lack expression of these alleles or carry DRB1*0402 alone have a much higher prevalence of anti-TPO (A.R. 23.1 and 15.8%, respectively), suggesting that the absence of DQB1*0503 may predispose patients to the development of anti-TPO antibodies. Similarly, anti-Dsg1/3 patients have a higher anti-TPO A.R. (26.9%) than anti-Dsg1/3 (18.8%), anti-Dsg1/3 (14.3%), and anti-Dsg1/3 (3.9%) patients. Our data suggest that anti-TPO reactivity in PV is driven by genetic markers that may be in linkage disequilibrium with the established PV-susceptibility alleles and that this association drives the selection of a combination of anti-Dsg and anti-TPO antibodies, with anti-TPO filling the gap in active patients that do not carry the established PV-associated autoantibodies and/or are lacking the established PV-HLA-susceptibility alleles.

Anti-Thyroid Peroxidase Reactivity Is Heightened in Pemphigus Vulgaris and Is Driven by Human Leukocyte Antigen Status and the Absence of Desmoglein Reactivity.

HLA-DPB1*18:01:01:04 differs from HLA-DPB1*18:01:01:01 by four substitutions within intron 1.

Identification of a novel allele, HLA-DPB1*18:01:01:04, in an African American renal transplant candidate.

Although precursor-targeted immune-mediated anemia (PIMA) is thought to be caused by immune targeting of erythroid precursors (nucleated RBCs, nRBCs), its pathogenesis is unknown. Immunoglobulin G (IgG) or phosphatidylserine (PS) may promote nRBC destruction in PIMA.

Article	Year
Human lymphocyte antigen molecular typing: how to identify the 1250+ alleles out there. Archives of pathology & laboratory medicine\| PubMed ID: 11860300	2002
Possible pathogenic pathways in the adverse clinical events seen following ivermectin administration to onchocerciasis patients. Filaria journal\| PubMed ID: 14975062	2003
Characterization of major histocompatibility complex-associated peptides from a small volume of whole blood. Analytical biochemistry\| PubMed ID: 15113691	2004
MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura. Human immunology\| PubMed ID: 16216671	2005
PTPN22 1858T is not a risk factor for North American pemphigus vulgaris. Experimental dermatology\| PubMed ID: 21585555	2011
Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1. Veterinary clinical pathology\| PubMed ID: 22136477	2011
Emanuel Hackel: 1925-2012. Transfusion\| PubMed ID: 23110734	2012
PTPN22 profile indicates a novel risk group in Alopecia areata. Human immunology\| PubMed ID: 24055692	2014
Anti-Thyroid Peroxidase Reactivity Is Heightened in Pemphigus Vulgaris and Is Driven by Human Leukocyte Antigen Status and the Absence of Desmoglein Reactivity. Frontiers in immunology\| PubMed ID: 29675021	2018
Identification of a novel allele, HLA-DPB1*18:01:01:04, in an African American renal transplant candidate. HLA\| PubMed ID: 31943840	2020
Immunoglobulin G and phosphatidylserine in regenerative and nonregenerative immune-mediated anemias of dogs. Journal of veterinary internal medicine\| PubMed ID: 34716708	2021

John A. Gerlach

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