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The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Routine screening of blood donations with second-generation hepatitis hepatitis C virus (HCV) assays has substantially reduced the occurrence of posttransfusion hepatitis. However, following the development of third-generation assays, several studies indicated that these assays may identify HCV-infected individuals who are not identified by second-generation assays., Study Design and Methods: The sensitivity of a third-generation HCV enzyme-linked immunosorbent assay (ELISA-3) was compared with a second-generation ELISA (ELISA-2) in a side-by-side study of 9936 commercial blood donors. ELISA-reactive specimens were subjected to supplemental analysis by third-generation recombinant immunoblot assay and polymerase chain reaction., Results: ELISA-3 demonstrated greater sensitivity than ELISA-2, detecting 1 additional recombinant immunoblot assay-positive specimen per 2000 tested. ELISA-3 also detected 1 additional HCV-infectious polymerase chain reaction-positive unit among approximately 10,000 units screened., Conclusion: The incremental sensitivity achieved with ELISA-3 can be expected to eliminate approximately 20 infectious donations per week among those made by commercial donors in the United States. In accordance with previous studies, most of the improved sensitivity of ELISA-3 derives from its increased detection of anti-c33c (NS3), rather than from the inclusion of HCV antigen NS5.

Syrian hamsters show evidence of classical T-cell-mediated immune reactivity to acute virus infection as judged by primary foot pad swelling, kinetics of in vitro cytotoxic activity, and virus specificity of cytotoxic effector cells. In spite of this, no evidence of genetic restriction is observed among the variety of allodisparate inbred strains tested. This virus-induced, cell-mediated killing extends across strain barriers despite strong cellular and serologic alloreactivity among some of the strains utilized. To account for the apparent lack of genetic restriction, we currently favor the hypothesis that all hamsters examined thus far share at least one class I MHC antigen. Since these animals differ at hamster loci which elicit MLR, GVHR, acute SGR, CML, and alloantibody, we presume class II MHC polymorphism exists in this species. The presence of putative class II MHC polymorphism without detectable class I polymorphism is unusual among mammals examined to date, and of unknown biologic significance.

We describe two mouse monoclonal antibodies reactive with lipopolysaccharide derived from the J5 mutant of Escherichia coli O111:B4. These antibodies react with purified lipopolysaccharide derived from rough mutants of E. coli and Salmonella typhimurium and also with lipopolysaccharide associated with both smooth- and rough-phenotype, gram-negative bacteria. Both antibodies appear to bind determinants present in the lipopolysaccharide core region, and this reactivity is inhibited in the presence of polymyxin B. Although their patterns of reactivity differ, both antibodies exhibit extensive serological cross-reactivity with a variety of gram-negative bacteria. Reagents of this type should prove useful in animal models to delineate the requisite affinity, epitope specificity, immunoglobulin class, etc., needed for the prevention and treatment of gram-negative bacteremia.

An intrastrain cross-reactive idiotype, CRIA, is associated with a large proportion of the anti-p-azophenylarsonate (anti-Ar) antibodies of A/J mice. The present experiments, in which the methods of direct and reverse passive cutaneous anaphylaxis (RPCA) were used, indicate that IgE anti-Ar antibodies are produced in A/J mice upon stimulation with a protein-Ar conjugate and that a large proportion of these antibodies express CRIA. The use of monoclonal anti-CRIA for RPCA eliminated the strong nonspecific reactions previously observed. The results provide a basis for studying factors that regulate the switch to IgE biosynthesis during an immune response.

To determine if radiolabeled specific antibodies directed against bacterial antigens could be used to detect sites of infection, gamma camera imaging studies were performed in animals infected with Pseudomonas aeruginosa. Murine monoclonal antibodies (Mabs) directed against Fisher Immunotype 1 Pseudomonas aeruginosa and a nonmicrobial, nonmammalian haptene, p-arsanilic acid, were labeled with 125I by the lodogen-Bead method. Unilateral, deep thigh infections were created by innoculation with 2 X 10(8) Fisher Immunotype 1 P. aeruginosa. Twenty-four hours later, one of the radiolabeled antibodies was injected intravenously at a dose of 0.25 mg/kg (100-150 microCi). Serial gamma imaging was then carried out beginning at 4 hr and at approximately 24-hr intervals thereafter. Beginning as early as 4 hr postinjection, the area of inflammation could be visualized with either the specific or nonspecific Mab, with the images continuing to intensify until 24-48 hr postinjection. At 48 hr, the contrast between lesion and background with the nonspecific Mab began to fade, while the contrast in the specific Mab-generated images continued to intensify until approximately 192 hr postinjection. Clear-cut differentiation between specific and nonspecific Mab-generated images was possible by 72 hr postinjection. We conclude that specific immune imaging of localized infection with Mab's directed against specific microbial antigens is possible and should be clinically useful. In addition, images created by the localization of immunoglobulin non-specifically at the site of inflammation in the first 24-48 hr postinjection may also provide useful information as to the anatomic location of hidden abscesses.

In vivo expression of the type 8 capsular polysaccharide (CP) of Staphylococcus aureus was studied in the rat model of aortic valve endocarditis. An enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies to type 8 CP was sensitive to 0.16 ng/ml in serum. All (23 of 23) animals infected with the prototype type 8 strain Becker had CP detected in one or more serum samples. Peak CP levels ranged from 0.16 to 240 ng/ml (median, 25 ng/ml). Among the 14 rats alive on day 2, antigenemia and bacteremia were significantly correlated (r = .71, P less than .005). CP was also present in serum of rats infected with three additional type 8 bacteremic isolates. All serum samples from animals infected with the type 5 prototype strain were negative in the type 8 ELISA. These studies document that the type 8 CP of S. aureus, previously identified only in vitro, is produced and released during in vivo infection and can be detected in serum of infected animals.

Previous reports have shown the A/J anti-para-azophenylarsonate (anti-Ar) antibodies that share a major cross-reactive idiotype (CRI) comprise a family of closely related but nonidentical molecules. Serological studies with CRI+ monoclonal anti-Ar antibodies have suggested the presence of a conserved idiotypic determinant within the family. The present study utilized monoclonal ant-idiotypic determinant within the family. The present study utilized monoclonal anti-idiotypic antibodies to define further the nature of the conserved idiotypic determinant. It was found that 8 of 10 CRI+ monoclonal antibodies possess an idiotypic determinant reactive with each of three monoclonal anti-idiotypic antibodies. In addition, approximately 60% of CRI+ serum anti-Ar antibodies reacted with one of the monoclonal anti-idiotypic preparations. The monoclonal anti-idiotypic antibodies react with an idiotope in the region of the hapten-binding site, as indicated by the ability of free haptens to inhibit idiotype-anti-idiotype interactions. Finally, two of three monoclonal anti-idiotypic antibodies suppressed the subsequent production of CRE+ serum anti-Ar antibodies when administered before antigen, without significantly affecting the total anti-Ar response.

This competitive immunoassay, based on inhibition by antigen of the idiotype-anti-idiotype interaction, detects adenosine deaminase binding protein (ABP), and involves use of monoclonal anti-idiotype antibodies prepared to a monoclonal antibody specific for ABP. The conditions for this new type of competitive immunoassay are investigated. This competitive immunoassay is as sensitive and reproducible as an earlier described "sandwich"-type immunoassay for ABP (Clin Chem 31: 679-683, 1985). Evaluation of urine samples from normal subjects and from patients showed increased concentrations of ABP in patients with renal disease.

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRIA and CRIC , characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c X A/J)F1 ( CAF1 ) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF1 mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRIC was markedly enhanced in mice suppressed for CRIA.

Staphylococcus aureus has been classified into at least eight different capsular types by using polyclonal rabbit antisera specific for their associated capsular polysaccharides. We produced and characterized monoclonal antibodies reactive with two serologically distinct capsular types, types 5 and 8, which account for more than 70% of all S. aureus bacteremias. These type-specific, monoclonal antibodies reacted with S. aureus clinical isolates possessing the homologous capsular type and exhibited no cross-reactivity against S. aureus clinical isolates possessing the heterologous capsular type, nontypeable S. aureus clinical isolates, Staphylococcus epidermidis clinical isolates, or a variety of gram-negative organisms. The anti-type 8 monoclonal antibodies also reacted with purified capsular polysaccharide derived from the prototype type 8 S. aureus strain.

The identity of the effector cell(s) mediating vaccinia virus-induced cytotoxic activity in Syrian hamsters undergoing acute virus infection has been investigated. Two different approaches have been utilized in this regard. Although T cells do not mediate vaccinia virus-induced cytotoxic activity directly, studies employing adult thymectomized, x-irradiated, bone-marrow-reconstituted hamsters lacking discernible T cell reactivity demonstrated that the presence of an intact thymus and/or functional T cells were required for the in vivo development of a significant portion of vaccinia virus-induced cytotoxic activity. In addition, incorporation of aggregated gamma-globulins as well as anti-immunoglobulin reagents into the in vitro 51Cr release assay inhibited a significant proportion of the cytotoxic activity mediated by spleen cells obtained from acutely infected hamsters possessing an intact thymus. Both approaches have yielded information consistent with the idea that a sizable portion of vaccinia virus-induced cytotoxic activity in the Syrian hamster is effected by K cells mediating antibody-dependent cell-mediated cytotoxicity (ADCC). The significance of this observation is discussed with regard to hamster viral immunity in general.

A direct (antiglobulin) solid-phase enzyme immunoassay for the detection of antibody to hepatitis B core antigen (anti-HBc) is described. The assay utilizes recombinant hepatitis B core antigen as the solid-phase 'capture' reagent and a mixture of monoclonal antibodies specific for human IgG and IgM conjugated to horseradish peroxidase as the 'detector' reagent. The direct assay demonstrated excellent sensitivity and specificity when compared with a commercially available competitive enzyme immunoassay. The direct assay format lends itself to a confirmatory assay for anti-HBc by addition of monoclonal anti-HBc to the reaction mixture. Feasibility of the confirmatory assay for anti-HBc was demonstrated using specimens reactive for anti-HBc as documented by both the direct and competitive assays.

The participation of hamster T cells in a variety of putative MHC-determined reactions was studied utilizing a well-characterized, highly selective goat anti-hamster thymocyte (G alpha HT) serum. Hamster lymphoid cell suspensions treated with G alpha HT lose much of their capacity to induce local graft-versus-host reactions and to function as responder cells in mixed lymphocyte reactions. In contrast to the participation of hamster T cells in alloimmune reactions (MLR and GVHR), virus-induced, cytotoxic activity in hamsters undergoing acute virus infection is not T-cell-mediated. This latter finding was rather surprising in view of the major role played by cytotoxic T effector cells in comparably infected mice and rats. These results suggest that, although hamsters are able to respond to putative class II MHC disparities in allogeneic reactions, MHC-encoded molecules, presumably class I, are not utilized for induction of effective cytotoxic activity in response to acute virus infection in this species. The implications of these findings are discussed in relation to our present understanding of the hamster MHC.

By using polyethylene glycol 1540, BW5147 AKR T lymphoma cells were fused with splenocytes from A/J mice treated so as to induce suppressor T cells specific for azobenzenearsonate (ABA). Of 576 microwells originally seeded, 132 demonstrated growing cell clones, 4 of which produced an ABA-binding supernatant factor. When tested in vivo for suppression of delayed-type hypersensitivity to ABA, two of these cell lines, A4 and F12, were shown to produce suppressive supernatant factors. Fluorescence analysis of the F12 cells with appropriate antisera demonstrated this T cell hybrid to be Thy 1.2+, Lyt 1+,2-, and surface immunoglobulin negative, the surface marker phenotype of conventional ABA-specific suppressor T cells. This cloned suppressor cell line, F12, produces a culture supernatant factor that is suppressive at dilutions up to 1:100 and has provided material for genetic and immunochemical analysis.