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Two monoclonal antibodies (MoAb). 9-1 (anti-CD2) and 3G8 (anti-CDI6), were previously shown to enhance the cytotoxic activity of human natural killer (NK) cells. The present study examined the effect of 9-1 and 3G8 with different effector and target cells to determine whether they activate NK cells through a common mechanism. Analysis of purified lymphocyte subpopulations demonstrated that the CD3+ CD16+ CD3&mn; NK effector cell population is enhanced by both antibodies, while purified CD2+ CD16&mn; CD3+ T cells are not activated by either antibody. Although both antibodies enhance killing of K-562 and Daudi, killing of T-cell lines is enhanced by 9-1 and inhibited by 3G8. In constrast, killing of the promyelocytic cell line. U-937 is inhibited by 9-I and enhanced by 3GK. On NK-susceptible cells the pattern of enhancement with 3GH. an IgGI MoAb, is consistent with the pattern of target cell expression of an Fc reccptor, FcR II. known to hind IgGI antibodies. This suggests that 3G8 may cross-link effector and target cells through CD16 on the effectors und FcR II on these targets. This could activate NK killing by a mechanism similar lo anti body-dependent cellular cytotoxicity reactions (ADC'C) with the MoAb in the reverse orientation. The failure of 3G8 F(ab')- fragments to enhance NK killing. further supports the reverse ADCC mechanism of enhancement by 3G8. The pattern of enhancement mediated by 9-1. an IgG3 MoAb. is not correlated with any target cell Fcreceptor known to bind lgG3 MoAb. The effect of 9-1 may result, instead, from its binding to the unique 9-1 epitope on the CD2 molecule involved in CD2-medialed T-cell activation, as previously described. Alternative mechanisms, including activation of NK killing by 9-1 mediated cross-linking of CD2 and CD16 on the effector cells, have also been discussed. [ABSTRACT FROM AUTHOR]

Background: Membranes surrounding the fetus play a crucial role in providing a physical and immunological barrier between a semiallogeneic fetus and mother during pregnancy. In this study, we tested whether cotransplantation of fetal membranes (FMs) and allogeneic donor cells would improve the retention and function of allografts in mice., Methods: Intact and enzyme-digested membranes obtained from E18-E19 pregnant mice were subcutaneously cotransplanted with 10F7MN hybridoma cells that are of BALB/cByJ (Balb) origin and secrete anti-human CD235a antibody. Cells were transplanted into C57BL/6J (B6, allogeneic), Balb (syngeneic), and FVB/NJ (third-party) mice. Serum was collected after 1 and 3 weeks of cell transplantation and tested using flow cytometry for the presence of anti-human CD235a antibody. Immunosuppressive functions of membranes were further investigated by analyzing the cytokine profile of supernatants collected from allo-reactive mixed lymphocyte reactions (MLRs) using a multiplex cytokine assay., Results: B6 mice transplanted with 10F7MN cells along with membranes syngeneic to the host had significantly higher levels of CD235a antibody when compared to B6 mice that received cells without membranes, allogenic membranes, or third-party membranes. Syngeneic membranes significantly inhibited T-cell proliferation in the presence of allogeneic stimuli and suppressed the release of Th1-cytokines such as IFNγ, TNFα, and IL-2 in MLRs. Additionally, increases in the levels of Th2-cytokines were found in MLRs containing membrane-derived cells., Conclusions: Our study highlights the potential use of syngeneic FMs to act as potent cell-carriers that could improve graft retention as well as graft-specific immunoprotection during allograft transplantation., Competing Interests: The authors declare no conflicts of interest.

Sickle cell disease (SCD) is a common, life-threatening genetic disorder that is best managed when diagnosed early by newborn screening. However, SCD is most prevalent in low-resource regions of the world where newborn screening is rare and diagnosis at the point-of-care is challenging. In many such regions, the majority of affected children die, undiagnosed, before the age of 5 years. A rapid and affordable point-of-care test for SCD is needed. The diagnostic accuracy of HemoTypeSC, a point-of-care immunoassay, for SCD was evaluated in individuals who had SCD, hemoglobin C disease, the related carrier (trait) states, or a normal hemoglobin phenotype. Children and adults participated in low-, medium- and high-resource environments (Ghana [n = 383], Martinique [n = 46], and USA [n = 158]). Paired blood specimens were obtained for HemoTypeSC and a reference diagnostic assay. HemoTypeSC testing was performed at the site of blood collection, and the reference test was performed in a laboratory at each site. In 587 participants, across all study sites, HemoTypeSC had an overall sensitivity of 99.5% and specificity of 99.9% across all hemoglobin phenotypes. The test had 100% sensitivity and specificity for sickle cell anemia. Sensitivity and specificity for detection of normal and trait states were >99%. HemoTypeSC is an inexpensive (<$2 per test), accurate, and rapid point-of-care test that can be used in resource-limited regions with a high prevalence of SCD to provide timely diagnosis and support newborn screening programs., (© 2018 Wiley Periodicals, Inc.)

Gram-negative bacteria (GNB) that express CTX-M β-lactamases have become a serious threat to the clinical management of GNB infections. While antibody-based platforms have been successfully used in research settings to study and detect other β-lactamases-including SHV, CMY, and TEM enzymes-there is currently a lack of antibody-based tools to detect the CTX-M enzymes. Here we describe the development of an anti-CTX-M sandwich ELISA based on a pair of monoclonal antibodies (mAbs)-mAb 6101-33 and mAb 6101-19-used as the capture and detection antibody, respectively. This antibody pair detected CTX-M variants from group 1 (CTX-M-15), group 2 (CTX-M-2), group 8 (CTX-M-8), and group 9 (CTX-M-14) that were expressed by a training set of clinical GNB isolates. The limit of detection for this sandwich ELISA was 30ng of recombinant CTX-M-15, and CTX-Ms expressed by 10 6 lysed CFU of GNB. When tested against a blinded panel of 78 clinical isolates, the sandwich ELISA demonstrated a sensitivity of 96% and a specificity of 100%. The mAb pair did not cross-react with bacteria that contained other β-lactamases, including TEM, SHV, OXA, KPC, NDM, CMY, and DHA. In conclusion, we developed a highly sensitive and specific sandwich ELISA, capable of detecting CTX-M enzyme production in GNB pathogens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sickle cell disease (SCD) is a significant healthcare burden worldwide, but most affected individuals reside in low-resource areas where access to diagnostic testing may be limited. We developed and validated a rapid, inexpensive, disposable diagnostic test, the HemoTypeSC ™ , based on novel monoclonal antibodies (MAbs) that differentiate normal adult haemoglobin (Hb A), sickle haemoglobin (Hb S) and haemoglobin C (Hb C). In competitive enzyme-linked immunosorbent assays, each MAb bound only its target with <0·1% cross-reactivity. With the HemoTypeSC ™ test procedure, the sensitivity for each variant was <5·0 g/l. The accuracy of HemoTypeSC ™ was evaluated on 100 whole blood samples from individuals with common relevant haemoglobin phenotypes, including normal (Hb AA, N = 20), carrier or trait (Hb AS, N = 22; Hb AC, N = 20), SCD (Hb SS, N = 22; Hb SC, N = 13), and Hb C disease (Hb CC, N = 3). The correct haemoglobin phenotype was identified in 100% of these samples. The accuracy of the test was not affected by Hb F (0-94·8% of total Hb) or Hb A 2 (0-5·6% of total Hb). HemoTypeSC ™ requires <1 μl of whole blood and no instruments or power sources. The total time-to-result is <20 min. HemoTypeSC ™ may be a practical solution for point-of-care testing for SCD and carrier status in low-resource settings., (© 2016 John Wiley & Sons Ltd.)

Urinary tract infections (UTIs) are frequently encountered in clinical practice and most commonly caused by Escherichia coli and other Gram-negative uropathogens. We tested RapidBac, a rapid immunoassay for bacteriuria developed by Silver Lake Research Corporation (SLRC), compared with standard bacterial culture using 966 clean-catch urine specimens submitted to a clinical microbiology laboratory in an urban academic medical center. RapidBac was performed in accordance with instructions, providing a positive or negative result in 20 min. RapidBac identified as positive 245/285 (sensitivity 86%) samples with significant bacteriuria, defined as the presence of a Gram-negative uropathogen or Staphylococcus saprophyticus at ≥10(3) CFU/ml. The sensitivities for Gram-negative bacteriuria at ≥10(4) CFU/ml and ≥10(5) CFU/ml were 96% and 99%, respectively. The specificity of the test, detecting the absence of significant bacteriuria, was 94%. The sensitivity and specificity of RapidBac were similar on samples from inpatient and outpatient settings, from male and female patients, and across age groups from 18 to 89 years old, although specificity was higher in men (100%) compared with that in women (92%). The RapidBac test for bacteriuria may be effective as an aid in the point-of-care diagnosis of UTIs especially in emergency and primary care settings., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Some in vitro-generated allospecific T-cell clones can kill target cells bearing specific antigen, whereas others can only proliferate in response to that antigen. The mechanism of target lysis by clones that exhibit antigen-specific cytotoxicity is thought to involve the exocytosis of lytic granules, which contain the pore-forming protein perforin. Here, CD4+, CD8+, and CD4-8- T-cell clones, positive for CD3 and the alpha/beta T-cell receptor, were tested for their ability to lyse the mouse-anti-human CD3 hybridoma OKT3; this hybridoma has been shown to trigger the cytolytic mechanism in cytotoxic T cells regardless of their clonal specificity. We found that all in vitro-generated allospecific T-cell clones can efficiently lyse the OKT3 targets whether or not they can kill alloantigen-bearing lymphoblastoid B-cell line targets. Furthermore, all tested clones contained perforin. The OKT3 hybridoma was not lysed by perforin-negative, CD3+ leukemic T-cell lines or by CD3- NK clones. Thus, the presence of perforin in T-cell clones correlated with their ability to lyse OKT3 targets, but not with their ability to lyse alloantigen-bearing targets. These results demonstrate that T-cell clones that are nonlytic when activated by specific antigen nevertheless contain a complete lytic mechanism and also support the proposed central role in perforin in that mechanism.

Antigen-specific cytotoxic killer lymphocytes (CTLs) represent one of the major effector functions of the immune system. It is well established that, as a consequence of TCR recognition of the antigen-bearing target cell, resting T lymphocytes develop into fully active antigen-specific CTLs. In contrast, natural killer (NK) cells are immediately lytic upon contact with an appropriate target cell. The lytic machinery of CTLs and NK cells is thought to include the contents of their cytoplasmic granules, in particular the pore-forming protein perforin. Here we report direct cytolytic activity by resting peripheral CD3+CD8+ T cells as a result of TCR-CD3 binding to the target cell; the murine OKT3 hybridoma (anti-human CD3) was used as a target. The cytotoxicity was more pronounced in the CD8+CD45RO+ population, which contains 'memory' T cells, than in the reciprocal CD8+CD45RA+ subset; CD8+CD4- mature thymocytes were non-cytotoxic. The cytolytic potential of these populations correlated with the presence or absence of perforin. The results demonstrate that the cytolytic machinery of T cells develops post-thymically and can be immediately triggered by TCR-CD3 stimulation.

Perforin is a cytolytic protein of natural killer (NK) cells and cytotoxic T cells (CTL). Purified perforin has been shown to cause cell lysis and to form stable pores in the target cell membrane, but its relevance to cytolysis in vivo is not clear. The gene for human perforin has been cloned, but monoclonal antibodies (mabs) have not been available. In order to study further its role in cytotoxicity, we have generated mabs to different regions of human perforin. Four mabs were produced from mice immunized with hybrid proteins comprising E. coli TrpE protein at the N-terminus and different regions of human perforin at the C-terminus. These proteins were made using the pATH expression plasmids into which fragments of perforin cDNA were subcloned. Monoclonal antibody PA1 was made from a mouse immunized with a hybrid protein containing the C-terminal 240 amino acids (AA) of perforin, PE1 - the N-terminal 118 AA, and PB1 and PB2 - the central 199 AA. The three plasmid constructs contained non-overlapping cDNA segments which covered the entire sequence of perforin. All mabs reacted with the immunizing hybrid protein, but not with the other hybrid proteins, indicating that at least three epitopes are recognized by this set of mabs. All mabs immunoprecipitated a molecule of about 68 kd from lysates of metabolically-labelled cytolytic large granular lymphocytic leukemia cells, but not from control lysates of non-cytolytic promyelocytic U937 cells. These mabs should be of use in determining structure-function relationships for perforin.