Potential of Membranes Surrounding the Fetus as Immunoprotective Cell-Carriers for Allogeneic Transplantations.

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.

Point-of-care screening for sickle cell disease in low-resource settings: A multi-center evaluation of HemoTypeSC, a novel rapid test.

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.

Monoclonal antibody-mediated detection of CTX-M ß-lactamases in Gram-negative bacteria.

Gram-negative bacteria (GNB) that express CTX-M ß-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 ß-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 106 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 ß-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.

A rapid, inexpensive and disposable point-of-care blood test for sickle cell disease using novel, highly specific monoclonal antibodies.

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 A2 (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.

Performance of a New Rapid Immunoassay Test Kit for Point-of-Care Diagnosis of Significant Bacteriuria.

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.