Reactivity of microhemagglutination, fluorescent treponemal antibody absorption, Venereal Disease Research Laboratory, and rapid plasma reagin tests in primary syphilis.

Seroreactivity of sera from 109 patients with first-infection primary syphilis was 98.2% in the fluorescent treponemal antibody absorption test, 92.7% in the rapid plasma reagin 18-mm circle card test, 72.5% in the microhemagglutination test (MHA-TP), and 72.5% in the Venereal Disease Research Laboratory test. Seroreactivity of sera from 18 patients with primary syphilis with documented previous infection(s) was 100% in the fluorescent treponemal antibody absorption test, the rapid plasma reagin 18-mm circle card test, and the MHA-TP test and 88.9% in the Venereal Disease Research Laboratory test. The MHA-TP test failed to confirm reactivity in 13 of 79 sera which were reactive in the Venereal Disease Research Laboratory test and in 24 of 101 sera which were reactive in the rapid plasma reagin 18-mm circle card test. Testing another production lot of MHA-TP reagents resulted in even poorer correlation. The reactivity of the MHA-TP test in primary syphilis appeared to vary with the sensitivity of the production lot of reagents.     

Cell-mediated immune responses between HLA-identical siblings: recognition of antigenic changes associated with acute myelogenous leukaemia.

Cell-mediated immune reactions between a patient suffering from acute myelogenous leukaemia (AML) and an HLA-identical sibling were studied in order to characterize the in vitro reactions in MLC and CML prior to bone marrow transplantation. Our results indicated that antigenic differences were detectable between the blasts and the remission lymphocytes. While the normal sibling did not respond in MLC to her HLA-identical sister's remission lymphocytes, there was an anti-blast response. This proliferative response, however, did not lead to the development of detectable cytotoxic cells capable of destroying blast cells. Unrelated individuals, on the other hand, responded strongly both in MLC and CML to the allogeneic tumour blasts and remission lymphocytes of the patient and the lymphocytes of the healthy sibling. The kinetics and magnitude of the MLC response to blast cells was different from that to remission lymphocytes. This response indicated that the blast cells expressed antigenic differences which were recognized in MLC by both the HLA-identical sibling and unrelated individuals. Furthermore, these tumour cells were capable of sensitizing allogeneic, but not syngeneic lymphocytes to become cytotoxic, though they seemed to be more resistant to destruction in CML than normal cells.     

A simple rosette assay for demonstration of complement receptor sites using complement-coated zymosan beads.

In the present article we describe a simple rosette assay for detection of C3 receptor-bearing B lymphocytes with complement (C)-coated zymosan (Zy) beads. Zy coated with murine or human C bound to a distinct population of human and mouse lymphocytes as well as to the majority of lymphoblastoid cells of several human established cell lines. Rosette formation was also observed with human red blood cells, with human monocytes and neutrophils. Experiments with anti-immunoglobulin sera, with other B and T cell markers and with mouse thymocytes proved that the capacity to bind C-coated Zy is primarily a feature of B lymphocytes. The following findings suggested that C-coated Zy is bound via receptor sites for the activated components of C3: (a) Zy coated with C3-deficient serum failed to bind, (b) comparable percentages of various lymphoid and nonlymphoid cells formed rosettes with C-coated Zy as well as with antibody and C-coated sheep red blood cells, and (c) antibodies against human or murine C3 inhibited the binding of C-coated Zy.     

Effect of delayed specimen processing on cytomegalovirus antigenemia test results.

Blood samples held at either 4 degrees C or room temperature for 1 day had similar mean decreases in number of cytomegalovirus antigenemia-positive cells (52 to 55%) and similar false-negative test results (13 to 14%). After 2 days, samples held at 4 degrees C showed no further decline, whereas samples held at room temperature had a mean 81% decrease in positive cells, a 32% false-negative rate, and a more marked deterioration in cell morphology.     

Coxsackievirus B-3 myocarditis in Balb/c mice. Evidence for autoimmunity to myocyte antigens.

Male Balb/c mice inoculated with a heart-adapted variant of Coxsackievirus, group B, type 3 (Nancy) (CVB3M), develop extensive myocarditis and cytolytic activity to primary cultures of uninfected and infected myocytes. To elucidate the mechanisms of myocyte injury in myocarditis, two distinct cytolytic T-lymphocyte (CTL) populations were isolated by immunoadsorption of lymph node cells to glutaraldehyde-fixed uninfected and infected myocyte monolayers. One population preferentially adsorbed to and lysed uninfected myocytes (autoreactive CTLs), while the other adsorbed to and lysed CVB3M-infected myocytes (virus-specific CTL). Neither CTL population adsorbed to monolayers of HeLa, L929, or umbilical cord endothelial cells, or to myocytes infected with a related but nonmyocarditic Coxsackievirus B-3 variant ( CVB3o ). While both autoreactive and virus-specific CTLs induced myocarditis in vivo, the lesions caused by autoreactive CTLs were more extensive and necrotizing than those of virus-specific cells. These results support the hypothesis that CVB3 -induced myocarditis results, in part, from autoimmunity to myocyte antigens.     

Threshold pressure for the pressure-dependent renin release in the autoregulating kidney of conscious dogs

1. The effect of varying renal artery pressure between 160 and 40 mm Hg on renal blood flow and renin release was studied in seven conscious foxhounds under β-adrenergic blockade receiving a normal sodium diet (4.1 mmol/kg/day). Pressure was either increased by bilateral common carotid occlusion or reduced in steps and maintained constant by a control-system using an inflatable renal artery cuff. Carotid occlusion itself had no influence on renal blood flow and renin release when renal artery pressure was kept constant and the β-receptors in the kidney were blocked.
2. Between 160 mm Hg and resting pressure there was no change in renal blood flow; between resting blood pressure and the lower limit of autoregulation (average 63.9 mm Hg) renal blood flow increased slightly (average 7%) indicating a high efficiency of renal blood flow autoregulation.
3. The relationship between renal artery pressure and renin release could be approximated by two linear sections:a low sensitivity to a pressure change (average slope: ?0.69 ±0.26ng AI/min/mm Hg) was found above a threshold pressure (average: 89.8±3.3 mm Hg) and a high sensitivity to a pressure change (average slope: ?64.4±20.8 ng AI/ min/mm Hg) was observed between threshold pressure and 60 mm Hg. There was no further increase of renin release between 60 and 40 mm Hg.
4. It is concluded that within the autoregulatory plateau the kidney of a conscious β-blocked dog receiving a normal sodium diet releases only negligible amounts of renin until renal artery pressure falls below a threshold pressure of 90 mm Hg which is close to the animals resting systemic pressure. Since beyond that a decrease of systemic pressure by as little as 1.3 mm Hg below threshold can raise resting renin release (84.8±29.8 ng/min) by 100%, it is suggested that systemic blood pressure tends to stabilize at a level at which renin release is minimal.

     

Functional folding of a cytoplasmic single-chain variable fragment and its use as elutable protein purification tag

The variable fragment (Fv) of the monoclonal B1-8 antibody recognizes 3-nitro-4-hydroxy-phenylacetate (NP) and 5-iodo-NP (NIP) allowing for the affinity purification of the respective B cell antigen receptor with NP-sepharose and its specific elution with NIP-capronic acid (NIPcap). We generated an intracellular single-chain B1-8 Fv (iscFv), fused it to the N-terminus of the regulatory subunit (p85alpha) of phosphatidylinositol-3-kinase (PI3K) (isc-p85alpha), and examined the potential of this iscFv to serve as an intracellular elutable protein purification tag. The isc-p85alpha fusion protein could be specifically affinity-purified from the lysates of transfected Drosophila S2 cells with NP-sepharose and eluted with NIPcap, indicating the functional folding of the iscFv in the reducing environment of the cytosol. Furthermore, co-purification of the catalytic subunit of PI3K (p110) was achieved from lysates of co-transfected S2 cells as well as RBL-2H3 mast cells stably expressing isc-p85alpha. This indicates that the iscFv part of isc-p85alpha does not negatively influence p85alpha folding and interaction with p110. Moreover, successful incorporation of the p85alpha-moiety of isc-p85alpha into endogenous protein complexes in mast cells suggests the use of isc-containing fusion proteins for the native purification, elution, and analysis of intracellular signaling complexes.     

CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein

A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera. Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses. The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls. The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the respective antigens. Due to these limitations and despite the potential clinical relevance large-scale autoantibody tests are established for only a few of these tumor antigens. Here we describe an enzyme-linked immunosorbent assay, Crude lysate ELISA (CrELISA), suitable for antigens identified by expression screening based on crude lysates of antigen-expressing bacteria. This assay permits sensitive and specific autoantibody seroscreening without the need of laborious and time-consuming cloning, expression and purification of recombinant proteins. CrELISA is robust and provides a versatile high throughput procedure for the rapid evaluation of multiple antigens in large-scale serology.     

Lymphoid cells in afferent and efferent intestinal lymph: lymphocyte subpopulations and cell migration.

Gut wall emigrating cells have been characterized in the intestinal lymph. The intestinal lymph duct was cannulated in 6-month-old minipigs. Under non-restraining conditions the efferent lymph from the mesenteric lymph nodes was collected in seven normal animals. Lymph coming directly from the gut (afferent lymph) was also collected in 18 pigs after resection of the mesenteric lymph node chains 3 months previously. The intestinal lymph flow was similar in both groups (around 18 ml/h). The lymphoid cell yield was 1.2 +/- 1.0 x 10(6)/h in control animals, while in mesenteric lymph node resected pigs it was around 20 times higher (26.2 +/- 17.6 x 10(6)/h). In the gut-derived lymph 76.5 +/- 8.8% T lymphocytes were observed (CD4+, 48.1 +/- 15.5%; CD8+, 53.6 +/- 12.7%). The percentage of immunoglobulin-positive cells was lower (IgM+, 10.1 +/- 4.5; IgA+, 1.7 +/- 1.1). In 14 mesenteric lymph node resected pigs a mean of 5.6 +/- 3.1 x 10(8) lymphocytes from the gut lymph were labelled in vitro with a fluorescent dye and retransfused. The labelling index of fluorescent cells in the intestinal lymph increased rapidly and remained at a high level until 44 h after cell transfusion. A four-to-ten times lower labelling index was found in the spleen, various lymph nodes and Peyer's patches. Most of the recovered lymphocytes were T cells. This model provides access to the cell pool leaving the gut wall, thus allowing an examination of its role in the gastrointestinal tract and other mucosal-lined organs.     

X-linked mixed deafness (DFN3): cloning and characterization of the critical region allows the identification of novel microdeletions

We have found that the microsatellite marker AFM207zg5 (DXS995)maps to all previously described deletions which are associatedwith X-linked mixed deafness (DFN3) with or without choroideremiaand mental retardation. Employing this marker and pHU16 (DXS26)we have identified two partially overlapping yeast artificialchromosome clones which were used to construct a complete 850kb cosmid contig. Cosmids from this contig have been testedby Southern blot analysis on DNA from 16 unrelated males withX-linked deafness. Two novel microdeletions were detected inpatients which exhibit the characteristic DFN3 phenotype. Bothdeletions are completely contained within one of the known DFN3-deletions,but one of them does not overlap with two previously describeddeletions in patients with contiguous gene syndromes consistingof DFN3, chorolderemia, and mental retardation. Assuming thatonly a single gene is involved, this suggests that the DFN3gene spans a chromosomal region of at least 400 kb.