Rosette Technique Research Articles

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Isolation of T Cells Using Rosetting Procedures.

This unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes, and macrophages). Neuraminidase- and 2-aminoethylisothiouronium bromide (AET)-treated SRBC are used for rosetting because of enhanced binding to T cells. It should be noted that use of the rosetting technique to obtain purified T cells or purified non-T cells by negative selection has largely been superceded by other techniques such as panning and immunomagnetic beads.

Real-Time Weld Zone Strain/Slope Measurements Using the Interferometric Strain/Slope Rosette Technique

A laser-based optical technique, the Interferometric Strain/Slope Rosette (ISSR) technique, was used to measure real-time deformations in welding heat-affected zones. This was accomplished by measuring simultaneously real-time in-plane strains, out-of-plane slopes and weld plate temperature from both carbon and stainless steel plates. All strain/slope results demonstrated a strong sensitivity to such welding parameters as temperature, weld length and heat input. Accuracy of the measurements was studied. The strain/slope responses were also proven to be predictable, with measured strains comparable to released strains measured by resistance strain rosette/hole-drilling and ISSR/ring-core cutting.

Study of structural defects in ZnGeP2crystals by X-ray topography based on the Borrmann effect

The first study of structural defects in a ZnGeP2semiconducting nonlinear optical crystal has been carried out by X-ray topography, based on the Borrmann effect, and the effect of anomalous transmission of X-rays on ZnGeP2crystals has been examined. It is shown that the rosette technique of defect study under conditions of the Borrmann effect, developed earlier for elementary semiconductors, can be applied to the study and identification of defects in ZnGeP2. Features of contrast from individual edge and screw dislocations, microdefects, and coherent and semi-coherent microinclusions were considered. Defect identification was carried out by comparison of the experimental intensity contrast with simulated images of the defects.

E rosette-forming lymphocytes in multiple sclerosis patients. Basic protein stimulation of rosette-forming cells

"Total" and "active" T cells were enumerated using sheep red blood cell (SRBC) rosetting technique. The percentages of "total" and "active" T lymphocytes were not significantly different in multiple sclerosis patients as compared to other neurological disorders and/or healthy controls. However, the number of "avid" T cells binding more than three SRBC was significantly increased in MS patients. Active E rosette test has shown hypersensitization to myelin basic protein in 50% MS patients and 31% OND patients. All healthy controls studied, except one had negative response.

Lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in multiple sclerosis.

Lymphocytes subpopulations in cerebro-spinal fluid (CSF) and peripheral blood (PB) from multiple sclerosis (MS) patients were studied. PB of MS patients contains the same prevalence of E and EA rosette forming cells compared with controls, consisting of patients affected by various "nonimmunological" neuropsychiatric diseases. Cytochemical identification by the method of acid esterases in PB demonstrated in MS a prevalence of lymphocyte subpopulations similar to controls, and a relatively high percentage of macrophages compared with other methods, especially in MS patients: this may partially account for variable results obtained by various authors with the rosette technique. In CSF a significant decrease of total T, and particularly of T gamma cells, was found. Since T gamma lymphocytes have a suppressor effect on B cell proliferation and Ig synthesis, their decrease could be related with Ig hypersynthesis commonly found in the central nervous system of MS patients.

Monocyte heterogeneity in angiotensin-converting enzyme induction mediated by autologous T lymphocytes.

The ability of monocyte subpopulations to be induced selectively by T lymphocytes to synthesize enhanced levels of angiotensin-converting enzyme (ACE) was examined using an in vitro model employing normal peripheral blood monocytes and T lymphocytes. Separation of monocytes into subpopulations on the basis of buoyant density indicated no difference in the ability of the resulting monocyte subpopulations to produce basal levels of ACE when cultured in the absence of T lymphocytes. However, the subpopulations differed significantly in their ability to synthesize enhanced levels of ACE in response to the presence of autologous T lymphocytes; low-density monocytes were induced by T lymphocytes to synthesize three-fold more ACE than were high-density monocytes. Surface antigen labelling using MoAbs demonstrated that the low-density monocyte subpopulations also had a significantly higher percentage of Leu-M2+ monocytes compared with the high-density monocyte subpopulations. When monocytes were separated on the basis of the presence of the Leu-M2 antigen using an immune rosetting technique, T lymphocytes were able to induce significantly elevated levels of ACE in the Leu-M2+ enriched monocyte subpopulation but were unable to induce ACE beyond basal levels in the Leu-M2(+)-depleted monocyte subpopulation. These results demonstrate that monocytes are heterogeneous with respect to their ability to be induced by T lymphocytes to synthesize ACE. This raises the possibility that selective accumulation of a monocyte subpopulation in the granulomatous inflammation of sarcoidosis may be one of the factors required for elevated ACE synthesis in the resulting granuloma epithelioid cells.

A rapid rosetting method for separation of hemocyte sub-populations of Drosophila melanogaster

Hemocytes, cellular elements of the innate immune system in insects, play a crucial role in the cellular and humoral immune response. Although a significant amount of information has been collected on their differentiation and function, our understanding of hemocyte development is far from complete. Their characterisation is mostly based on morphological criteria. However, molecular markers were recently identified, defining functional subsets by the aid of monoclonal antibodies. Isolated subsets of hemocytes, in sufficient quantity and purity could help to analyse their development in vitro and also to further define their molecular characteristics. Here we describe an antibody-based rosetting technique for the physical separation of Drosophila hemocyte sub-populations. We have applied anti-hemocyte antibodies coupled to sheep red blood cells for separation. The method relies on the formation of rosettes between hemocytes and sheep erythrocytes, sensitised with discriminative anti-hemocyte monoclonal antibodies. Using this method the rosetting and non-rosetting hemocytes can be separated from each other by gradient centrifugation. Rosette-forming cells from the pellet and non-rosetting cells from the interface can be isolated in high recovery. The method can be used for functional and molecular characterisation of hemocyte sub-populations. The procedure is sensitive, reproducible and easy to perform.

Using the Rosette Technique for Canada Thistle (Cirsium arvense) Control in Row Crops

Clopyralid applied to Canada thistle rosettes has provided better control in the following growing season than applications to bolted plants. The objectives of this research were to determine if using cultivation to prevent plants from bolting prior to herbicide application (the rosette technique) could be successfully incorporated into a row crop production system and to evaluate the effect of Canada thistle growth stage on the absorption and translocation of14C-clopyralid. Canada thistle control 8 mo after postharvest herbicide treatment (MAFT) using the rosette technique was similar to control when using conventional in-crop plus postharvest herbicide treatments in corn and soybean. Glyphosate and clopyralid plus 2,4-D were the most consistent postharvest herbicide treatments for Canada thistle control 8 MAFT in corn and soybean. Corn yields were similar, but soybean yields were slightly lower when Canada thistle was controlled using cultivation compared to conventional herbicide treatments.14C-clopyralid translocation to Canada thistle roots and lower shoot parts was greater when clopyralid was applied to the rosette stage than when applied to bolted Canada thistle plants. The increased translocation probably accounts for the increased Canada thistle control observed in the field. Incorporating the rosette technique into a weed management program should allow growers to control Canada thistle with less herbicide input than do standard practices.

Application of interferometric strain rosette to residual stress measurements

An interferometric strain rosette (ISR) technique is extended to residual stress measurements. The ISR technique is based on diffraction and interference of laser light reflected from three micro-indentations depressed in a specimen surface. Three in-plane strain components between the three micro-indentations can be measured simultaneously. Therefore the ISR enables a determination of two normal and one shear strain components. For many applications, the ISR is superior to a resistance strain rosette due to its short gage length and non-contacting nature. By applying an ISR to a material surface, residual stresses at the location of the ISR can be obtained through measurement of residual strains relieved via hole-drilling. Since the gage length can be as short as 50 μm, the ISR is capable of recording high strain gradients and it allows the strains close to the hole to be measured. The size of the hole can be small and precise location is not required. Since the ISR technique is non-contacting, it may be used to measure residual stresses in hostile environments.

The interferometric strain rosette technique

An interferometric strain rosette can be used to measure three in-plane strains. The strain rosette consists of three microindentations produced on an object surface. Upon illuminating the indentations with laser light, each pair of indentations acts as a two-point source generating a pair of Young's interference fringe patterns. When the object is deformed, the distance between the indentations is altered. By measuring the change of spacing of the Young's fringes, the strain in the direction of the separation of the indentations is measured. Three indentations are at the vertexes of an equilateral triangle to constitute a strain rosette. Each pair of the microindentations enables measurement of an axial strain in the indentation separation. The rosette measures simultaneously three in-plane strains in the directions of the triangular sides. As three in-plane strains are measured, the in-plane shear and two normal strains can be found. Compared with a resistance strain rosette, the interferometric strain rosette has great features such as non-contacting and a short gage length. In addition, the interferometric strain rosette can measure large elastoplastic strains and is applicable to measurements at elevated temperatures. The theory with experimental evaluation is presented. Measurement sensitivity of the technique is discussed. Potential applications and limitations of the technique are to be described as well.

Modular Tibial Augmentations in Total Knee Arthroplasty

Proximal tibial bony deficiencies are not uncommon in primary and revision total knee arthroplasty. Modular tibial augmentations were introduced to address these deficiencies. Alterations in strain distribution as a result of medial wedge and block augmentations were evaluated for a modular total knee arthroplasty system in 6 fresh frozen anatomic specimen tibias. Full-field strain patterns were examined using photoelastic coating methods, and high strain regions were evaluated using strain gage rosette techniques. The total knee arthroplasty installations were tested in static physiologic axial and torsional load configurations. The relative effects of sequential wedge and block augmentations compared with the nonaugmented case were statistically analyzed. There were no overall statistical differences in the 3 treatments in terms of maximal (principal) strains. A secondary analysis that evaluated specific location and load pattern combinations established several minor statistical differences along with insights into the manner in which each construct loads the proximal tibia. Although metal wedge augmentation commonly is used, block augmentation seems to be an appropriate alternative from a strain distribution standpoint in cases in which the block geometry better approximates the bony defect.

Fetal erythroblasts from maternal blood identified with 2,3-bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y-specific probes.

Different types of fetal nucleated cells can be found in maternal blood, providing the possibility of non-invasive prenatal diagnosis. For this purpose, we have studied fetal erythroblasts. We discovered that haemoglobin-containing cells treated with 2,3-bisphosphoglycerate (BPG) can be visualized by a peroxidase reaction, which at the same time visualizes an in situ hybridization (ISH) signal, specific for the X, Y or 21 chromosome. In order to prove that the BPG-positive cells were erythroid, an anti-glycophorin A (GPA) antiserum combined with a staphylococcal rosette technique was used. To enrich for erythroblasts, leukocytes were depleted from maternal blood by treatment with anti-CD45 monoclonal antibody and passage over an anti-mouse IgG-coated glass bead column. To evaluate the potential of the method for clinical use, we studied maternal blood samples from 18 women referred to us for prenatal diagnosis between 6 and 19 weeks of gestation. Erythroblasts were found in 13 out of 14 normal pregnancies. Erythroblasts with a Y-signal were found as early as 9 weeks of gestation, but at 6 weeks the Y-signal was seen in BPG-negative cells only. These cells showed an epithelioid morphology indicating that they were cytotrophoblasts. The BPG-ISH method provides a simple technique for identifying erythroblasts and simultaneously visualizing a desired probe.

Histamine receptors on bovine peripheral blood lymphocytes.

Histamine receptors on bovine peripheral blood lymphocytes (PBL) were detected by three different methods: a rosetting technique, binding to histamine-bearing Sepharose beads and immunofluorescence staining. The rosetting technique used histamine-rabbit serum albumin (H-RSA) conjugated to bovine red blood cells to detect histamine receptors and this showed that 10.8% of bovine PBL were positive. A method using H-RSA conjugate coupled Sepharose beads also detected histamine receptor bearing PBL but was not quantitative. The indirect immunofluorescence method, by which the subpopulation of histamine receptor bearing lymphocytes can be easily double stained to concurrently identify the B cell marker, revealed that PBL, the B cell and T cell fraction of bovine PBL contained 18.4, 52.8 and 9.3% histamine receptor bearing cells, respectively. This method was found to be more stable and more easily quantifiable than the other two methods. Blocking tests using the histamine H1 receptor antagonist diphenhydramine and the histamine H2 receptor antagonist cimetidine suggested that bovine PBL have both H1 and H2 receptors on their surfaces. Addition of histamine into cultures of PBL at the concentration range 10(-6) to 10(-3) M suppressed the response of PBL to the mitogen phytohemagglutinin. The histamine induced suppression of mitogenesis could be reduced partially by the H2 receptor antagonist cimetidine, but not by the H1 antagonist diphenhydramine. It is possible that histamine induced suppression of PBL mitogenesis was mediated by H2 receptors on T cells.

Regulation of VCAM-1 Expression and Involvement in Cell Adhesion to Murine Microvascular Endothelium

The present studies were undertaken to examine the regulation of murine VCAM-1 expression and the involvement of this molecule in adhesive processes occurring on the surface of microvascular endothelium. Flow cytometric analyses revealed that murine microvascular endothelium (MME) in culture constitutively expresses VCAM-1 and that stimulation of MME by TNF, IL-1, or LPS, but not by PMA or staurosporine, strongly increased the surface expression of this cell adhesion molecule. Stimulation of VCAM-1 expression by TNF may be diminished by ionomycin as well as by inhibitors of protein kinases (H-7 and sangivamycin). However, TGF-β, which strongly inhibited the adhesiveness of endothelium, had little effect on the expression of VCAM-1. A newly developed adhesion assay, based on the rosette technique, allowed us to distinguish between the adhesive properties of an individual endothelial cell and those of endothelial cell monolayers and demonstrated that inhibition of binding by TGF-β resulted primarily from its influence on the adhesive properties of individual cells. Studies on the inhibition of cell binding by monoclonal antibodies against mouse VCAM-1 and mouse VLA-4 indicated that VCAM-1 plays a dominant role in mediating the adherence of a variety of cell types, including murine splenocytes and thymocytes, P815 mastocytoma cells, PT 18 mast/basophil cells, human Molt-4 cells, and human eosinophils, to cytokine-activated MME.

A flow cytometric rosetting assay for the analysis of Fc receptors and C3 receptors on HSV-infected cells

A sensitive and reproducible flow cytometric assay was developed for the analysis of Fcγ and C3b(i) receptors on HSV-infected cells. The method is based on a rosette technique using fluorochrome-labeled erythrocytes sensitized with IgG or C3b(i). A comparison of flow cytometric and microscopic quantitation demonstrated that the binding of EIgG, EC3b(i) to HSV-infected cells were correlated. Flow cytometric analysis provides the opportunity to study simultaneously the distribution of E per HSV-infected cell and the total binding of E to the whole population of HSV-infected cells. Receptor activity and HSV glycoprotein cell surface expression were shown to be correlated in a linear fashion. The assay could be applied to other FcγR- and C3b(i)R-bearing cells.

Chorionic villus sampling for fetal Rh typing: Clinical implications

The prenatal Rh typing of red blood cells obtained by chorionic villus sampling was performed with an immune rosette technique. Ten Rh-negative pregnant women undergoing chorionic villus sampling at 9 to 11 weeks' gestation were studied at a large referral hospital. Accurate Rh phenotyping may be done on red blood cells obtained from chorionic villi weighing 2 to 8 mg. The preparations were shown to be greater than 90% fetal in origin as demonstrated by radial immunodiffusion quantitation of fetal hemoglobin-containing cells. Of the 10 fetuses typed in the first trimester nine of the pregnancies were carried to term. In all cases typing of red blood cells confirmed the antenatal fetal red cell typing. This study shows that antenatal Rh phenotyping may be performed as early as 9 to 11 weeks' gestation. This technique may be used in the management of pregnancies complicated by Rh incompatibility.

Interaction between rat peritoneal macrophages and sensitised erythrocytes: Dependence on IgG subclass, antibody density and the degree of hapten conjugation

The interaction between macrophages (MØ) and antibody sensitised target cells was studied by the use of rat peritoneal macrophages, TNP hapten conjugated sheep red blood cells (SRBC) and homologous antibodies of subclasses IgG1 and IgG2a. Under optimal conditions, the great majority of the MØ formed rosettes with IgG1-sensitised antibodies while a maximum of 50% was achieved when target cells were sensitised by IgG2a. Using a double rosette technique, the major part of rosette-forming cells was found to bind both of the isotypes. IgG1-mediated rosette formation was observed at very low degrees of sensitisation as opposed to IgG2a-mediated target cell binding. Not only the amount of bound antibody but also the degree of hapten conjugation (epitope density) appear to influence the ratio of rosette-forming cells. IgG1-mediated rosette formation was partially inhibited by monomeric IgG1 and more efficiently by soluble ovalbumin (OVA)-anti-OVA complexes involving IgG1-type antibodies, while IgG2a mediated rosette formation was inhibited by OVA-anti-OVA complexes containing IgG2a type antibodies, and less efficiently by complexes involving IgG1. No inhibition was found by monomeric IgG2a. Based on the present data, we propose that two types of receptors are involved in the interaction of MØ and target cells coated by IgG1 and/or IgG2a type antibodies. One requires a multiple antibody-receptor interaction, binding both subclasses at overlapping binding sites; the other is able to interact with IgG1 and does not depend on the multiplicity of interactions.

Evidence of allograft related transplantation antigen binding cells for detection of cardiac allograft rejection—Preliminary report

In 8 heart transplant recipients in follow-up checks we have evaluated the binding of transplantation antigen loaded methacrylate-carrier to peripheral mononuclear cells (rosette technique). All patients were treated with a triple-drug regimen for immunosuppression, consisting of steroids, azathioprine and cyclosporine A. The increase of the number of these antigen binding cells over a limit value of 30 RFC/103 MNC is a reliable sign of a beginning immune reaction against the graft. The evidence is given by comparing the results with these of situation in endomyocardial biopsies (EMB). The test is predictive 3 to 6 days before infiltrating cells are visible in biopsies and donor independent (use of antigen mixture from cadaver spleens).

Induction of HLA-DR Expression in Endometrial Epithelial Cells by Endometrial T-Cells: Potential Regulatory Role of Endometrial T-Cellsin Vivo

Endometrial epithelial cells express HLA-DR molecules of the major histocompatibility complex in vivo adjacent to aggregates of T-cells in the endometrial stroma. To test whether HLA-DR expression on endometrial epithelium is mediated by T-cells, endometrial T-cells were isolated from human endometrium by the sheep red blood cell rosetting technique. In contrast to the resting T-cells from peripheral blood and similar to the peripheral blood T-cells activated with Concanavalin-A, endometrial T-cells formed colonies in vitro. Direct addition of the endometrial T-cells to epithelial cell cultures derived from autologous glands induced both morphological changes as well as HLA-DR molecules in the epithelial cells. The intensity of the immunostained HLA-DR molecules in the epithelial cells as well as the percentages of the HLA-DR-positive epithelial cells correlated with the number of T-cells added to the epithelial cell cultures. T-Cells were bound to the HLA-DR-positive epithelial cells as single cells or aggregates, and they were not found bound to the HLA-DR-negative epithelial cells. The supernatant of the endometrial T-cells induced expression of HLA-DR molecules and morphological changes in cultures of HLA-DR-negative epithelial cells. This expression could be inhibited by a neutralizing antiserum to interferon-gamma. These findings are consistent with the hypothesis that endometrial T-cells are activated and suggest that the expression of HLA-DR molecules in glandular epithelium in vivo is mediated by the interferon-gamma secreted by the endometrial T-cells.