Cell adhesion molecule expression in the sheep thymus

Zhao, Ting C.; He, Chunbo; Su, Mei; West, Charles A.; Swanson, Scott J.; Young, Alan J.; Mentzer, Steven J.

doi:10.1016/s0145-305x(01)00006-4

Cited by 4 publications

(3 citation statements)

References 50 publications

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“…This distance measure can be expressed as a “dissimilarity index” with lower distance measures reflecting greater similarity and higher distance measures reflecting greater dissimilarity (Figure 4B). To validate the clustering result, antibodies recognizing the same target molecule—independently confirmed by biochemical and genetic studies (15,25)—were examined. Mab recognizing class I, LFA-1 and β1 integrin demonstrated high similarity indices (Figure 4B: yellow, green and red).…”

Section: Resultsmentioning

confidence: 99%

Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman species

Pratt¹,

Zeng²,

Ravnic³

et al. 2009

Cytometry Pt A

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Monoclonal antibodies (Mab) are an important resource for defining molecular expression and probing molecular function. The characterization of Mab reactivity patterns, however, can be costly and inefficient in nonhuman experimental systems. To develop a computational approach to the pattern analysis of Mab reactivity, we analyzed a panel of 128 Mab recognizing sheep antigens. Quantitative single parameter flow cytometry histograms were obtained from five cell types isolated from normal animals. The resulting 640 histograms were smoothed using a Gaussian kernel over a range of bandwidths. Histogram features were selected by SiZer-an analytic tool that identifies statistically significant features. The extracted histogram features were compared and grouped using hierarchical clustering. The validity of the clustering was indicated by the accurate pairing of externally verified molecular reactivity. We conclude that our computational algorithm is a potentially useful tool for both Mab classification and molecular taxonomy in nonhuman experimental systems. ' 2009 International Society for Advancement of CytometryKey terms other animals; antibodies; antigens/epitopes THE focus of the early human leukocyte differentiation antigens (HLDA) workshops was the use of monoclonal antibodies (Mab) and flow cytometry to identify new molecules (1-7). The workshops characterized the reactivity patterns of large panels of antibodies against a panel of cell populations. For practical purposes, most of the expression analyses were performed with widely available cell lines. Because multipassaged cell lines generally represented uniform cell populations, the results of the analyses were typically binary; that is, the antibody either reacted with the cell line or it did not. Although the histograms derived from cell lines were typically amenable to parametric analyses, the antibody reactivity was effectively summarized as the percentage of cells with fluorescence intensity greater than the negative control. This ''percent positive'' data were subjected to hierarchical clustering to identify ''clusters of differentiation'' or CD specificities (1). In human biology, the ability to identify the gene corresponding to the target antigen has rendered this workshop approach to identify new molecules obsolete. Mab, however, continue to be an important resource for defining molecular expression and probing molecular function.Despite the utility of Mab in biologic investigations, attempts to develop antibody panels in other species have had limited success. A practical problem is that the HLDA approach requires widely available cell lines-a resource that is uncommon in most species. An alternative approach is to use isolated cell populations, such as peripheral blood, thymocytes, and splenocytes. These cell populations, however, are nonuniform. Flow cytometry analyses with most Mab produce histograms that cannot be effectively clustered using ''percent positive'' data. Furthermore, the visual inspection method of pairwise comparison ...

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Section: Resultsmentioning

confidence: 99%

Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman species

Pratt¹,

Zeng²,

Ravnic³

et al. 2009

Cytometry Pt A

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“…No obstante, es importante destacar que si bien la mayor población leucocitaria se observó a este nivel, determinado mediante el contraste nuclear con hematoxilina, esta población no correspondió a células inmunomarcadas con la IHQ. Al respecto, Zhao et al (2001) descri-bieron en ovinos la presencia de pro-timocitos, que expresan CD3 + , marcador de superficie presente solo en linfocitos T, que mantienen esta positividad hasta su madurez; asimismo describieron los timocitos TN CD3 -, CD4y CD8que corresponden a una etapa muy inmadura de los timocitos, donde no expresan estos marcadores de superficie. Es así, que la población cortical observada, no inmunomarcada, correspondería a una población de timocitos en etapa inmadura.…”

Section: Discussionunclassified

Estudio histoquímico e inmunotipificación de poblaciones leucocitarias del timo de alpacas (Vicugna pacos) menores a 60 días de edad

Mamani

Andrade

Pitot

et al. 2019

Rev. investig. vet. Perú

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El objetivo del estudio fue identificar las poblaciones leucocitarias en el timo posnatal de alpacas menores de 60 días de edad. Se realizó la eutanasia a seis alpacas Huacaya en la provincia de Huanta, Ayacucho, Perú. Se obtuvieron muestras de timo que fueron embebidas en OTC y congeladas inmediatamente en nitrógeno líquido y conservadas a -70 ºC hasta su procesamiento. Se prepararon secciones congeladas y se fijaron a la lámina con el adhesivo organosilano. Se realizó la inmunohistoquímica con anticuerpo primario de ratón y secundarios de conejo. La reacción se evidenció empleando el complejo avidina-biotina-peroxidasa y se contra-coloreó con Hematoxilina. Se emplearon anticuerpos monoclonales específicos para la identificación de las subpoblaciones leucocíticas del timo; se evaluaron cinco campos visuales de forma aleatoria a un aumento de 400X. La distribución y densidad de la tinción se clasificó como negativa (-), inconstante (±), ligera (+), moderada (++) y fuerte (+++). Los resultados muestran que la mayor población de células leucocitarias se ubicó en la corteza del timo y correspondieron a células inmaduras que no expresan marcadores de superficie; así mismo, la mayor población de células inmunomarcadas, en maduración, se ubicaron en la médula del timo, predominando los linfocitos TCR αβ, con valores bajos de linfocitos TCR γδ, linfocitos CD8, linfocitos B y monocitos/macrófagos, con valores inconstantes de linfocitos CD4. En la corteza del timo se observaron valores bajos de linfocitos TCR αβ y linfocitos CD8, y valores inconstantes de monocitos/macrófagos, sin evidencias de linfocitos TCR γδ, linfocitos CD4 y linfocitos B.

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“…The monoclonal antibodies used in these experiments were murine monoclonal antibodies specific for the following specificities: CD4 (17D) (Mackay, 1988), CD8 (A51) (Ellis et al, 1986), macrophages (clone VPM64; Biosource, Camarillo, CA), CD11b (Du-87; Young, manuscript in preparation), CD40 (M2/61) (Zhao et al, 2001). …”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Dynamic deformation of migratory efferent lymph‐derived cells “trapped” in the inflammatory microcirculation

West

Young

et al. 2002

Journal Cellular Physiology

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The cellular immune response depends on the delivery of lymphocytes from the lymph node to the peripheral site of antigenic challenge. During their passage through the inflammatory microcirculaton, the migratory cells can become transiently immobilized or "trapped" in small caliber vessels. In this report, we used intravital microscopy and temporal area mapping to define the dynamic deformation of efferent lymph-derived mononuclear cells trapped in the systemic inflammatory microcirculation. Mononuclear cells obtained from the efferent lymph draining the oxazolone-stimulated microcirculation were labeled with fluorescent dye and reinjected into the feeding arterial circulation. Intravital video microscopy observed thousands of cells passing through the microcirculation; 35 cells were "trapped" in the oxazolone-stimulated microcirculation. Temporal area maps of the trapped cells demonstrated dramatic slowing and deformation. The cells were trapped in the microcirculation for a median of 8.90 sec (range 5-17 sec) prior to returning to the flow stream. During this period, the cells showed sustained movement associated with both antegrade locomotion (mean cell velocity = 7.92 microm/sec; range 1.16-14.23 microm/sec) and dynamic elongation (median cell length = 73.8 microm; range 58-144 microm). In contrast, efferent lymph-derived cells passing unimpeded through the microcirculation demonstrated rapid velocity (median velocity = 216 microm/sec) and spherical geometry (median diameter = 14.6 microm). Further, the membrane surface area of the "trapped" cells, calculated based on digital image morphometry and corrosion cast scanning electron microscopy, suggested that the fractional excess membrane of the cells in the efferent lymph was significantly greater than previous estimates of membrane excess. These data indicate that transient immobilization of efferent lymph-derived mononuclear cells in the systemic inflammatory microcirculation is rare. When "trapping" does occur, the shape changes and sustained cell movement facilitated by excess cell membrane may contribute to the return of the "trapped cells" into the flow stream.

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Cell adhesion molecule expression in the sheep thymus

Cited by 4 publications

References 50 publications

Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman species

Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman species

Estudio histoquímico e inmunotipificación de poblaciones leucocitarias del timo de alpacas (Vicugna pacos) menores a 60 días de edad

Dynamic deformation of migratory efferent lymph‐derived cells “trapped” in the inflammatory microcirculation

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