Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices.

Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin-fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEK WT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition.

Macrophage requirement for induction of in vitro anti-2,4,6-trinitrophenyl response in low-affinity receptor lymphocytes.

The possibility that the requirement for macrophages in the induction of an immune response is related to the antigen (Ag)-binding capacity of the lymphocyte populations that undergo activation has been examined. Spleen cells from mice immunized with 2,4,6-trinitrophenyl (TNP)-conjugated sheep red blood cells (TNP-SRBC) were filtered on a TNP-substituted Bio Gel column. The excluded cells were shown to be mainly low-affinity anti-TNP cells by using an anti-TNP plaque-forming assay and assessing the avidity of the plaque forming cells (PFC). In order to obtain a low-affinity anti-TNP precursor cell population, lymphocytes from nonimmunized mice were filtered as above. Unfiltered and filtered excluded nonimmune cells were cultured in the presence of Ag-pulsed macrophages or with the supernatants of the pulsed macrophage cultures. The filtered cells produced a specific antibody response only in the presence of the Ag-pulsed macrophages, while the total (nonfiltered) lymphocyte population contained PFC when cultured with free antigen. Determination of the avidity of the PFC confirmed the presence of low-affinity cells in the filtered population and the high affinity of the antibody-producing cells cultured in the absence of macrophages. The induction of a response in low-affinity lymphocytes appeared to require the presence of macrophages rather than that of a soluble factor present in the supernatant. It is suggested that the Ag-presenting role of macrophages are essential for the induction of the immune response in low-affinity cells, while high-affinity lymphocytes could be directly activated by free Ag.

[T and B lymphocytes in peripheral blood during acute infections]. / Estudio de linfocitos T y B en sangre periférica durante infecciones agudas.

A longitudinal study was carried out in 40 patients with typhoid fever, 21 with infectious hepatitis and 15 with amebic liver abscess in order to determine percentual values of lymphocytes T and B in relation with age and nutritional state as compared to a normal control group. In patients with typhoid fever, a decrease of percentual values of lymphocytes T was observed starting on the second week of evolution with intensification on the third week, predominating in patients less than seven years of age and in malnourished cases (p less than 0.001) and late recovery in the group of patients less than seven years old. In patients with acute infectious hepatitis, there was depression of lymphocytes T that persisted to the eighth week and was independent of age, but more severe in the malnourished (p less than 0.001). In cases with amebic liver abscess, depression of lymphocytes T was observed during the first six weeks of evolution without relation with age or the state of nutrition, but with increased slowliness in recovery in patients under seven years. There were no disturbances in lymphocytes B nor in cutaneous response to dinitrofluorobencene (DNFB) in the three groups of patients.

[Study of the immune system in relatives of children with dermatomyositis]. / Estudio del sistema inmune en familiares de niños con dermatomiositis.

In view of the fact that relatives of patient with other connective tissue diseases may show immunologic abnormalities, we investigated cellular (PPD, SK-SD, candidine and diphteria-tetanous toxoid cutaneous tests) and humoral (IgG, IgM, IgA. CH50, C3, rheumatoid factor and ANA) immune responses in first degrees relatives (parents and siblings) of children with dermatomyositis. Of the 53 subjects tested, 19 had high values of one or two immunoglobulines; however, the mean Ig value was not different from that of the general population (p greater than 0.1), CH50, C3, rheumatoid factor and ANA were normal or negative. Cutaneous tests made on 46 subjects did not reveal abnormalities. Therefore our conclusion is that first degree relatives of children with dermatomyositis have not significant immunologic abnormalities.

[Rosette forming lymphocytes as prognostic indices in idiopathic thrombocytopenic purpura]. / Linfocitos formadores de rosetas como índices pronósticos en la trombocitopenia idiopática.

The study included 12 patients with acute ITP to whom subpopulations of rosette forming lymphocytes were determined in peripheral blood and in serial form, the number of platelets to establish a correlation as prognostic index in the evolution of the disease. No correlation was found between the progressive increment of platelets in the evolution of the disease. However, in patients with a tendency to remission, the percentage of E(T) rosette forming lymphocytes was low and normal that of EAC (B) lymphocytes. Dissimilarly, it was seen that in patients without tendency to remission, the percentage of T and B rosette forming lymphocytes was within normal limits. This seems to show that determination of the subpopulations of lymphocytes may be useful as a prognostic index in this stage of the disease.

[Rosette-forming peripheral lymphocytes, in children with measles]. / Linfocitos periféricos formadores de rosetas, en niños con sarampión

During measles infection in human beings, depression of cellular immunity appears. The authors studied several groups of children with measles complaining of various degrees of malnutrition in terms of rosette forming lymphocytes in peripheral blood; marked reduction of T lymphocytes were seen in all of the groups during the acute stage of the disease, that persisted up to two weeks after the exanthematous stage. When comparison was established of eutrophic and 1st degree malnourished children with those affected with 3rd degree malnutrition, differences were found in the recovery of the number of rosett-E forming cells.