Influence of mechanical stress on cell viability.

The cartilage is a hydrated connective tissue in joints that withstands and distributes mechanical forces. The chondrocytes utilize mechanical signals to regulate their metabolic activity through complex biological and biophysical interactions with the extracellular matrix (ECM). The aim of this work was to study the influence of mechanical stress on cells behavior cultured in 3D biosystems (alginate and alginate supplemented with hyaluronate). After mechanical stimulation, cell viability and cell death process were the main studied parameters. Our results indicated that viability and cell cycle progression were inhibited under mechanical stimulation, as far as the extracellular matrix was not yet synthesized. In contrast, on day 21, the mechanical stimulation had positive effect on these parameters.

Effect of cyclic stretching and foetal bovine serum (FBS) on proliferation and extra cellular matrix synthesis of fibroblast.

It is well known today that mechanical forces are one of the important factors that induce a variety of cellular responses including morphological changes, protein synthesis, and gene expression and which are involve in tissue remodelling. We studied the effect of uniaxial cyclic stretching on the proliferation, collagens, and tenascin C mRNA expression of fibroblasts under different concentrations of foetal bovine serum. Proliferation was studied by cell cycle analysis, mRNA expression of collagen and tenascin C was studied by RT-PCR. Human fibroblasts were grown in silicon sheet coated with 1% gelatin. Cyclic stretching (5% elongation) was applied at 0.5 Hz (30 cycle/min), for 24 h with two concentrations of the serum (0.5%, 10% FBS). We showed that stretching enhances the synthesis of collagen and tenascin C, but do not act on the proliferation. In contrast, higher concentration of serum enhances the proliferation. These findings suggest that both mechanical stretching and serum concentration can modulate proliferation and extra cellular matrix synthesis in human fibroblasts.

Back-pack mice as a model of renal mesangial IgA dimers deposition.

Mesangial IgA in IgA nephropathy are dimers with a J chain but no poly-Ig receptor. This molecular structure has led to the hypothesis that these IgA are issued from the lamina propria of mucosal areas, reaching the kidney by way of the peripheral blood. The availability of hybridomas producing IgA dimers provided an opportunity to test this hypothesis in a new experimental model of IgA nephropathy. Mice were injected subcutaneously (back-pack mice) or intraperitoneally with hybridoma cells secreting either monoclonal IgA dimers, or monoclonal IgA monomers. The influence of immune complex formation was also tested in both these models. Renal IgA deposition was investigated 12 days after the injection of hybridoma cells. Backpack mice developed highly vascularized subcutaneous tumors. Mesangial IgA deposits were observed only in dimeric IgA hybridoma back-pack animals. No significant staining was observed in glomeruli from animals injected with hybridoma cells producing monomeric IgA. None of the hybridomas induced mesangial deposition when injected intraperitoneally. This animal model demonstrates the capacity of circulating IgA dimers to spontaneously form mesangial deposits and contributes to confirm the involvement of abnormalities of mucosal immunity in the pathogenesis of IgA nephropathy.

Assessment by flow cytometry of peripheral blood leukocyte enzymatic activities in HIV patients.

Leukocyte enzymatic activities are important in non-specific protection against bacterial infections, but traditional methods for the detection of intracellular enzymatic activities rely on cumbersome and complex assays. The development of specific substrates, which become fluorescent upon degradation of the biomolecule after its passive entry into intact cells, permits a simplified evaluation of leukocyte enzymatic activities. We have used this method to assess intracellular elastase, collagenase and cathepsin D activities of peripheral blood leukocytes using flow cytometry in a series of HIV patients and healthy controls. Monocytes displayed the highest enzymatic activities for the three proteases tested. In HIV-infected patients, the collagenase and cathepsin D activities of monocytes were significantly lower, whereas the elastase and cathepsin D activities of polymorphonuclear cells were elevated. Slightly higher elastase activity was detected in the lymphocytes of patients. This study demonstrates the feasibility of this new method for the study of intracytoplasmic enzymatic activities. Significant variations were observed in the peripheral blood of HIV-infected patients and different patterns were especially evident in monocytes and polymorphonuclear cells.

Anti-myelin oligodendrocyte glycoprotein B-cell responses in multiple sclerosis.

Humoral auto-immunity to the myelin oligodendrocyte glycoprotein (MOG) is likely involved in the pathogenesis of multiple sclerosis (MS). In 44 MS patients and 30 controls, Ig-producing B cells were identified by their isotype and as MOG-specific spot-forming cells (SFC). Peripheral anti-MOG antibodies were assayed in ELISA as well as anti-butyrophilin antibodies to investigate for molecular mimicry. MS patients had significantly higher levels of IgA- and MOG-SFC than controls, as well as significantly higher antibody responses to MOG and butyrophilin. These data provide added support for the implication of anti-MOG humoral immunity in the pathophysiology of MS, and suggest a balance of systemic (anti-self) and mucosal (environment-modulated) immune reactions in an attempt at regulating the pathogenic specific immune response.

Increased usage of TCR V-beta8 in kidney transplant recipients with aberrant immune reconstitution and clinical complications.

BACKGROUND: The efficiency of immunosuppressive drugs prescribed after organ transplantation is mostly monitored through clinical and biological signs of organ rejection or infection. However, it may be expected that some patients develop subtle alterations of their reconstituting immune system, not immediately associated with clinical events. Identification of such anomalies could be useful to alert clinicians for possible future complications. METHODS: A systematic follow-up of peripheral lymphocyte subsets, performed in a cohort of 89 kidney transplant recipients, identified severely skewed CD4/CD8 ratios in 32 patients. For 19 patients, the expression of specific T cell receptor fragments was examined using a panel of 10 monoclonal antibodies. Abnormal control of spontaneously Epstein Barr virus-infected B cells was tested by investigating for the generation of spontaneous lymphoblastoid cell lines in 17 cases. The incidence of rejection and infectious episodes was monitored. RESULTS: A bias in T cell receptor fragments usage was detected in 14/19 cases, involving Vbeta8 in all cases. Spontaneous lymphoblastoid cell lines of Epstein Barr positive B blasts developed in 9 of 17 cases. Eleven patients had early rejection episodes and 16 presented with viral primo-infection or reactivation. The incidence of rejection and infectious episodes was higher in the group of 32 patients who developed such abnormal patterns than in the 57 who did not. CONCLUSION: Transient bias in the T cell receptor repertoire may be observed during immune reconstitution after kidney transplantation, perhaps related to abnormal lymphocyte functions and associated to an impaired control of rejection and/or infectious agents.

Automated cell count in flow cytometry: a valuable tool to assess CD4 absolute levels in peripheral blood.

The enumeration of lymphocyte subsets in absolute counts has long relied on different methods applied separately to whole blood cell count, lymphocyte differential appreciation, and flow cytometric evaluation of lymphocyte subsets percentages. The development of multicolor labeling methods inflow cytometry now allows a more homogeneous appreciation of several cell subsets among gated lymphocytes. The use of internal calibrators, such as microbead suspensions, also permits a direct appreciation of subsets in absolute counts in a single-platform method. These methods were compared with a traditional multiplatform method of assessing absolute counts of lymphocyte subsets in a pilot study in which all manipulations were performed by 1 person and in a full-scale larger study performed in the normal working conditions of a hospital laboratory. Microspheres seem to be a reliable tool to perform absolute count enumeration inflow cytometry, but several precautions in the sample preparation and flow cytometric analysis are required.

Longitudinal study of rheumatoid arthritis patients discloses sustained elevated serum levels of soluble CD106 (V-CAM).

OBJECTIVE: To appreciate the evolution of serum angiogenic and/or adhesion molecules levels during a long term follow-up of rheumatoid arthritis (RA) patients. METHODS: Serum levels of 5 soluble adhesion/angiogenesis glycoproteins (VEGF, CD31, CD54, CD62E, CD106) were measured in Elisa in samples collected over 6 years in a cohort of 43 RA patients with monitored clinical parameters of disease activity and severity. RESULTS: RA patients had significantly higher levels (p < 0.0001) of sCD106 (VCAM-1) than control subjects. Conversely, the levels of soluble VEGF, CD31, CD54 and CD62E were normal or lower than normal. No statistically significant time effect was noted. No effect either was noted as related to the therapeutic agents taken by the patients. CONCLUSION: The sustained elevated serum levels of sCD106 observed here imply that this molecule might be related to the chronicity and progression of RA.

Mouse lung immune response after acute exposure to flour dust.

To approach the physiopathology of the mucosal immune response in flour-mill and bakery workers, the authors developed a mouse model, which allowed them to study immune responses induced in the deep lung by acute inhalation of flour dust. In situ quantification of T lymphocytes (Thy1-2) and T-cell subsets (CD4 and CD8), macrophages, B lymphocytes, and immunoglobulin A plasma cells in the lungs of all animals revealed significant modifications of these cell compartments as early as 3 d after exposure; within 10 d of exposure, levels returned to baseline. The data suggest that the lung could be a sensitive target for inhaled xenobiotics, which might generate rapid immune local modifications that result in the maintenance of homeostasis.

Altered partition of T cell subsets in the peripheral blood of healthy workers exposed to flour dust.

Occupational exposure to organic compounds can induce obvious immunological disorders or more subtle modifications. We investigated peripheral blood lymphocyte subsets in 34 bakers and 82 millers exposed to wheat flour dust, and 51 salt factory workers. Significantly decreased levels of CD4+, CD8+, CD57+ and CD8+/57+ cells were noted in mill workers, and of CD57+ cells in bakers. CD29+ and CD4+/CD29+ cells were significantly lower in millers, CD4+/CD45RA+ cells higher in all exposed workers. The lower numbers of positive cells noted in millers appeared associated to significantly higher (p < 0.001) levels of CD29 and CD45RA expression as measured by fluorescence intensity. These data are opposite to those previously reported in asthmatic workers exposed to flour dust. Since the individuals tested here were clinically healthy, the alterations of T-cell subsets observed could be interpreted as a successful attempt at immunoregulation maintaining homeostasis.

Airway responsiveness to methacholine, respiratory symptoms, and dust exposure levels in grain and flour mill workers in eastern France.

Our goal was to assess the relation between dust exposure levels and the respiratory health status of workers in grain and flour mills in eastern France. We studied 118 male workers from 11 mills and 164 unexposed male controls. Dust concentration was measured by personal sampling methods. Outcome variables included respiratory symptoms, routine pulmonary function tests, and indices of airway responsiveness to methacholine. A great within- and between-area variability of inhalable dust concentration was found in all mills. A dose-response relationship was observed between dust exposure levels and chronic respiratory symptoms, suggesting that exposure to grain and flour dust may lead to chronic bronchitis. A significant relation was found between dust exposure and airway hyper-responsiveness; this finding is important since it has been hypothesized that the latter abnormality may lead to or be a predisposing factor in subsequent chronic, irreversible airflow obstruction.

Kinetics of local immune responses in mouse Peyer's patches after respiratory exposure to flour.

Peyer's patches play a major role in the initiation of mucosal immune responses since most environmental antigens gain access to the digestive tract and are therefore liable to achieve contact with their specialized dome epithelium. We investigated kinetics of immunophenotypic modifications induced in the cells from murine Peyer's patches in a model of intensive controlled exposure to flour dust. Three groups of mice were placed in an atmosphere enriched in wheat flour for 3, 6 or 10 consecutive days. Although Ig bearing B cell numbers did not vary, the numbers of IgA-containing plasma-cells increased significantly during the course of exposure. A significant and increasing imbalance of the CD4/CD8 ratio was noted, as early as after 3 days of exposure related both to increasing numbers of CD4+ and decreasing numbers of CD8+ cells. These data indicate a rapid kinetics of immune responses in Peyer's patches in an original model of controlled respiratory challenge.

Discriminative immunophenotype of bronchoalveolar lavage CD4 lymphocytes in sarcoidosis.

The diagnosis of pulmonary sarcoidosis relies in part on the observation of alveolar CD4+ lymphocytosis. However, this criterion is not fully discriminative because this anomaly is also found in other types of lung diseases. Among other possible distinctive criteria, we investigated the expression of lymphocyte-addressing molecules, which could differ according to the pathophysiology of lung diseases. We investigated CD103 (alpha(E)beta7 integrin, CD103-beta7), reported to be both expressed on intra-epithelial lymphocytes in mucosal areas, including bronchi, and possibly involved in the recruitment of alveolar lymphocytes. The expression of CD103 was examined on bronchoalveolar lavage lymphocytes from 93 consecutive patients, including 34 patients with CD4+ lymphocytosis. For all patients, the expression of CD19, CD3, CD4, CD8, CD57, LFA1, DR, and CD103 was assessed by flow cytometry. Sarcoidosis seemed remarkably characterized by the lack of CD103 expression on the predominant CD4+ subset. Statistically significant differences were found between patients with sarcoidosis, with other types of CD4+ lymphocytosis, and with other lung disorders in the CD103+ cell levels and in the CD103/CD4 ratio. Combined use of the CD4/CD8 ratio (> 2.5) and the CD103/CD4 ratio (< 0.31) to assess bronchoalveolar lavage lymphocytes is a promising new tool for the diagnosis of sarcoidosis.

Immunohistological analysis of macrophages, B-cells, and T-cells in the mouse lung.

BACKGROUND: Numerous studies have described the anatomy of the large lymphoid aggregates of bronchus-associated lymphoid tissue (BALT) in rabbits and rats. Less work has been performed on other immunocompetent areas of the respiratory tract, and available data again mostly describe rabbit or rat tissues. Little is known therefore of the microanatomy of the mouse lung immune system. METHODS: We report a study, devised in order to establish the immunohistological characteristics of normal healthy mice lungs, performed on whole lungs from 22 mice of various strains and/or ages. Snap frozen tissues were serially sectioned and analysed using histochemistry and immunohistological techniques. Scattered macrophages, IgA plasma cells, B and T cells were enumerated in each sample. RESULTS: The largest population was that of macrophages. B-cells were numerous in all mice but 3 adults. T-cells were always present, L3T4+ often more numerous than Lyt2+ cells. Small lymphoid aggregates, composed of B or T cells (L3T4+ and Lyt2+) were seen in all mice, in the vicinity of a bronchiole and a vein. In 12/22 mice, a peculiarly elongated para-esophageal lymph node with large peripheral B-cell nodules and medullary T-cells was observed. In the five strains of mice studied, large variations were noted, affecting all the cell types studied, and related either to age or strain. CONCLUSION: Besides providing a qualitative description and quantitative analysis of immunocompetent cells, this work reports age and strain-related variations in these cells' distribution. These data could be relevant for studies involving the analysis of mice respiratory immune responses to environmental antigens.