Control of cell cycle progression in human mesothelioma cells treated with gamma interferon.

Recombinant human interferon gamma (r-hu-IFNgamma) exerts both antitumoral activity in the early stages of human malignant mesothelioma and a cytostatic effect in human mesothelioma (HM) cell lines in vitro. The antiproliferative effect of interferons (IFNs) reported in a variety of cells has been attributed to several mechanisms. In order to progress in the understanding of HM cell growth modulation by r-hu-IFNgamma, modifications of cell cycle progression and expression of key cell cycle regulator proteins in response to r-hu-IFNgamma were examined. Nine HM cell lines were studied, including one resistant to the antiproliferative effect of r-hu-IFNgamma. Except in the resistant cell line r-hu-IFNgamma produced an arrest in the G1 and G2-M phases of the cell cycle, associated with a reduction in both cyclin A and cyclin dependent kinase inhibitors (CDKIs) expression. Moreover cyclin B1/cdc2 activity was decreased. The present study provides the first evidence of a G2-arrest in r-hu-IFNgamma-treated HM cell lines and indicates that HM cell lines, despite their tumorigenic origin still support cell cycle control. The cell cycle arrest induced by r-hu-IFNgamma seems to depend on cyclin regulation through p21(WAF1/CIP1)- and p27(Kip1)-independent mechanisms and is not directly related to the induced DNA damage.

Absence of SV40 large T-antigen expression in human mesothelioma cell lines.

Simian virus (SV) 40 and SV40-like DNA sequences have recently been detected in several types of human tumors, including malignant mesothelioma. However, the presence of SV40 DNA sequences is not sufficient to account for its possible role in tumor development because the viral proteins must be expressed and ultimately impair the function of relevant cell proteins, such as p53 and pRb. In this study we investigated SV40 large T antigen (SV40 Tag) protein expression in mesothelioma cell lines, established in our laboratory, by Western blotting, immunoprecipitation, and immunocytochemistry using Tag-specific mouse monoclonal antibodies (mAbs) Ab-1 (or Pab 419). By Western blotting of cell extracts, none of the mesothelioma cell lines expressed detectable amounts of SV40 Tag. However, we found that Ab-1 as well as Pab-101, another SV 40 Tag-specific mAb, may generate false-positive signals due to the fact that both antibody preparations are contaminated by a protein of similar size (90 kD) as SV40 Tag and react with the various secondary horseradish peroxidase- conjugated antimouse immunoglobulin Gs tested. The present study suggests that immunodetection of SV40 Tag protein may be puzzling because this contaminating Taglike protein may bind to particular cell structures, thereby generating false-positive signals.

The target antigens of naturally occurring human anti-beta-galactose IgG are cryptic on porcine aortic endothelial cells.

The identification of the xeno-antigens/xeno-antibodies combinations involved in pig-to-human xenograft rejection is an essential step for understanding this process and for the development of procedures to prevent it. Although it is widely accepted that the terminal disaccharide Galalpha1,3Gal-R is by far the major epitope recognized by human natural antibodies reactive with pig tissues, there is also evidence that other carbohydrate epitopes might be important in xenograft rejection. In an attempt to further improve our knowledge of the repertoire of human natural antibodies with anti-pig specificity we sought to determine whether naturally occurring human anti-beta-galactose IgG could interact with porcine aortic endothelial cells (PAEC). Histochemical analysis of porcine aorta sections revealed that the carbohydrate structures recognized by the anti-beta-galactose IgG are present on endothelial cells but in a cryptic form that can be unmasked by sialidase treatment. These structures were also found to be cryptic in cultured PAEC. In addition we demonstrated that PAEC may adsorb fetal calf serum (FCS) glycoproteins when cultured in FCS-supplemented medium, a process susceptible to generating artifactual observations in carbohydrate antigens analysis. In conclusion, despite their abundance, human anti-beta-galactose IgG do not represent a primary concern in pig-to-human xenotransplantation as the carbohydrate structures to which they bind are normally masked by sialic acid residues on porcine endothelial cells. However, whether these cryptic epitopes might be exposed on endothelial cells from genetically engineered animals should be further investigated because, if so, additional approaches will be needed to suppress their interaction with human anti-beta-galactose IgG.

Imbalances in serum proinflammatory cytokines and their soluble receptors: a putative role in the progression of idiopathic IgA nephropathy (IgAN) and Henoch-Schönlein purpura nephritis, and a potential target of immunoglobulin therapy?

Following recent experimental data suggesting an aggravating effect of circulating proinflammatory cytokines on the histological lesions of IgAN, we studied changes in serum proinflammatory cytokines and their soluble receptors and antagonists in patients treated with polyvalent immunoglobulins (15 with severe nephropathy who had indicators of poor prognosis: heavy proteinuria, hypertension, altered renal function and Lee's histological grade III or IV; and 14 with moderate forms of IgAN who had permanent albuminuria > 300 mg/day and < 2000 mg/day, Lee's histological grade II and a glomerular filtration rate > 70 ml/min) in comparison with healthy controls (n = 20) and patients with non-IgA nephritides (n = 50). These were measured by means of specific immunometric assays before and after 9 months of immunoglobulin therapy. Total tumour necrosis factor (TNF) serum and IL-6 levels were elevated in IgAN patients before therapy, relative to controls, and normalized after immunoglobulin therapy. Levels of soluble TNF receptor of type I (sR55) and type II (sR75) increased on immunoglobulin therapy. TNF index alpha-55,75 used to assess biologically available TNF-alpha (ratio of total TNF-alpha divided by levels of soluble TNF receptors sR55 and sR75) was elevated before therapy and was below healthy control values after 9 months of immunoglobulin administration. Levels of serum IL-1 receptor antagonist were low prior to immunoglobulin administration in patients with severe forms of IgAN, and normalized on therapy. Serum interferon-gamma was unmodified. The histological activity index correlated with serum total TNF-alpha, TNF index alpha-55,75 and serum IL-6 levels, whereas proteinuria correlated with serum total TNF-alpha and TNF index alpha-55,75 but not with serum IL-6. These data suggest that the overproduction of proinflammatory cytokine is unbalanced by their natural antagonists in IgAN and Henoch-Schönlein syndrome. This process may play a role in the progression of the disease and be one of the targets of immunoglobulin therapy.

Shedding of CD44 from PMA-differentiated U-937 cells is enhanced by treatment with mineral particles.

In this report, we show that enhanced shedding of CD44 might contribute to the down-regulation of this receptor observed after phagocytosis of MnO2 particles by PMA-differentiated U-937. The apparent Mr of the soluble CD44 detected in culture supernatants was slightly lower than that of the membrane form suggesting that shedding resulted from proteolytic cleavage. Increased shedding of CD44 was also noted with other mineral particles (chrysotile and DQ12) but to a lower extent whereas some (TiO2 and amosite) had no effect on this process. These results indicate that shedding enhancement was particle-specific rather than a general consequence of phagocytosis. The ability of the particles to enhance CD44 shedding was not directly dependent on their cytotoxic potency. Different patterns of reactivity were noted with CD11b, suggesting that the underlying mechanisms are specific.

Human malignant mesothelioma cell growth: activation of janus kinase 2 and signal transducer and activator of transcription 1alpha for inhibition by interferon-gamma.

Intrapleural injections of recombinant human IFN-gamma have shown some efficacy in reducing tumor growth in early stages of diffuse malignant mesothelioma (DMM). Here, we have addressed the potential therapeutic effect of IFN-gamma in DMM by investigating the activation of the JAK/STAT signaling pathway in seven human mesothelioma cell lines (HMCLs) that were differentially responsive to the antiproliferative activity of IFN-gamma. We showed that janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1) were phosphorylated on tyrosine residues within 15 min in all the HMCLs in which IFN-gamma (500 units/ml) inhibited proliferation. In addition, STAT1 binding activity to the gamma-activated sites DNA sequence was detected within 15 min in electrophoretic mobility-shift assay analysis, and IFN regulatory factor-1 RNA expression was observed within 6 h in the more responsive cells (72.7-95.2% inhibition of DNA synthesis after 72 h of treatment). Conversely, in several HMCLs, absent or limited growth suppressive effect (less than 22% inhibition of DNA synthesis) was associated with alterations in expression or activation of JAK2 or STAT1 or, downstream, with low induction of IFN regulatory factor-1 RNA expression and/or STAT1 protein expression following IFN-gamma treatment. These data suggest that at least part of the IFN-gamma effect on proliferation of HMCLs is mediated directly through activation of the JAK/STAT1 signaling pathway, and it could account for the antitumoral activity reported in DMM patients treated with IFN-gamma.

Analysis of cell cycle disruptions in cultures of rat pleural mesothelial cells exposed to asbestos fibers.

The control of DNA integrity in mammalian cells is important to maintain the cell homeostasis and prevent neoplastic transformation. Control of cell division and cell death permits repair or elimination of damaged cells. Since asbestos fibers can produce DNA damage, chromosome alterations and apoptosis in several sorts of cells, including mesothelial cells, it was interesting to investigate cell cycle disturbances in rat pleural mesothelial cells (RPMC) treated with asbestos fibers. Cell cycle analyses were performed in RPMC exposed to crocidolite (10 and 20 microg/cm2) and chrysotile (5 and 10 microg/cm2) for different times (4 to 48 h). Both fiber types entailed a G2/M accumulation in agreement with a delay in the mitosis course. Chrysotile fibers produced a G0/G1 accumulation associated with a time-dependent p53 and p21 expression. Crocidolite exposure resulted in a delay in the G1/S transition paralleling a low rate of p53 expression. These results are in agreement with a DNA damaging potential of asbestos fibers since similar results were found following RPMC exposure to gamma rays. In asbestos-treated RPMC, a low rate of apoptosis was found suggesting that RPMC may follow a DNA repair pathway that could contribute to the formation of DNA lesions. In addition, the cell cycle disturbances at the G2/M checkpoint suggest that genetically altered cells have progressed through the cycle and support the already published findings on the ability of asbestos fibers to impair cell division.

Alterations in protein glycosylation in PMA-differentiated U-937 cells exposed to mineral particles.

Carbohydrate moieties of cell glycoconjugates play a pivotal role in molecular recognition phenomena involved in the regulation of most biological systems and the changes observed in cell surface carbohydrates during cell activation or differentiation frequently modulate certain cell functions. Consequently, some aspects of macrophage response to particle exposure might conceivably result from alterations in glycosylation. Therefore, the effect of mineral particles on protein glycosylation was investigated in phorbol myristate acetate (PMA)-differentiated U-937. Jacalin, a lectin specific for O-glycosylated structures, showed a global increase in O-glycosylation in particle-treated cells. In contrast, no significant modifications were observed with concanavalin A, a lectin that recognizes certain N-glycosylated structures. The sialic acid-specific lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin and the galactose-specific lectin Ricinus communis agglutinin revealed a complex pattern of alterations in glycoprotein glycosylation after crystalline silica or manganese dioxide treatments. Expression of sialyl Lewis(x), a glycosylated structure implicated in leukocyte trafficking, could not be detected in control or treated cells. This finding was consistent with the decrease in sialyl Lewis(x) expression observed during PMA-induced differentiation. In conclusion, various treatments used in this study induced quantitative as well as qualitative changes in protein glycosylation. Whether these changes are due to glycosidase release or to an alteration in glycosyltransferase expression remains to be determined. The potential functional implications of these changes are currently under investigation.

Inhibition of acid sialidase by inorganic sulfate.

Sulfated glycosaminoglycans are known to inhibit mammalian acid-active sialidase. Although the inhibition depends clearly on the presence of sulfate groups on these macromolecules, there was no information on the intrinsic inhibitory potency of inorganic sulfate. In this study, we demonstrate that inorganic sulfates inhibit acid-active Mu-Neu5Ac sialidase of U937 cells. This inhibition was found to be reversible and it appeared to be of the mixed competitive type. Sulfate-induced inhibition was also observed in other cells as well as with other substrates such as sialyl lactose and bovine mixed brain gangliosides. We conclude that the intrinsic inhibitory potency of sulfate groups may be significantly involved in the inhibition of acid-active sialidase by sulfated glycosaminoglycans. In addition, inorganic sulfate by its apparent potency to selectively inhibit acid sialidases might constitute an interesting tool for the characterisation of the minor forms of sialidases occurring in mammalian cells.

Further identification of human plasma glycoproteins interacting with the galactose-specific lectin Jacalin.

In this report we show that Jacalin binds the heme-binding protein hemopexin and the C4b-binding protein sgp120 in human plasma. The interaction of Jacalin with hemopexin confirms that a single O-linked oligosaccharide is sufficient to mediate binding of a protein to this lectin. Retention of sgp120 by immobilized Jacalin demonstrated that this protein was O-glycosylated and, therefore, clearly different from another C4b-binding protein, the complement protein C2 which is physicochemically similar but exclusively N-glycosylated. In addition, Jacalin was also shown to bind several proteolytic enzymes which remain to be identified.

Immunomodulation with low-dose immunoglobulins for moderate IgA nephropathy and Henoch-Schönlein purpura. Preliminary results of a prospective uncontrolled trial.

Recently, our group has shown that a 3-month course of intravenous immunoglobulin (2 g/kg/monthly) followed by 6 months of intramuscular immunoglobulins (IMIG, 16.5%, 0.35 ml/kg every 15 days) was able to slow or to stop the decline in the glomerular filtration rate, to reduce proteinuria, hematuria, leukocyturia and the histological index of activity on renal biopsy in patients with severe forms of IgA nephropathy (IGAN) and Henoch-Schönlein purpura (HSP). The aim of this open prospective trial was to evaluate the efficacy and safety of low-dose immunoglobulin therapy in moderate IGAN and HSP with permanent proteinuria. Fourteen patients with moderate IGAN [idiopathic IGAN: n = 11; chronic idiopathic HSP: n = 3] and permanent albuminuria were treated with polyvalent IMIG (16.5%) for 9 months (0.35 ml/kg once a week for 1 month, followed by 0.35 ml/kg every 15 days for a further 8 months). Eligibility criteria in the study were Lee histological stage I, II or III, albuminuria between 300 and 2,000 mg/day and a glomerular filtration rate > 70 ml/min/1.73 m2. IMIG were well tolerated and only 1 patient withdrew from the trial. No viral, renal or immunological side effects were observed. IMIG induced a significant decrease in albuminuria as well as in the histological activity index in the 11 cases in which a follow-up biopsy was performed. There was also a decrease in serum IgA, serum beta 2-microglobulin and IgA immune complex levels, and an increase in serum IgG1 levels. Twelve of the 13 evaluable patients improved during treatment.

Initial characterization of human thymocyte sialidase activity: evidence that this enzymatic system is not altered during the course of T-cell maturation.

1. The sialidase activity of human thymocyte was examined by a fluorogenic assay. 2. These studies revealed that human thymocyte sialidase activity is essentially acid-active and membrane-bound since 59.6% and 33% of the total activity was recovered in the lysosome-enriched and microsomal fractions, respectively. 3. A weak activity was also detected in the cytosolic fraction. 4. However, the acidic optimum pH of this soluble sialidase was at variance with the general concept of mammalian soluble sialidases which are known to be optimally active at more neutral pH. 5. This acidic soluble sialidase seems to be a general characteristic of the human T-cell lineage since examination of mature circulating T-cells revealed that they contain a soluble sialidase activity similar to that observed in thymocytes. 6. Analysis of mature and immature thymocyte subpopulation obtained by differential PNA agglutination indicated that this enzymatic system was not altered during the course of thymic maturation. 7. These results suggest that unlike in T-cell activation where changes in the level of sialidase activity were shown to influence the extent of cell surface sialylation and thereby the cell physiology, this enzymatic system seems not to be involved in the fluctuation of cell surface sialic acid content observed during thymic maturation.

High-dose immunoglobulin therapy for severe IgA nephropathy and Henoch-Schönlein purpura.

OBJECTIVE: To determine if polyvalent IgG is promising therapy for severe IgG nephropathy. DESIGN: Open prospective cohort study. SETTING: Referral nephrology unit. PATIENTS: 11 adult patients with severe IgA nephropathy (9 who had idiopathic disease and 2 who had Henoch-Schönlein purpura) and indicators of poor prognosis. INTERVENTION: Patients were given high-dose immunoglobulins (2 g/kg each month) for 3 successive months, followed by intramuscular immunoglobulins (preparation content, 16.5%; 0.35 mL/kg every 15 days) for another 6 months. MEASUREMENTS: Histologic changes were analyzed by comparing pre- and post-therapy renal biopsy specimens blindly, using an activity index (14-point scale), a sclerosis index (10-point scale), and a semiquantitative immunofluorescence test of immune deposits. Proteinuria, hematuria, leukocyturia, enzymuria, and global renal function (creatinine and polyfructosan clearances) were evaluated before and after intervention. RESULTS: Proteinuria (median level before intervention, 5.20 g/d; median level after intervention, 2.25 g/d), hematuria, and leukocyturia decreased substantially. The decrease in glomerular filtration rate was greatly slowed or stopped (median rate of decline in glomerular filtration before, -3.78 mL/min per month; after, 0 mL/min per month). The histologic index of activity (median index before, 5; after, 2) and the staining intensity of glomerular IgA and C3 deposits also decreased. Immunoglobulin therapy was well tolerated. CONCLUSIONS: Immunoglobulin therapy may be effective in treating severe IgA nephropathy and protecting renal function. However, prospective controlled trials must confirm these preliminary results.

Histone-reactive IgA antibodies in adult IgA nephropathy and other primary glomerulonephritis.

The levels of histone-reactive IgA antibodies in the sera of adult patients with IgA mesangial glomerulonephritis, membranous glomerulonephritis, membranoproliferative glomerulonephritis and idiopathic nephrotic syndrome (minimal change disease+segmental glomerulosclerosis+IgM nephropathy) were evaluated by an enzyme-linked immunosorbent assay. Increased levels of IgA antibodies to all five major histones (H1, H2A, H2B, H3, H4) were found in all four disease groups when compared to normal controls. These histone-reactive IgA antibodies were restricted to the IgA1 subclass and their levels did not correlate with the levels of total serum IgA, nor with serum creatinine, creatinine clearance, and 24-hour proteinuria. Increasing ionic strength resulted in only partial inhibition of the binding to histones and, in individual patients, levels of reactivity with individual histones were usually correlated. This study shows that elevated levels of IgA antibodies reactive with self antigens are present in primary glomerulonephritis and extends previous observations indicating that anomalies of the IgA system occur in various forms of primary glomerulonephritis and are not limited to IgA nephropathy.

Determination of the sialic acid linkage specificity of sialidases using lectins in a solid phase assay.

A procedure for the determination of activity and linkage specificity of sialidases is described. The sialoglycoprotein fetuin is coated onto a microtiter plate and incubated with sialidases from different sources. Enzymatic activities and linkage specificities are then determined by a sandwich method which measured the binding of different lectins to fetuin. The lectins used were peanut agglutinin (PNA) from Arachis hypogaea, which binds specifically the galactose beta-1-3-N-acetylgalactosamine structures that are unmasked following sialidase treatment of fetuin, the lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for alpha-2-6 and alpha-2-3 bound sialic acids, respectively, and the slug agglutinin from Limax flavus (LFA) that is specific for N-acetyl and N-glycolyl neuraminic acids. Increased PNA and decreased LFA, SNA, and MAA lectin binding correlated with sialidase-induced desialylation of the substrate. In this report, the assay was used to determine the activities and specificities of influenza, Vibrio cholerae, and Arthrobacter ureafaciens sialidases.

Differentiation between vascular permeability factor and IL-2 in lymphocyte supernatants from patients with minimal-change nephrotic syndrome.

Immunotherapy of cancers with recombinant IL-2 induces a vascular leak syndrome which is mainly due to an increase in vascular permeability. A lymphokine, the vascular permeability factor (VPF), which increases vascular permeability, has been characterized in minimal-change nephrotic syndrome (MCNS) and appeared very similar to IL-2. Here we have undertaken a further characterization of VPF in order to determine how closely related this factor was to human IL-2. Both the IL-2 bioassay and Western blot analysis of the MCNS lymphocyte concentrated supernatants with high VPF activity revealed the presence of low quantities of IL-2. Preparative isoelectrofocusing (IEF) of concentrated supernatants resolved each lymphokine in a separate peak, with apparent pIs of 5.2 for VPF and 7.5-10.1 for IL-2. Since a sensitive IL-2 ELISA failed to exhibit any significant antigenic presence of IL-2 in the IEF fractions with the highest VPF activity, we conclude that VPF activity of the concentrated supernatants is not related to IL-2 nor to a biologically inactive form of IL-2. When concentrated supernatants were subjected to preparative SDS-PAGE, VPF activity was recovered within low mol. wt material (1-12 kD). Immunoadsorption experiments gave definite proof since the complete removal of IL-2 from concentrated supernatants did not affect the VPF activity. Although high amounts of IL-2 increased vascular permeability, our experiments clearly demonstrate that VPF is a lymphokine distinct from IL-2.

Sialidase in the guinea pig pulmonary parenchyma. Increased activity in the cytosolic and microsomal subcellular fractions after stimulation with Bacillus Calmette Guérin.

The sialidase activity was assayed in the guinea pig pulmonary parenchyma after removal of bronchoalveolar cells by washing. After differential centrifugation of the crude tissue homogenate, sialidase activities were measured in the subcellular fractions using the fluorogenic substrate 2-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminate. Sialidase activities were found in the lysosomal-enriched (17,000 x g pellet), in the microsomal (105,000 x g pellet) and in the cytosolic (105,000 x g supernatant) fractions. Microsomal and lysosomal forms of sialidase had an optimum activity at pH 3.6-3.8, whereas the optimum for the cytosolic form was pH 4.6. The activity of all three forms was inhibited by Cu2+, whereas 1 mM Zn2+ and 0.5 mM Ca2+ activated the lysosomal and the cytosolic forms, respectively. In the crude homogenate taken from lungs of Bacillus Calmette Guérin-(BCG-) stimulated guinea pigs, the sialidase activity was increased by 43% (p = 0.025) 3 weeks after the end of the treatment. The cytosolic (+246%) and microsomal (+51%) sialidase activities were significantly increased, whereas the lysosomal sialidase activity was not changed significantly by BCG stimulation.

Anticarbohydrate autoantibodies to sialidase-treated erythrocytes and thymocytes in serum from patients with pulmonary sarcoidosis.

PURPOSE: The presence of immunoglobulins and complement in sarcoid granulomata and bronchoalveolar lavage from patients with sarcoidosis suggests that humoral mechanisms may be of importance in granuloma formation. To test this hypothesis, we examined the possibility that antibodies to specific tissue carbohydrates causing alterations and/or dysfunction of immunocompetent cells might be present during sarcoidosis. Because we had previously shown the presence of sialidase activity in bronchoalveolar lavage from these patients, we have looked for the presence of antibodies that recognize sialidase-treated erythrocytes (mostly antigalactose) in the serum of patients with sarcoidosis. Since thymocytes are spontaneously recognized by peanut agglutinin, a lectin that binds galactose, the reactivity of serum from sarcoidosis patients with normal or neuraminidase-treated thymocytes has also been studied. PATIENTS AND METHODS: Serum samples were obtained from the venous blood of patients with biopsy-proven sarcoidosis, most of whom had no extrathoracic symptoms. The mean patient age was 31 years, with a range from 21 to 57 years. There were 12 women and 19 men, and 10% of the patients were smokers. Sarcoidosis was classified as recent if symptoms had been present for less than 1 year and chronic if symptoms had been present for longer than this. Control serum samples were obtained from patients with idiopathic pulmonary fibrosis (n = 9) and from healthy volunteers (n = 15). Furthermore, serum from patients who had previously had sarcoidosis but in whom cures had been achieved was also studied (n = 6). RESULTS: Sialidase-treated erythrocytes were lysed in autologous serum upon incubation at 37 degrees C providing that the serum came from a patient with active disease. Serum from either normal volunteers or patients with resolved sarcoidosis had no significant cytotoxic activity. Lysis proceeded through activation of the classical complement pathway following fixation of autoantibodies. These antibodies were predominantly of the IgM class. They were able to agglutinate neuraminidase-treated thymocytes, whereas untreated thymocytes did not fix the antibodies. Carbohydrate inhibition experiments demonstrated that these antibodies are mostly galactose specific. As this sugar is located immediately below the sialic acid residues in the carbohydrate moiety of membrane glycoconjugates, it is unmasked following sialidase treatment. CONCLUSION: Since galactose has been shown to be present on the membrane of certain subsets of immunocompetent cells (e.g., lymphocytes and macrophages either spontaneously or after stimulation), it is possible that antigalactose antibodies may affect the metabolism of these cells, leading to some of the immune dysfunctions that are observed during sarcoidosis.