Survivin overexpression in head and neck squamous cell carcinomas as a new therapeutic target (Review).

Head and neck squamous cell carcinoma (HNSCC) is the sixth most commonly diagnosed cancer worldwide. It has poor clinical outcome due to intrinsic or acquired drug resistance. Deregulation of both apoptosis and autophagy contributes to chemotherapy resistance and disease progression. A new member of the inhibitors of apoptosis protein (IAP) family, namely survivin, is selectively overexpressed in tumors, including HNSCC, but not in normal tissues. Thus, it is considered a tumor biomarker. Here, we reviewed survivin expression and function in tumor progression focusing on its nodal role in the regulation of cell apoptosis and autophagy. Based on literature data, survivin targeting may be envisaged as a novel therapeutic strategy.

Autophagy: A New Mechanism of Prosurvival and Drug Resistance in Multiple Myeloma.

Autophagy is an intracellular self-degradative process that balances cell energy source and regulates tissue homeostasis. In physiological condition, autophagy funnels cytoplasmic constituents to autophagolysosomes for degradation and is an alternative way for cell-death behavior. Here, we inspected autophagy as a prosurvival mechanism essential for drug resistance in multiple myeloma (MM). Accordingly, autophagy inhibitors used in association to conventional anti-MM drugs might enforce the effect against resistant MM plasma cells and render autophagy a new therapeutic target.

Belimumab restores Treg/Th17 balance in patients with refractory systemic lupus erythematosus.

Belimumab, a specific inhibitor of the soluble B lymphocyte stimulator (BlyS), is the first biological drug approved by the United States Food and Drug Administration for the treatment of patients with active systemic lupus erythematosus (SLE) refractory to standard therapy. Given that an imbalance between regulatory T cells (Treg) and interleukin (IL)-17A-secreting T cells (Th17) has been reported in various autoimmune disorders, we assessed the frequency of both Treg and Th17 peripheral blood populations before and after belimumab administration in 20 patients with active SLE refractory to standard therapy. After six months of treatment, the mean SELENA-SLEDAI score as well as the mean anti-double-stranded DNA antibody titers were significantly decreased. In addition, we observed a significant increase in Treg percentages and a parallel, significant decrease in Th17 percentages, accompanied by significantly reduced serum levels of IL-21. In vitro studies showed that Treg purified from belimumab-treated patients were fully functional and displayed a suppressor function similar to that of Treg purified from healthy donors. Belimumab can restore Treg/Th17 balance in SLE patients with uncontrolled disease activity, and this results in decreased flare rate and reduced glucocorticoid dosage.

JAM-A as a prognostic factor and new therapeutic target in multiple myeloma.

Cell adhesion in the multiple myeloma (MM) microenvironment has been recognized as a major mechanism of MM cell survival and the development of drug resistance. Here we addressed the hypothesis that the protein junctional adhesion molecule-A (JAM-A) may represent a novel target and a clinical biomarker in MM. We evaluated JAM-A expression in MM cell lines and in 147 MM patient bone marrow aspirates and biopsies at different disease stages. Elevated JAM-A levels in patient-derived plasma cells were correlated with poor prognosis. Moreover, circulating soluble JAM-A (sJAM-A) levels were significantly increased in MM patients as compared with controls. Notably, in vitro JAM-A inhibition impaired MM migration, colony formation, chemotaxis, proliferation and viability. In vivo treatment with an anti-JAM-A monoclonal antibody (αJAM-A moAb) impaired tumor progression in a murine xenograft MM model. These results demonstrate that therapeutic targeting of JAM-A has the potential to prevent MM progression, and lead us to propose JAM-A as a biomarker in MM, and sJAM-A as a serum-based marker for clinical stratification.

Halting pro-survival autophagy by TGFß inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients.

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) ß. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFß mediates bort-induced autophagy, and its blockade by LY2109761, a selective TßRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies.

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-ß, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Sjögren's syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB.

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren's syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

Blockade of the Ras pathway by manumycin, a farnesyltransferase inhibitor, overcomes the resistance of myeloma plasma cells to Fas-induced apoptosis.

Ras activation (by point mutation or binding of IL-6) is frequently observed in multiple myeloma (MM). As farnesylation of Ras protein by farnesyltransferase is a critical step for Ras functional activity, farnesyltransferase inhibitors (FTI) have emerged as potential anti-cancer agents. Manumycin, a natural FTI, prevents proliferation and induces apoptosis of myeloma cells refractory to Fasand drug-induced cell death. Fas pathway analysis showed that Fas-resistant apoptosis of Fas-positive myeloma cells parallels FLIP (FLICE/caspase-8-inhibitory protein) expression. Treatment of fresh purified myeloma cells, myeloma cell clone-2 and U266 cell line with manumycin induced down-regulation of FLIP expression with concomitant expression of Apo 2.7 antigen, the marker of early apoptosis. Down-regulation of FLIP mRNA levels in drug-treated cells was associated to suppression of the transcription factor NF-kappaB that plays a central role in chemoresistance, survival and proliferation of myeloma cells. Further analysis showed that manumycin-induced apoptosis involved caspases activation and was prevented by the addition of caspases specific inhibitors. Finally, pretreatment of Fas-resistant/FLIP-positive cells with manumycin sensitised them to Fas-triggered apoptosis. Overall results indicate that manumycin-induced apoptosis involves Fas pathway. FTIs may thus be proposed as a promising class of anti-cancer agents which can boost the cytotoxic effect of conventional drugs by overcoming NF-kappaB activation and Fas-resistant apoptosis.

Combined cyclosporin-A /prednisone therapy of patients with active uveitis suppresses IFN-gamma production and the function of dendritic cells.

In this study, we assessed the Th1/Th2 polarization of the immune response and the involvement of dendritic cells (DC) and Th1 lymphocytes in the pathogenesis of uveitis. Thirty-seven patients with chronic idiopathic uveitis were enrolled: 21 of them had active uveitis and the remaining 16 were in complete remission. Patients with active uveitis were characterized as follows: 5 had intermediate uveitis, 5 panuveitis and the remaining 11 posterior uveitis. Thirteen healthy subjects were also studied as controls. Patients with active uveitis were treated with cyclosporin-A (CsA) associated to low doses of prednisone (PDS) and studied at baseline and after 6 months of therapy. Analysis of cytokine-producing CD3+ lymphocytes revealed a strong Th1 polarization of the immune response in patients with active uveitis. Th1 lymphocytes paralleled serum IL-12 levels and the response to therapy, which greatly reduced both IFN-gamma+/CD3+ lymphocytes and serum IL-12 levels, associated with a general clinical improvement. In vitro studies demonstrated that DC from untreated patients with active uveitis were mature and functionally active. In fact, they showed a higher ability to stimulate cell proliferation of allogeneic T cells in primary mixed lymphocyte reaction (MLR) and produced larger amounts of IL-12 than DC from CsA/PDS-treated patients and those in remission. These results demonstrate that CsA/PDS therapy impairs the capacity of mature DC to secrete IL-12 and inhibits their MLR activity.

Multiple myeloma and mycosis fungoides in the same patient: clinical, immunologic, and molecular studies.

The coexistence of multiple myeloma (MM) and mycosis fungoides (MF), and hence of both B- and T-cell neoplastic clones, is a rare event. We describe a case of plaque-stage MF associated with IgG lambda MM. The clinical onset of MF preceded that of MM, supporting the hypothesis that MF predisposed to the development of the B-cell proliferative disorder. The skin tumor cells were found to originate from CD4(+) T-lymphocytes, characterized by a V beta 8 receptor and no proviral human T-lymphotropic virus (HTLV)-I monoclonal integration. They also displayed a polarized Th1-type cytokine profile containing mRNAs for interleukin (IL)-2, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-beta, but not for IL-4, IL-5, or IL-10. The CD8(+)/CD57(+) suppressor-cytotoxic subpopulation was significantly increased in the peripheral blood and was associated with MM progression. Expression of p16INK4A, a gene from the INK family that inhibits cyclin-dependent kinases and regulates the cell cycle, was lacking in MF cells and bone marrow (BM) plasma cells. The p16INK4A gene was silenced by hypermethylation, suggesting that it plays a role in the early phase of the pathogenesis of both diseases. Thus, a lymphoid precursor cell presumably contains aberrations that induce the development of both clonal diseases in a multistep process.

Deregulated cytokine network and defective Th1 immune response in multiple myeloma.

Intracellular cytokine production by peripheral blood mononuclear cells (PBMC) was analysed in 51 patients with multiple myeloma (MM), 22 with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy subjects, as a parameter of immunological dysfunction in MM. An increased proportion of T cells and HLA-DR+ cells producing IL-6 was observed in MM patients with active disease (at diagnosis and relapsing) compared with patients in remission and with MGUS, whereas no difference of IFN-gamma+, IL-2+ PBMC between patients and controls was evident. Determination of serum cytokine levels demonstrated that the imbalanced IL-6 production by T cells and the defective anti-tumour Th1 cell activity were related to elevated levels of IL-6 and IL-12. In vitro studies of PHA- and anti-CD3/anti-CD28 MoAbs stimulation of PBMC demonstrated the ability of lymphocytes from MM patients to differentiate towards the Th1 subset in the presence of rIL-12. By contrast, addition of exogenous rIL-6 impaired IFN-gamma production by rIL-12-prompted T cells. Inhibition of Th1 polarization of the immune response by IL-6 was direct on T cells and not mediated by dendritic cells (DC). Evaluation of the ability of MM-derived DC to stimulate cell proliferation of allogenic T lymphocytes and produce IL-12 in vitro, in fact, suggested that MM-derived DC were functionally active. Taken as a whole, these results indicate that a deregulated cytokine network occurs in active MM. They also suggest that increased IL-6 production by peripheral T lymphocytes contributes to the immune dysfunction observed in MM, and enables tumour cells to escape immune surveillance by preventing the anti-tumour Th1 immune response.

Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis.

In this study, flow cytometry was used to evaluate interleukin-6 (IL-6) production by bone marrow mononuclear cells from 47 patients with multiple myeloma (MM) in different clinical stages and 15 patients with monoclonal gammopathy of undetermined significance. In patients with MM, autocrine IL-6 production paralleled the clinical disease stage. The largest proportion of syndecan-1(+)/IL-6(+) cells was detected in patients with resistant relapse or primary refractory disease, suggesting that tumor progression involves expansion of myeloma cells producing IL-6. The authors assessed autocrine IL-6 production and in vitro proliferation and apoptosis of myeloma cells in 6 myeloma cell clones (MCCs) and in 2 myeloma cell lines, namely IM-9 and U-266-1970, which showed different sensitivities to the addition of exogenous IL-6. Autocrine IL-6 production was observed in IL-6-independent MCC-2, MCC-3, and MCC-5 cloned from patients with aggressive disease and in the IM-9 cell line. In contrast, IL-6-dependent MCC-1, MCC-4, and MCC-6 were syndecan-1(+) and IL-6(-). Blocking experiments with anti-IL-6 monoclonal antibody from clone AH65, which binds IL-6-IL-6Ralpha complexes, prevented cell proliferation of IL-6(+) MCCs. Flow cytometry evaluations after propidium iodide staining revealed different susceptibilities of MCCs to cell death. IL-6-producing MCCs showed minimal spontaneous and dexamethasone-induced apoptosis, whereas a regular amplitude of apoptosis occurred in the IL-6(-) MCCs. These data provide evidence that autocrine IL-6 reflects a highly malignant phenotype of myeloma cells. In fact, autocrine IL-6 production and deregulated apoptosis may induce expansion of selective IL-6(+) myeloma cells resistant to spontaneous and drug-induced cell death.

Molecular mechanisms of augmenter of liver regeneration as immunoregulator: its effect on interferon-gamma expression in rat liver.

BACKGROUND: We have shown that the administration of exogenous Augmenter of Liver Regeneration protein in intact rats i) regulates mitochondrial gene expression by inducing the transcription and translation of the nuclear-encoded mitochondrial transcription factor A, and ii) inhibits the lytic activity of liver-resident Natural Killer cells. AIMS: The present investigation was carried out to study the effect, in intact rats, of exogenous administration of Augmenter of Liver Regeneration protein on Interferon-gamma, a cytokine produced by activated Natural Killer cells and known to control the expression of mitochondrial transcription factor A, a nuclear gene responsible for mitochondrial metabolism. METHODS: Interferon-gamma was measured as messenger RNA in liver-derived mononuclear leukocytes and as protein in liver-derived Natural Killer cells after a single injection of Augmenter of Liver Regeneration protein. RESULTS: The data obtained demonstrate that: i) in intact rats, Augmenter of Liver Regeneration protein administration induces a reduction of Interferon-gamma in the liver-resident Natural Killer cells and ii) the administration of Interferon-gamma in 70% hepatectomized rats is followed by a significant reduction both of the mitochondrial transcription factor A expression and of liver regeneration. CONCLUSIONS: These data demonstrate the pivotal role of Augmenter of Liver Regeneration as Growth Factor and as immunoregulator by controlling, through Interferon-gamma levels, the mitochondrial transcription factor A expression and the lytic activity of liver-resident Natural Killer cells.

Th1 polarization of the immune response in Behçet's disease: a putative pathogenetic role of interleukin-12.

OBJECTIVE: To investigate whether immunologic abnormalities in patients with Behçet's disease (BD) are related to abnormalities of the Th1/Th2 ratio. METHODS: Th1/Th2 cytokine production by peripheral blood lymphocytes (PBL) from 31 patients with BD, 11 patients with inflammatory arthritis, and 10 healthy blood donors was evaluated by intracellular immunofluorescence staining. Serum interleukin-12 (IL-12) levels were measured using an enzyme amplified-sensitivity immunoassay. The effect of recombinant IL-12 (rIL-12) on spontaneous and Fas-mediated apoptosis of phytohemagglutinin (PHA)-stimulated PBL was evaluated by flow cytometry using propidium iodide (PI) staining and a bromodeoxyuridine (BrdU)/PI procedure. RESULTS: Intracellular immunofluorescence staining of IL-2, IL-4, and interferon-gamma (IFNgamma) in CD3+ lymphocytes from BD patients demonstrated a strong polarization of the immune response toward the Th1 pathway that correlated with the progression of BD. Peripheral Th1 cells were significantly increased in patients with active disease (n = 14) as compared with those in patients in complete remission (n = 17), patients with inflammatory arthritis, and normal donors. In addition, serum IL-12 levels were correlated with peripheral Th1 lymphocytes and disease progression. Apoptotic analysis revealed that PHA-activated PBL from patients with active disease were highly sensitive to spontaneous and Fas-mediated activation-induced cell death. However, addition of rIL-12 to complete medium prevented this spontaneous and Fas-induced apoptosis and enhanced the proliferation of Th1 lymphocytes. CONCLUSION: Taken together, these results indicate that a strong Th1 immune response occurs in active BD and suggest that IL-12 plays a substantial part in the pathogenesis of BD. By preventing spontaneous and Fas-induced cell death, in fact, it results in an abnormal growth of autoreactive Th1 lymphocytes that could contribute to the prolonged inflammatory autoimmune condition of BD.

Fas/Fas ligand (FasL)-deregulated apoptosis and IL-6 insensitivity in highly malignant myeloma cells.

IL-6 is a growth factor which interferes in the apoptosis of malignant plasma cells. Here we explore its role in the spontaneous and Fas/FasL-regulated apoptosis of seven myeloma cell clones (MCC). MCC-2 and -7 were constitutively defective in Fas antigen in the presence of large membrane exposure of FasL, and showed a high rate of cell proliferation irrespective of the presence of IL-6. Cytofluorimetric analysis following propidium iodide (PI) staining revealed a minimal extent of spontaneous apoptosis, as in other IL-6-insensitive, though Fas-positive MCC, namely MCC-3 and -5. By contrast, a regular amplitude of apoptosis occurred in the remaining IL-6-dependent clones. Their propensity to cell death, as well as their FasL membrane expression, were promptly down-modulated by the cytokine, whereas no substantial effect was detected in IL-6-independent MCC. Furthermore, we investigated the quantitative secretion of FasL. Both [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) cytotoxicity assay and PI staining of WC8 lymphoblasts from a Fas-transfected mouse lymphoma, incubated with supernatants from MCC, showed a variable cytocidal property, thus confirming the cellular release of FasL. However, a significant elevation of FasL secretion occurred in both Fas- MCC, whereas molecular cloning and sequencing of Fas revealed the presence of a splicing variant, namely Fas Exo4,6Del, in the cDNA from both MCC-3 and -5, which were previously demonstrated to be unresponsive to Fas stimulation. Taken together, these data provide evidence that concurrence of IL-6 insensitivity and deregulation of apoptosis in myeloma cells reflects a high malignancy grade. It is suggested that the secretion of Fas splicing variants in Fas+ plasma cells, as well as the over-production of FasL in Fas- myelomas, are differential mechanisms by which myeloma cells escape host immune surveillance.

CD8+/CD57 cells and apoptosis suppress T-cell functions in multiple myeloma.

The aim of this study was to evaluate the role of CD8+/CD57+ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8+/CD57+ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR+ and Fas+. Removal of CD8+/CD57+ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8+/CD57+ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8+/CD57+ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8+/CD57+ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8+/CD57+ population. Our results indicate that both the immunosuppressive effect of CD8+/CD57+ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.

Functional Fas-ligand expression on T cells from HIV-1-infected patients is unrelated to CD4+ lymphopenia.

Recent studies have demonstrated that the expression of Fas by peripheral T cells from HIV-1+ patients is deregulated and increases the susceptibility of these cells to undergo apoptosis. Here, we show that secretion of Fas-ligand (L), the complementary agonist of Fas, is abnormally upregulated in CD4+ cells from HIV-1-infected individuals, particularly during the non-lymphopenic stages of the disease. An increase of soluble Fas-L occurred in T cell cultures from 26 patients with a number of CD4+ cells higher than 400/microliter, whereas it was almost undetectable in cultures from 21 severely lymphopenic patients (CD4+ < 200/microliter). The MTT test, cytofluorimetric analysis of cellular DNA, cytotoxicity, and proliferative assays using the Fas-transfected WC8 mouse lymphoma confirmed the cytocidal capability of T cell supernatants from non-lymphopenic patients. Double-fluorescence analysis revealed that the majority of CD4+ cells (approximately 90%) in these cultures secreted Fas-L in the presence of high intracellular gamma-interferon and low Bcl-2. In contrast, the CD8+/Fas-L+ population was comparably decreased (approximately 55%). Molecular cloning of Fas-L revealed a substantial expression of Fas-L mRNA in cells from non-lymphopenic patients compared with patients with advanced disease and healthy controls. Since CD4+ cells of Th1 phenotype are impaired during HIV-1 infection and show high cellular expression of Fas-L, it is conceivable that excess Fas-L during the early or non-lymphopenic phase of the disease increases the extent of apoptosis in these cells by the Fas/Fas-L pathway. The defective expression of the ligand in severely lymphopenic stages could be explained by exhaustion of this mechanism as the disease progresses.

IgG M-components in active myeloma patients induce a down-regulation of natural killer cell activity.

Natural killer cell activity and related cell surface markers of peripheral blood lymphocytes were studied in 73 patients with multiple myeloma, 25 with monoclonal gammopathy of undetermined significance and 20 normal controls. Natural killer cell number was significantly higher in both multiple myeloma and monoclonal gammopathy patients than in controls, whereas the natural killer activity of multiple myeloma patients was inversely related to their disease status. Incubation of peripheral blood lymphocytes or natural killer cells with IgG myeloma proteins purified from several patients induced a down-modulation of basic natural killer activity. This inhibitory effect of monoclonal IgG was dose dependent and significantly stronger in patients with active (at diagnosis and at relapse) than stable multiple myeloma or in normal controls. Addition of exogenous recombinant interleukin-2 restored natural killer cell activity against K562 target cells, indicating that natural killer cells were able to recover their functions. However, recombinant interleukin-2-stimulated natural killer cells were responsive to down-modulation of monoclonal IgG. These data suggest that impaired natural killer cell function in active multiple myeloma is caused by the inhibitory effect of M-component.

Antiphosphatidylserine antibodies in human immunodeficiency virus-1 patients with evidence of T-cell apoptosis and mediate antibody-dependent cellular cytotoxicity.

Serum reactivities to a panel of phospholipid antigens, including cardiolipin (CL), phosphatidylserine (PS), sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, were measured by enzyme-linked immunosorbent assay in 196 human immunodeficiency virus-l+ (HIV-1+) patients with CDC II to IVC clinical disease. Significant levels of IgG to CL, PS, or both were observed in 23 patients lacking evidence of thrombophilic events or any peculiar clinical feature of HIV-1 infection. Fluorescence-activated cell sorting analyses showed that in vitro apoptosis of T cells was increased in patients with high serum anti-PS IgG, whereas the overexpression of Fas/Apo-1 marker was detected in all patients regardless of their antiphospholipid reactivities. Macrophages from patients with significant titers of anti-PS IgG antibodies were not activated by the presence of apoptotic CEM lymphoblasts or by purified anti-PS IgG from the same patients. By contrast, these antibodies greatly improved the effector functions of autologous macrophages in antibody-dependent cellular cytotoxicity (ADCC) assays using 51Cr-labeled CEM cells, whereas polyspecific IgG were unable to induce an equivalent cytotoxicity in all instances. An increasing effect on ADCC was also observed in tests using macrophages from healthy controls to CEM coated with anti-PS IgG. These results support a potential correlation of anti-PS specificity with T-cell apoptosis in HIV-1 infection. Because PS is exteriorized by apoptotic lymphocytes, its persistence may stimulate antibodies which cooperate with macrophages in the clearance of dead cells by an enhanced ADCC mechanism. This interpretation could explain the absence of thrombophilia in HIV-1+ patients with serum elevations of antiphospholipid reactivities.

Overexpression of Fas antigen on T cells in advanced HIV-1 infection: differential ligation constantly induces apoptosis.

OBJECTIVES: To investigate Fas in peripheral lymphocytes from HIV-1-positive patients at different disease stages with respect to the extent of apoptosis. DESIGN: The study included analysis of Fas involvement in T-cell apoptosis observed during HIV-1 infection. Because ligation of Fas can result in costimulation of proliferation or the induction of apoptosis in uninfected cells, we evaluated the effect on T cells of Fas activation by monoclonal antibodies (MAb) of different specificity from both UB2 and CH11 clones and activation by the Fas ligand (Fas-L). METHODS: Fas was measured by FACS in peripheral blood and in phytohaemagglutinin (PHA)-driven cultures derived from 59 HIV-1-positive individuals with different Centers for Disease Control and Prevention stages. The percentage of apoptotic cells was detected by propidium iodide cell staining. The effect of Fas ligation was assessed in peripheral T cells from patients and healthy controls by a proliferative test measuring the 3H-thymidine uptake. RESULTS: FACS analysis revealed that Fas was predominantly expressed in advanced disease, although it was promptly exposed in PHA cultures from asymptomatic individuals. In several instances, Fas overexpression was associated with substantial subdiploid DNA content in cells from severely lymphopenic patients. The proliferative assay showed a significant inhibition of 3H-thymidine uptake in T cells from all patients following Fas ligation by the immunoglobulin (Ig) G1 MAb from the UB2 clone. This was in contrast to the apparent cell activation detected in controls and the weak suppression observed in Fas-positive cell lines. In addition, the IgM anti-Fas and recombinant Fas-L concentrations inducing a moderate inhibition of fresh T cells from controls strongly depressed the proliferative rate of cells from patients. CONCLUSIONS: Our data suggest that Fas overexpression parallels the progression of the disease and that the increased susceptibility of T cells from HIV-1-infected individuals to undergo apoptosis may include a Fas pathway. Functionally exhausted T cells in advanced HIV-1 infection are primed to apoptosis because of their high sensitivity to Fas stimulation even using the IgG1 MAb, which is unreactive to the death domain of Fas. This suggests that the increased sensitivity of Fas is apparently unrelated to its trimeric ligation and supports the hypothesis that Fas pathway plays a role in increasing the lymphocyte apoptosis during the disease.