Free light chains a novel biomarker of cardiovascular disease. A pilot study.

OBJECTIVE: Atherosclerosis and ischemic heart disease (IHD) are the major cause of morbidity and mortality but their inflammatory pathogenesis is still unclear. In this scenario, the role of serum free light chains (sFLC) has never been fully evaluated. The aim of the present study is to assess the clinical and pathogenetic role of sFLC in patients with IHD and to propose their use as a new biomarker for cardiovascular disease. PATIENTS AND METHODS: We enrolled 117 patients, divided into 5 cohorts: 15 healthy controls, non-diabetic and without ischemic heart disease; 19 patients with type 2 diabetes (T2DM), without ischemic heart disease at recruitment; 39 patients with stable chronic angina; 27 patients with NSTEMI, 17 patients with acute STEMI. Serum sFLC and high-sensitive C-reactive protein (hs-CRP) were measured. Patients also underwent a transthoracic echocardiographic study. RESULTS: sFLC were higher in patients with IHD and T2DM. However, we did not find statistically significant differences in sFLC concentration among subgroups. No correlation resulted between sFLC and hs-CRP levels. The median value of the sFLC κ/λ ratio in the population was 0.63, therefore stratifying it into two groups according to their levels. We found that an increase in left ventricular ejection fraction at 12 months was detected in 77% of patients with κ/λ ratio higher than 0.63 and 25% of patients with κ/λ ratio lower of 0.63 (p=0.016, OR=10.0 [1.8-55.6]). CONCLUSIONS: Our study suggests that the sFLC, produced by the B-lymphocytes in the context of generalized immune activation, could play a pathogenetic role in acute coronary syndromes and that they could represent a novel risk biomarker of cardiovascular disease.

Enhanced production of tumor necrosis factor-alpha and interleukin-6 due to prolonged response to lipopolysaccharide in human macrophages infected in vitro with human immunodeficiency virus type 1.

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages.

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.

Bile acids with differing hydrophilic-hydrophobic properties do not influence cytokine production by human monocytes and murine Kupffer cells.

Bile acids have been proposed to exert immunological effects of potential pathogenic or therapeutic relevance, yet the experimental evidence remains preliminary. We reexamined the effects of a variety of bile salts with differing hydrophilic-hydrophobic properties on the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) from monocytes and Kupffer cells. Monocytes from healthy human donors and Kupffer cells from 5-week-old mice were incubated for up to 18 hours with or without varying concentrations of bile salts and lipopolysaccharide (LPS). Monocyte viability was > or = 95% with up to 250 mumol/L sodium ursodeoxycholate and < or = 90% with 200 mumol/L chenodeoxycholate, decreasing sharply at higher concentrations. Kupffer cells were more vulnerable, particularly to chenodeoxycholate (viabilities of 25% and 0% at concentrations of 100 mumol/L and 200 mumol/L, respectively). In monocytes incubated in the presence of 20% fetal calf serum, neither ursodeoxycholate and chenodeoxycholate, nor a variety of other unconjugated and conjugated bile acids, tested up to their maximal noncytotoxic concentrations, influenced the IL-6 and TNF alpha production, at any level of LPS stimulation. Similar to monocytes, incubation of murine Kupffer cells with ursodeoxycholate and chenodeoxycholate did not influence cytokine release. In contrast, the addition of 10 nmol/L dexamethasone to monocytes significantly decreased TNF-alpha and IL-6 release (69 +/- 11% and 48 +/- 15%, respectively). When monocytes were incubated with 200 mumol/L chenodeoxycholate in the presence of lower concentrations of fetal calf serum (10% and 5%, respectively) a significant inhibition of cytokine release was observed, whereas incubation with ursodeoxycholate did not cause any effect. Flow cytometry using fluoresceinated LPS showed that chenodeoxycholate does not interact with the CD14 receptor, thus excluding the possibility of an interference with the LPS uptake by monocytes. Incubation with [14C]-chenodeoxycholate showed that the intracellular bile acid uptake was inversely related to the concentration of fetal calf serum, being negligible (< 3 fmol/cell) at the highest level. In conclusion, bile acids with widely different hydrophobicities are incapable of influencing the release of IL-6 and TNF alpha by monocytes and Kupffer cells, provided they are studied at noncytotoxic concentrations and in the presence of physiological amounts of proteins.

In vitro inhibition of the replication of human immunodeficiency virus type 1 by beta-mercaptoethylamine (cysteamine).

This study investigates the effects of cysteamine alone and in association with zidovudine or didanosine on the replication of human immunodeficiency virus type 1 (HIV-1). More than 90% viral inhibition was obtained by 200 microM cysteamine in lymphocytes and 100 microM cysteamine in macrophages against 4 primary isolates and 2 laboratory strains of HIV-1. Polymerase chain reaction analysis demonstrated that cysteamine interferes with early steps of HIV-1 replication, before proviral DNA formation. The use of cysteamine in conjunction with zidovudine or didanosine brought about an additive antiviral effect without concomitant increases in toxicity. The concentrations of cysteamine that are effective against HIV-1 in vitro have been well tolerated over long periods by patients under treatment for cystinosis, an inherited disorder. These observations suggest that cysteamine alone or in combination with zidovudine or didanosine could be a new potential treatment of HIV-1 infection.

Human immunodeficiency virus-induced cell death in cytokine-treated macrophages can be prevented by compounds that inhibit late stages of viral replication.

The basis of the cytopathic effect induced by a laboratory strain and several clinical isolates of human immunodeficiency virus (HIV) in human macrophages cultured in the presence of macrophage colony-stimulating factor was studied. Infected macrophages die of necrosis, the consequence of the production of mature virions in infected cells. Cell death can be prevented by antiviral compounds that interfere with the assembly and budding of virions. Programmed cell death (apoptosis), a potential mechanism of HIV-mediated cell death in CD4 T lymphocytes, does not occur in infected macrophages as shown by electron microscopy, cytofluorometric and gel electrophoretic DNA analysis, and nuclear fluorescent staining by Hoechst and terminal dUTP-nick-end-labeling (TUNEL) assay. The data suggest that macrophage killing by HIV may occur in vivo. Thus, combination therapies that include compounds that inhibit the cytopathic effect of HIV in macrophages should be considered for AIDS patients.

Macrophage colony-stimulating factor enhances the susceptibility of macrophages to infection by human immunodeficiency virus and reduces the activity of compounds that inhibit virus binding.

The effects of macrophage colony-stimulating factor (M-CSF) on CD4 receptor expression, susceptibility to human immunodeficiency virus type 1 (HIV) infection, and anti-HIV activity of dextran sulfate and soluble-CD4 were studied in cultured, human primary macrophages. M-CSF stimulated macrophage cells to express the CD4 receptor, and this resulted in an increase of both the number of CD4+ cells and the density of the receptor on the cell surface. M-CSF also significantly enhanced the susceptibility of macrophage cells to HIV infection. Interestingly, the anti-HIV activity of dextran sulfate and soluble-CD4 (two compounds that interfere with HIV-CD4 binding with different mechanisms) was reduced 100-fold and fivefold, respectively, in M-CSF-treated macrophages. Human blood concentrations of M-CSF are reported to be similar to those used in this work (1,000 U/mL); thus, it is conceivable that also in vivo this cytokine may modify the susceptibility of macrophages to HIV and the ability of dextran sulfate and soluble CD4 to inhibit HIV replication. These results suggest that the in vitro study in M-CSF-treated macrophages of promising drugs inhibitors of HIV-CD4 binding could provide further insights into the potential efficacy of these compounds in patients.