No abstract

SUMMARYLactoferrin (LF), an iron-binding protein present in specific granules of neutrophils, is expressed on membrane after granulocyte activation. It may represent a target for anti-neutrophil cytoplasmic antibodies (ANCA) in patients affected by some immunomediated diseases. We recently produced two MoAbs, AGM 2.29 and AGM 10.14, that recognize two spatially distant epitopes of human LF. In this study we perform a cytometric analysis in order to evaluate the expression of LF on the surface of granulocytes obtained from freshly drawn blood or after purification, in both the presence and absence of stimuli. Our results demonstrate that LF is not constitutively expressed on membrane of circulating neutrophils. After priming with phorbol myristate acetate (PMA) or tumour necrosis factor-alpha (TNF-a), an increased mean fluorescence intensity (MFI) was obtained on neutrophils stained with polyclonal anti-LF antibodies and with AGM 2.29. The kinetics of LF expression during activation demonstrated a progressive increase in MFI within 45 min. No increase in MFI was documented when primed granulocytes were stained with MoAb AGM 10.14, thus indicating that the epitope recognized by AGM 10.14 is not exposed at the cell surface. Following membrane permeabilization, performed in order to analyse the binding of anti-LF MoAbs to cytoplasmic LF, a marked increase in MFI was obtained by staining granulocytes with both anti-LF MoAbs. Indirect immunofluorescence (IIF) analysis confirmed that AGM 2.29 and AGM 10.14 reacted with human granulocytes, showing a cytoplasmic pattern on formalin-acetone-fixed neutrophils and a perinuclear one on ethanol-fixed cells.

Lactoferrin is a multifunctional immunoregulatory protein, stored in specific granules of neutrophil granulocytes, from which it is released following cell activation. As activated neutrophils play a crucial role in the destruction of synovial joints in rheumatoid arthritis, we evaluated lactoferrin concentration in synovial fluid and sera from 21 patients with rheumatoid arthritis and 11 patients with osteoarthritis. We also measured lactoferrin levels in sera from 12 healthy controls. Lactoferrin was measured by a solid-phase inhibition immunoassay. Median lactoferrin levels were significantly higher in synovial fluid from rheumatoid arthritis than from osteoarthritis patients (P = 0.0002). In contrast, no significant difference was found between serum lactoferrin from patients with rheumatoid arthritis or osteoarthritis compared with normal controls. In patients with rheumatoid arthritis, lactoferrin concentrations were higher in synovial fluid than in sera (P = 0.036). In both rheumatoid arthritis and osteoarthritis no correlation was found between serum and synovial fluid lactoferrin (P = 0.51 and P = 0.5, respectively). In synovial fluid from patients with rheumatoid arthritis, lactoferrin concentrations correlated with neutrophil granulocyte count (P < 0.0001), but neither serum nor synovial lactoferrin levels correlated with disease activity (P = 0.32 and P = 0.25, respectively). In conclusion, lactoferrin is a reliable marker of neutrophil activation at sites of inflammation in rheumatoid synovitis, but does not represent a marker of disease activity.

Lipoprotein(a) (Lp[a]) is generally considered to be a risk factor for the development of cardiovascular disease, but little is known about the possible influence of obesity on the circulating levels of this lipoprotein. The present study was undertaken to examine this aspect in 136 menstrually active women by comparing the serum concentrations of Lp(a) between 72 obese and 64 age-matched nonobese women. Since an adverse effect of androgens and a protective effect of estrogens have been described for plasma lipoprotein profiles in obese women, the relation between the circulating levels of Lp(a) and those of these other hormones was also investigated in obese patients. In addition, other lipoproteins, anthropometric parameters (body mass index and waist-to-hip ratio), and insulin were evaluated. The levels of Lp(a) were not significantly different (Mann-Whitney U test x \ 3-59; p=0.0582 [NS]) between obese (rank sum, 5,367) and control (rank sum, 3,949) women; in addition, the percentage of patients with high Lp(a) levels (cutoff defined at 30 mg/dL) did not differ between the two groups (obese women, 30%; control, 21.8%; x 2 , 0.90; two-sided p=0.341 [NS]). Moreover, no correlation was found between Lp(a) and body mass index. Lastly, when the Lp(a) prevalence odds ratio for obesity was examined by adjusting the levels of this lipoprotein for age, triglycerides, total cholesterol, and high density lipoprotein cholesterol, the probability value (0.88) was far from significant. In obese women, no correlation was found between the logarithmically transformed Lp(a) concentrations and all the other variables evaluated in the study. In conclusion, the present study shows that the circulating levels of Lp ( 1 It is synthesized by the liver 2 and secreted directly into the systemic circulation.

Fifteen patients affected by new-onset Graves' disease with ophthalmopathy have been examined for lymphocyte subset phenotypes by two-colour cytometric analysis. CD5+ B cells were significantly increased in comparison to the normal population (43.2 +/- 20.6% versus 19.5 +/- 6.9%; p < 0.0009); total T cells showed a normal amount (70.4 +/- 9.6%), but the levels of THLADR+ cells were significantly increased in comparison to the control group (13.7 +/- 10.5% versus 4.7 +/- 3.2%; p < 0.007). The characterization of the subset suppressor-inducer CD4+ CD45RA+, evaluated as percentage of the total CD4+ T cells, showed a significant decrease in comparison with the control group (43.8 +/- 13.5% versus 52.3 +/- 6.5%; p < 0.03). Serum immunoglobulin evaluation showed a statistically significant increase of IgM (p < 0.05) and a significant decrease of IgA (p < 0.02). A statistical correlation between IgM levels and percentage of CD5+ B cells has been found (p < 0.04). The defect of suppressor-inducer T cell subset, along with the increase of CD5+ B cells may contribute to the pathogenetic mechanism of the disease in patients with a genetically-induced aberrant immune response.