18F-FDG uptake in main arterial branches of patients with large vessel vasculitis: visual and semiquantitative analysis.

OBJECTIVE: Over the last decade, the contribution of (18)F-FDG (FDG) PET/CT imaging to the diagnosis of large vessel vasculitis has been widely investigated. The aim of this study was to evaluate a more extensive role for PET/CT in grading vascular inflammation in patients with different clinical stages of disease. METHODS: The images of 66 PET/CT studies of 34 patients, performed at diagnosis and/or during follow-up were reviewed. FDG uptake in different regions of aorta and in its major branches was visually (regional Score: rS) and semiquantitatively (regional SUVmean: rSUV) assessed. The global vascular uptake was also evaluated for each study by summing all rSs (summed Score; sS) and averaging rSUVs (averaged SUV; aSUV). FDG uptake in 15 PET/CT studies of control age-matched subjects without signs or symptoms of vasculitis was also analyzed. RESULTS: Higher levels of regional and global FDG uptake were found at diagnosis in comparison with follow-up studies of 12 patients with complete longitudinal observation (p value range 0.0552-0.0026). In the latter group high values were generally observed when disease relapse or incomplete response to therapy (active disease) occurred, whereas lower uptake was found in studies of remitted patients (p = <0.01), whose FDG levels were similar to those of control subjects. At ROC analysis performed on all image dataset, optimal cut-off levels of regional and global FDG vascular uptake provided a good discrimination between 25 patients at diagnosis and 15 control subjects (aSUV greater than 0.697; PPV = 92.3; NPV = 92.9). Major overlap was observed among FDG levels of 21 patients with active disease and in remission (aSUV greater than 0.653; PPV = 58.3; NPV = 94.1). Similar performances of visual and semiquantitative analyses were found when areas under curves (AUCs) were compared. CONCLUSIONS: (18)F-FDG PET/CT has a promising role in grading inflammation in patients with large arteries vasculitis. Nevertheless, a cut-off based analysis of FDG vascular uptake is not sufficient to separate patients with active and inactive disease during follow-up.

An encapsulated juice powder concentrate improves markers of pulmonary function and cardiovascular risk factors in heavy smokers.

OBJECTIVE: Cigarette smoking is associated with reduced pulmonary function and increased risk factors for cardiovascular disease. This randomized placebo-controlled double-blind study evaluated the effects of two different combinations of mixed fruit and vegetable juice powder concentrate (Juice Plus+, NSA, Collierville, TN) on heavy smokers. METHODS: At baseline (T 0) and after 3 months' supplementation (T 1), pulmonary function parameters and cardiovascular risk factors-that is, plasma total homocysteine (tHcy) with related B vitamins and cysteine (tCys) concentrations-were assessed in 75 apparently healthy smokers (aged 49.2 ± 10.6 years, >20 cigarettes/d, duration ≥10 years) randomized into 3 groups: placebo (P), fruit/vegetable (FV) and fruit/vegetable/berry (FVB). RESULTS: T 0: most smokers showed abnormalities in tHcy and tCys concentrations. T 1: respiratory function was unchanged in P and slightly, but not significantly, improved in FV, whereas FVB showed a significant improvement in forced expiratory flow at 25% (FEF25; p < 0.0001 vs P and FV) and significant improvement in CO diffusion lung/alveolar volume (DLCO/VA). FV and FVB (50%) showed significant reduction in tHcy and tCys compared to T 0 ( p < 0.0001) and P ( p < 0.0001). CONCLUSIONS: At T 1, both supplemented groups, but to a greater extent the FVB group, showed improvements in some pulmonary parameters, cardiovascular risk factors, and folate status. The beneficial effects of Juice Plus+ supplementation could potentially help smokers, even if smoking cessation is advisable.

Serum BAFF concentrations in patients with Graves' disease and orbitopathy before and after immunosuppressive therapy.

INTRODUCTION: B cells are known to play a key role in the pathogenesis of autoimmune disease. B lymphocyte activating factor (BAFF), a member of TNF family, promotes autoantibody production by increasing B cell survival and proliferation. Serum BAFF concentrations have been found to be increased in systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome. OBJECTIVE: We have measured serum BAFF concentrations in patients with Graves' disease (GD) with or without Graves' orbitopathy (GO) and in active GO in relation to immunosuppressive treatment. METHODS: Forty-two patients and nine normal controls were studied. Thirty-four patients had GO, which was active in 23. Of these, nine were treated with rituximab (RTX) and 14 with i.v. methylprednisolone (MP). Serum BAFF concentrations were measured at baseline in all patients, at peripheral B cell depletion and repopulation after RTX, and after therapy with MP. RESULTS: Serum basal BAFF concentrations in GD patients were significantly higher when compared with normal controls (P = 0.0001), and no difference was observed in those with active or inactive GO. Serum BAFF concentrations were also significantly correlated with serum antithyroglobulin antibodies (P = 0.04) but not with sex, age, smoking habits, therapy for thyroid disease, and serum antithyroperoxidase antibodies and TSH receptor antibodies. After RTX, there was an increase of serum BAFF concentrations at the time of B cell depletion (P = 0.02) but also at B cell repopulation (P = 0.04). In patients treated with MP, serum BAFF concentrations decreased significantly after therapy (P < 0.01). CONCLUSIONS: We report that serum BAFF concentrations are elevated in patients with GD, in whom hyperthyroidism is known to be based on a B-cell-driven pathophysiological mechanism. In active GO, BAFF further increases after therapy with RTX as a consequence of the B cell depletion per se. The decrease of serum BAFF after iv steroids suggests that MP may exert an immunosuppressive effect by modifying B-cell-derived immune reactions.

Severity assessment of healthcare-associated pneumonia and pneumonia in immunosuppression.

The study compares the ability of the PSI (pneumonia severity index), CURB-65 (confusion, urea >7 mol·L(-1), respiratory rate ≥ 30 breaths·min(-1), blood pressure <90 mmHg systolic or ≤ 60 mmHg diastolic, and age ≥ 65 yrs), CURB and CRB-65 scales and the Severe Community-Acquired Pneumonia (SCAP) score to predict 30-day mortality in healthcare-associated pneumonia (HCAP) patients, and analyses differences in the demographics, aetiology and outcomes of community-acquired pneumonia (CAP), HCAP and pneumonia in immunocompromised patients. 629 consecutive patients admitted to a tertiary care university hospital were prospectively categorised as having CAP (n=322) or HCAP (n=307), and the HCAP patients were further sub-divided into those who were immunocompromised (n=219) or immunocompetent (n=88). The 30-day mortality rate was 9.0% in the CAP group and 24.1% in the HCAP group. In the HCAP group, the PSI and SCAP scores had similar prognostic power (area under the curve (AUC) of 0.68 and 0.67, respectively) and performed better than the CURB-65 score (AUC ≤0.62). Among the immunocompetent HCAP patients, the PSI and CURB-65 scores were more sensitive than the others at every threshold, whereas SCAP was more specific than both of these. In the immunocompromised group, the PSI was highly sensitive but poorly specific at all thresholds. Our results suggest that prognostic tools should be designed for subsets of HCAP patients.

Leukocyte subsets dynamics following open pulmonary lobectomy for lung cancer: a prospective, observational study.

This study was planned to observe prospectively the effects of standard open pulmonary lobectomy on leukocyte subsets and their connection with oncological outcome. Leukocyte subsets from 200 patients undergoing pulmonary lobectomy were analyzed: 151 patients had non-small-cell lung cancer, and 49 had non-malignant diseases. Blood samples were taken for leukocyte flow cytometry before and five, 30 and 60 days after operation. The end points were: observation of postoperative leukocyte subsets that are dynamic in patients with lung cancer vs. patients without malignant disease; correlations between leukocyte subsets trend and disease-free interval or survival; and identification of prognostic factors related to preoperative leukocyte subsets. Lymphocyte counts significantly decreased at five days after lobectomy while monocyte counts increased, and complete recovery of the preoperative leukocyte setting was documented at 30 and 60 days. The patients with lung cancer showed a significant low percentage of human leukocyte antigens on their monocytes before surgery (P=0.0017), followed by a peculiar disarrangement of leukocytes subsets compared with patients without malignant disease at the five-day control point. There was no correlation between leukocyte subset dynamics and disease free interval or survival. This study proves reductions of T-, B and natural killer cells, and the expression of DR on T-lymphocyte after pulmonary lobectomy; oncological patients were significantly less affected by surgery than non-cancer patients.

Serum hepcidin and macrophage iron correlate with MCP-1 release and vascular damage in patients with metabolic syndrome alterations.

OBJECTIVE: Increased body iron stores and hepcidin have been hypothesized to promote atherosclerosis by inducing macrophage iron accumulation and release of cytokines, but direct demonstration in human cells is lacking. The aim of this study was to evaluate the effect of iron on cytokine release in monocytes ex vivo and the correlation with vascular damage and to evaluate the relationship among serum levels of hepcidin, cytokines, and vascular damage in patients with metabolic syndrome alterations. METHODS AND RESULTS: Manipulation of iron status with ferric ammonium citrate and hepcidin-25 induced monocyte chemoattractant protein (MCP)-1 and interleukin-6 in human differentiating monocytes of patients with hyperferritinemia associated with the metabolic syndrome (n=11), but not in subjects with hemochromatosis or HFE mutations impairing iron accumulation (n=15), and the degree of induction correlated with the presence of carotid plaques, detected by echocolor-Doppler. In monocytes of healthy subjects (n=7), iron and hepcidin increased the mRNA levels and release of MCP-1, but not of interleukin-6. In 130 patients with metabolic alterations, MCP-1 levels, as detected by ELISA, were correlated with hepcidin-25 measured by time-of-flight mass spectrometry (P=0.005) and were an independent predictor of the presence of carotid plaques (P=0.05). CONCLUSIONS: Hepcidin and macrophage iron correlate with MCP-1 release and vascular damage in high-risk individuals with metabolic alterations.

Rituximab treatment in a patient with severe thyroid-associated ophthalmopathy: effects on orbital lymphocytic infiltrates.

Rituximab (RTX) has been shown in previous work to improve thyroid-associated ophthalmopathy (TAO), but very little data is available on the effects of RTX in the target tissues. We studied the effects of RTX on peripheral lymphocytes and on the intra-orbital infiltrates in one patient with severe TAO who was treated with two cycles of therapy. Intra-orbital tissues derived at decompression from 3 patients with moderate-severe and 1 with severe TAO, treated with standard immunosuppression, were studied as controls. Peripheral blood lymphocytes were analyzed throughout the study period, while intra-orbital tissue lymphocytes at decompression. In the patient treated with RTX visual field and acuity improved in response to peripheral CD 20+ cell depletion, although there was a proportion of persisting CD 19+ cells. After RTX re-treatment the patient's optic nerve function improved only transiently. The number of CD 20+ cells was lower in orbital tissues (0-1%) than in the peripheral blood (3%). A greater percentage of CD 19+ was observed in the orbits compared to the periphery, most of which were CD 19+5+ (80%). By immunohistochemistry, orbital tissues from all control patients showed CD 20+ and CD 3+ cells, independently of the duration of TAO and of the treatment with either steroids or radiotherapy. This is the first report on the therapeutic effect of RTX in active, severe TAO associated to the depletion of intra-orbital CD 20+ lymphocytes. After RTX, CD 19+5+ lymphocytes were shown to be 2-3 times more prevalent in the orbital infiltrates, compared to CD 20+ cells. Persistence of autoreactive cells is believed to be related to TAO relapse.

Erythroid differentiation and maturation from peripheral CD34+ cells in liquid culture: cellular and molecular characterization.

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation from BFU-E to mature erythrocytes both in normal and pathological conditions. Most of the available in vitro liquid cultures are from cell lines or are limited by the production of few erythroid cells mixed with myeloid cells. Here we describe an erythroid liquid culture system starting from CD34(+)-enriched cells obtained from peripheral blood. CD34(+) cells were cultured for 21 days in different conditions. Precisely stem cell factor (SCF, 20 ng/mL) and IL-3 (10 ng/mL) were added at starting point plus Epo (3 U/mL) at day 0 or 7 of culture with or without cyclosporine A (Cy; 1 mg/mL). In all the conditions, the highest recovery was obtained at day 14 of culture. Epo and Cy added at day 0 produced the highest cell expansion (170-fold mean amplification of the initial cell input by day 14) and recovery of erythroid cell. Sixty seven percent of the cells were GP(+) at day 7 and 97% by day 14 respectively. Most of the cells were proerythroblasts at day 7 and mature erythroblasts at day 14 (>90% were benzidine(pos)). The presence of Cy favoured erythroid differentiation and maturation and reduced the percentage of non-erythroid CD45(+) cells (2% with Cy versus 5% without Cy). Cells cultured with Epo and Cy reproduced erythropoiesis also at the molecular level. The results suggest that in 14 days different steps of human erythropoiesis from peripheral CD34(+) cells could be reproduced, with high recovery of highly purified erythroid cells. The high number and purity of erythroid cells produced from a small amount of peripheral blood make this method useful for studying either normal or pathological erythropoiesis.

Treatment of Graves' disease and associated ophthalmopathy with the anti-CD20 monoclonal antibody rituximab: an open study.

INTRODUCTION: Hyperthyroid Graves' disease (GD) is a B-cell-mediated condition caused by TSH receptor antibodies (TRAb), which decline when GD remits. Anti-CD20 monoclonal antibody rituximab (RTX) induces transient B-cell depletion that may potentially modify the active inflammatory phase of thyroid-associated ophthalmopathy (TAO). METHODS: Nine patients with GD, (seven with active TAO, two with mild lid signs) were studied. The trial was only approved as an open pilot study; thus we compared the effect of RTX therapy to that of i.v. glucocorticoids (IVGC) in 20 consecutive patients. Patients were treated with RTX (1000 mg i.v. twice at 2-week interval) or with IVGC (500 mg i.v. for 16 weeks). TAO was assessed by the clinical activity score (CAS) and severity was classified using NOSPECS (No signs or symptoms; Only signs (lid); Soft tissue involvement; Proptosis, Extraocular muscle involvement; Corneal involvement; Sight loss). Thyroid function and lymphocyte count were measured by standardized methods. RESULTS: All patients attained peripheral B-cell depletion with the first RTX infusion. Minor side effects were reported in three patients. Thyroid function was not affected by RTX therapy and hyperthyroid patients required therapy with methimazole. After RTX, the changes in the levels of thyroglobulin antibodies, thyroperoxidase antibodies and TRAb were neither significant nor correlated with CD20+ depletion (P = NS). CAS values before RTX were 4.7 +/- 0.5 and decreased to 1.8 +/- 0.8 at the end of follow-up (P < 0.0001) and more significantly compared with IVGC (P < 0.05). Proptosis decreased significantly after RTX both in patients with active TAO (ANOVA; P < 0.0001) and those with lid signs (ANOVA; P < 0.003). The degree of inflammation (class 2) decreased significantly in response to RTX (ANOVA; P < 0.001). Relapse of active TAO was not observed in patients treated with RTX, but occurred in 10% of those treated with IVGC, who also experienced adverse effects more frequently (45 vs 33% of patients). CONCLUSIONS: RTX positively affects the clinical course of TAO, independently of either thyroid function or circulating antithyroid antibodies, including TRAb. If our findings are confirmed in large controlled studies, RTX may represent a useful therapeutic tool in patients with active TAO.

Efficacy of rituximab treatment for thyroid-associated ophthalmopathy as a result of intraorbital B-cell depletion in one patient unresponsive to steroid immunosuppression.

One patient with Graves' hyperthyroidism and ophthalmopathy in its active phase and unresponsive to steroid, was treated with the anti-CD20 monoclonal antibody, rituximab (RTX), as part of an open study. The effect of RTX in the thyroid and the orbital tissues was studied. The ophthalmopathy responded to RTX therapy by ameliorating the eye signs with a decrease in the clinical activity score from 5 to 2 in 3 months, while the patient had peripheral B-cell depletion. Hyperthyroidism did not improve during the 6 months of B-cell depletion and serum TSH-receptor antibodies (TRAb) levels did not significantly change after RTX therapy. Therefore, the patient underwent total thyroidectomy and few B-cells were found in the thyroid tissue specimens. While the patient eye disease remained stable (clinical activity score = 2), we performed corrective orbital decompression and we found absence of lymphocytes in the orbital tissue specimens. We believe that RTX treatment in Graves' disease may cause amelioration of ophthalmopathy by depleting total lymphocytes population in the orbit. The persistence of Graves' hyperthyroidism suggests that a single cycle of RTX does not result in complete lymphocyte depletion in thyroid tissue and thus no decline in serum TRAb was observed.

Effect of adalimumab on neutrophil function in patients with rheumatoid arthritis.

Neutrophils are known to be targets for the biological activity of tumour necrosis factor (TNF)-alpha in the pathogenesis of rheumatoid arthritis (RA). Therefore, these cells may be among the targets of anti-TNF-alpha therapy. In this study we evaluated the effect of therapy with adalimumab (a fully human anti-TNF-alpha mAb; dosage: 40 mg subcutaneously every other week) on certain phenotypic and functional aspects of neutrophils obtained from 10 selected patients with RA and 20 healthy control individuals. Peripheral blood neutrophils were obtained at baseline and during anti-TNF-alpha therapy (2, 6 and 12 weeks after the first administration of adalimumab). All patients had been receiving a stable regimen of hydroxychloroquine, methotrexate and prednisone for at least 3 months before and during the study. Baseline neutrophil chemotaxis was significantly decreased in RA patients when compared with control individuals (P < 0.001). Two weeks after the first administration of adalimumab, chemotactic activity was completely restored, with no differences noted between patients and control individuals; these normal values were confirmed 6 and 12 weeks after the start of anti-TNF-alpha therapy. Phagocytic activity and CD11b membrane expression on neutrophils were similar between RA patients and control individuals; no modifications were observed during TNF-alpha neutralization. The production of reactive oxygen species, both in resting and PMA (phorbol 12-myristate 13-acetate)-stimulated cells, was significantly higher in RA patients at baseline (P < 0.05) and was unmodified by anti-TNF-alpha mAb. Finally, we showed that the activation antigen CD69, which was absent on control neutrophils, was significantly expressed on neutrophils from RA patients at baseline (P < 0.001, versus control individuals); however, the molecule was barely detectable on cells obtained from RA patients during adalimumab therapy. Because CD69 potentially plays a role in the pathogenesis of arthritis, our findings suggest that neutrophils are among the targets of anti-TNF-alpha activity in RA and may provide an insight into a new and interesting mechanism of action of anti-TNF-alpha mAbs in the control of inflammatory arthritis.

Regulatory CD8+ T cells control thyrotropin receptor-specific CD4+ clones in healthy subjects.

One of the mechanisms ensuring immunological unresponsiveness or tolerance depends on the action of CD8(+) lymphocytes. In this paper, we report that, in healthy subjects, a subset of CD8(+)CD28(-) T cells suppresses the specific response to TSH receptor (TSHR) of CD4(+) clones. Suppression was highly specific, required cell-cell interaction, and was not mediated by cytotoxicity. Co-incubation of CD8(+) and CD4(+) clones, followed by the removal of the CD8(+) cells from the cultures before testing CD4(+) responsiveness to TSHR, demonstrated that CD4(+) cells were anergic since they showed low response to the antigen and a significant impairment of IL-2 production. In CD8-mediated anergy induction, the T-cell receptor (TCR) on both CD4(+) and CD8(+) cells seems to play a role. Our results indicate that one of the mechanisms ensuring peripheral tolerance involve CD8(+)CD28(-) cells. A disregulation in the control of autoreactive clones by this subset might be important for the onset of autoimmune thyroid diseases.

Peripheral lymphocytes and intracellular cytokines in C282Y homozygous hemochromatosis patients.

BACKGROUND/AIMS: Several abnormalities in the immune status of hereditary hemochromatosis patients have been reported. We evaluated the peripheral blood lymphocytes phenotype and cytokine profile of CD8(+) and CD4(+) T cells in C282Y homozygous hereditary hemochromatosis patients compared to control subjects. METHODS: Peripheral blood lymphocytes from 17 asymptomatic patients and 14 control subjects were analyzed. We determined the distribution of lymphocyte subsets and investigated at single-cell level by flow-cytometry the potential of cytokines production. The frequency of cytokine (interferon gamma, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-4, IL-5, IL-10 and IL-13) producing cells was assessed in total T-lymphocytes, CD3(+)CD8(+) and CD3(+)CD4(+) subsets. RESULTS: The patients studied showed a significant decrease of total lymphocyte count, T CD4(+)CD3(+), CD28(+), CD8(+)CD28(+) lymphocytes and natural killer (NK) CD56(+)CD16(+)CD3(-) cells. The reduction of CD28(+) and CD8(+)CD28(+) lymphocyte count was inversely related to transferrin saturation index. An increase in the ability of T-cells to produce all the cytokines studied and a major increase in IL-4 and IL-10 production in the CD3(+)CD8(+) subset was found. Our results demonstrate that activated Th1 and Th2 lymphocytes coexist in the peripheral blood of hereditary hemochromatosis patients and that T-cytotoxic (Tc) 2 subset is more expanded than in control population. CONCLUSIONS: The association of a decreased number of T CD8(+) cytotoxic lymphocytes and NK cells, and the development of Tc2 cells in asymptomatic C282Y homozygous patients represents an imbalance in their immune function that might contribute to the high incidence of hepatocarcinoma.

Co-expression of the CD8 receptor in a human CD4+ T-cell clone influences proliferation, cytosolic Ca2+ release and cytokine production.

In normal human subjects a small proportion of peripheral blood T-cells simultaneously express both CD4 and CD8 differentiation antigens. In this study we characterized a subset of CD4+ clones, from a healthy donor, that is specific for the thyrotropin receptor (TSHR) and that showed cells co-expressing the CD8 receptor. To address whether the expression of the CD8 receptor on the cell membrane was associated to differences in the physiology of the T-cells, we isolated, from the same clone, CD4 single positive (SP) cells from those co-expressing CD4/CD8 receptors (DP cells) and stimulated them in vitro with antigen presenting cells (APC) carrying TSHR. The results demonstrated that CD8 co-expression has a profound effect on the physiology of T helper (Th) cells. In comparison to cells expressing the CD4 receptor alone, DP T-cells showed: (1) increased proliferation; (2) higher and more sustained release of free Ca2+ in the cytosol, under stimulus; (3) lower levels of IL-2 and IL-4 released in the supernatants; (4) increased amounts of IFN-gamma released.

Induction of CD69 activation molecule on human neutrophils by GM-CSF, IFN-gamma, and IFN-alpha.

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.