BackgroundPeriostin(PN), a matricellular protein, serves as a regulator of wound healing and fibrosis. PN-/- mice develop lower degrees of fibrosis when exposed to bleomycin(BLM), suggesting a possible role in the pathogenesis of Systemic Sclerosis(SSc)1. PN serum levels are increased in SSc and seem to be associated with skin disease severity2.ObjectivesTo evaluate the role of serum PN as a biomarker of SSc severity, and to determine PN tissue expression in SSc patients.MethodsPN serum levels were assessed by ELISA in 48 patients: 3 primary Raynaud’s, 12 early SSc, 11 SSc without organ involvement, 22 SSc with organ involvement. All SSc patients met 2013 ACR/EULAR criteria. PN serum levels were evaluated in 28 sex-/age-matched healthy-controls(HCs). Data regarding disease subtypes and organ involvement were correlated. PN skin expression was determined by immunohistochemistry on paired involved and uninvolved skin biopsy samples in 10 patients(4 lcSSc and 6 dcSSc) in combination with a-SMA, CD68, CD3, CD4, CD8, CD163, CD20 and CD131.ResultsPN serum levels were higher in SSc patients compared to HCs(32.7±8.0 vs 27.7±7.3 ng/ml, p<0.001). Its levels were comparable among different groups. No differences in PN serum levels were detected when comparing disease subtypes, disease duration, presence and extent of organ involvement, autoantibodies profile and current or previous treatment. Higher PN levels were found in SSc patients with active pattern at nailfold videocapillaroscopy and a history of digital ulcers(p<0.02). PN serum levels did not correlate with skin or lung disease extent. Skin samples from involved SSc skin showed high PN expression in the upper dermis and in the fibrotic area of the lower dermis(more evident in dcSSc), suggesting a role in skin fibrosis. In all SSc involved skin, PN was expressed in areas where ongoing fibroproliferation and macrophage/T lymphocytic infiltration occured, indirectly suggesting a pathogenic role in inflammation driven-fibrosis. Interestingly, an identical PN immunohistochemical expression was evident in uninvolved dcSSc skin, but not in lcSSc skin.ConclusionsIn our cohort PN serum levels are elevated in SSc patients but they do not correlate with disease features. Its postulated role as a severity biomarker needs to be further elucidated. The different immunohistochemical expression of PN in uninvolved skin from dcSSc and lcSSc patients suggests a possible pathogenic role in the progressive inflammation-driven fibrosis that characterise diffuse cutaneous involvement.References[1] Yang L, et al. Periostin facilitates skin sclerosis via PI3K/Akt dependent mechanism in a mouse model of scleroderma. PLoSOne2012. [2] Yamaguchi Y, et al. Serum Periostin levels are correlated with progressive skin sclerosis in patients with systemic sclerosis. Br J Dermatol2013.Disclosure of InterestNone declared

Background. Histamine release (HR) test has previously been shown to predict the presence of endogenous histamine-releasing factors in chronic spontaneous urticaria (CSU). Objectives and methods. Twenty CSU patients unresponsive to antihistamine treatment were enrolled in order to evaluate the correlations between HR test results and demographic features, quality of life, disease activity, clinical course, and autologous serum and plasma skin tests (ASST and APST). Results. All patients with positive HR test (9/9, 100%) had a more severe disease activity at onset (urticaria activity score, UAS > 2) when compared to negative HR test patients (5/11; p = 0.04). Quality of life questionnaires' results were not substantially different between HR positive and negative subgroups at baseline (p > 0.05), and results of HR test and ASST/APST did not co-segregate (p > 0.05). After 12 months, patients with a positive HR test had a significant reduction of disease activity (p = 0.003) whereas patients with a negative HR test did not (p > 0.05), leading to disease remission and antihistamine treatment withdrawal in 67% (6/9) of positive HR test patients versus 18% (2/11) of negative HR test patients (p = 0.027). Conclusions. Positive HR test may predict spontaneous CSU remission at 12 months.

RATIONALE: Sensitization to Profilin, a pan-allergen, is observed in 40% of grass allergic patients. So far the are no data regarding the clinical relevance of sensitization to profilin, in term of severity of symptoms in these patients. A purified natural date palm profilin (Pho-d-2) Skin Prick Test (SPT) has been recently developped. We investigated the prevalence of profilin sensitization and compared symptom and symptomatic drug scores during pollen season in patients with grass rhinoconjuctivitis in relation with profilin sensitization. METHODS: 50 consecutive patients with rhinoconjunctivitis and/or asthma due to grass pollen (28 men, mean age 34 years) were enrolled. SPT for grass and profilin were evaluated before pollen season 2008. Symptom score during peak pollen season (May 2008) was rated evaluating 6-item for nose and eye symptoms using a 4-point ordinal scale (0= no symptoms; 3=severe symptoms). Medication score was evaluated assessing the use of oral/topic antihistamine or corticosteroid drugs. RESULTS: All patients were positive for grass SPT. A total of 21 subjects (44%) were positive to profilin SPT (wheal diameter >3 mm: P+). In P+ patients, seasonal symptom score was significantly (P=0.01) higher in comparison with P- subjects (155 vs. 71). Medication score, mean(SD) was 39(28) in P+ patients and 13(16) in P- subjects (P=0.0025). CONCLUSIONS: Our study confirms that in grass allergic patients profilin sensitization evaluated by a specific SPT is detected in 40% of the patients. In these patients profilin sensitization is also associated with an higher symptom and medication score during pollen season.

RationaleWe evaluated three commercially available SPT (Product A: Lofarma, Product B: Stallergenes SA and Product C: Soluprick® SQ, ALK-Abellò) in a double-blind, randomized trial.Methods43 monosensitized patients (20 men) allergic to House dust mite (18 patients), Grass (11 patients) or Parietaria (14) were enrolled after written informed consent. Allergy was confirmed by history and IgE measurements. Patients were evaluated in two separate visits performed 15-days apart with all three SPT products using the relevant allergen. For each SPT product, two different batches were used. SPTs were performed with undiluted solution and with end point method using progressive dilutions. Histamine (H) (10 mg/mL) was used as positive control.ResultsH wheal mean (SD) area was 121 (77) mm2 and 116 (66) mm2 during first and second evaluation visits, (p = 0.76). With product A, B and C the wheal area at the first evaluation was: 87 (76), 103 (89) and 129 (88) mm2. A significant difference was observed between H and Product A (P = 0.03). No differences were observed between H and Product B and C. At second evaluation, the wheal area was: 109 (84) for product A (P = 0.05, vs. first evaluation), 125 (97) for product B (P = 0.04) and 125 (79) mm2 for product C (P = 0.68). With end point method, Product C showed higher potency in 59% of tests in comparison with Product B (28%) and Product A (16%) (P = 0.0001).ConclusionsOur trial results show that ALK Soluprick offers a greater batch-to-batch consistency and a higher potency in comparison with other commercially available SPT products. RationaleWe evaluated three commercially available SPT (Product A: Lofarma, Product B: Stallergenes SA and Product C: Soluprick® SQ, ALK-Abellò) in a double-blind, randomized trial. We evaluated three commercially available SPT (Product A: Lofarma, Product B: Stallergenes SA and Product C: Soluprick® SQ, ALK-Abellò) in a double-blind, randomized trial. Methods43 monosensitized patients (20 men) allergic to House dust mite (18 patients), Grass (11 patients) or Parietaria (14) were enrolled after written informed consent. Allergy was confirmed by history and IgE measurements. Patients were evaluated in two separate visits performed 15-days apart with all three SPT products using the relevant allergen. For each SPT product, two different batches were used. SPTs were performed with undiluted solution and with end point method using progressive dilutions. Histamine (H) (10 mg/mL) was used as positive control. 43 monosensitized patients (20 men) allergic to House dust mite (18 patients), Grass (11 patients) or Parietaria (14) were enrolled after written informed consent. Allergy was confirmed by history and IgE measurements. Patients were evaluated in two separate visits performed 15-days apart with all three SPT products using the relevant allergen. For each SPT product, two different batches were used. SPTs were performed with undiluted solution and with end point method using progressive dilutions. Histamine (H) (10 mg/mL) was used as positive control. ResultsH wheal mean (SD) area was 121 (77) mm2 and 116 (66) mm2 during first and second evaluation visits, (p = 0.76). With product A, B and C the wheal area at the first evaluation was: 87 (76), 103 (89) and 129 (88) mm2. A significant difference was observed between H and Product A (P = 0.03). No differences were observed between H and Product B and C. At second evaluation, the wheal area was: 109 (84) for product A (P = 0.05, vs. first evaluation), 125 (97) for product B (P = 0.04) and 125 (79) mm2 for product C (P = 0.68). With end point method, Product C showed higher potency in 59% of tests in comparison with Product B (28%) and Product A (16%) (P = 0.0001). H wheal mean (SD) area was 121 (77) mm2 and 116 (66) mm2 during first and second evaluation visits, (p = 0.76). With product A, B and C the wheal area at the first evaluation was: 87 (76), 103 (89) and 129 (88) mm2. A significant difference was observed between H and Product A (P = 0.03). No differences were observed between H and Product B and C. At second evaluation, the wheal area was: 109 (84) for product A (P = 0.05, vs. first evaluation), 125 (97) for product B (P = 0.04) and 125 (79) mm2 for product C (P = 0.68). With end point method, Product C showed higher potency in 59% of tests in comparison with Product B (28%) and Product A (16%) (P = 0.0001). ConclusionsOur trial results show that ALK Soluprick offers a greater batch-to-batch consistency and a higher potency in comparison with other commercially available SPT products. Our trial results show that ALK Soluprick offers a greater batch-to-batch consistency and a higher potency in comparison with other commercially available SPT products.

Tumour Necrosis Factor-alpha (TNF alpha) triggers maturation of dendritic cells (DC), a process which results, among other events, in downregulation of the ability of these cells to phagocytose antigens. We have studied the effects and mechanisms of action of nitric oxide (NO) on endocytosis, investigated with fluorescein-iso-thiocyanate (FITC)-labelled dextran, using human, monocyte-derived DC, both immature and after treatment with TNF alpha. NO, released by the NO donor DETA-NO, reversed the inhibition of endocytosis observed in DC exposed to TNF alpha. This effect of NO was mimicked by the membrane-permeant cyclic GMP analogue, 8-Br cyclic GMP, and prevented by inhibition of the soluble guanylate cyclase. The mechanism of action by NO on endocytosis is due to regulation of the generation of ceramide, a lipid messenger involved in the maturation process of DC initiated by TNFa. In particular, NO was found to inhibit, in a cyclic GMP (cGMP)-dependent way, ceramide accumulation triggered by TNF alpha. NO, however, was found to exert an inhibitory effect also downstream of this event, as NO donors reversed the inhibition of endocytosis induced by the cell-permeant C-2-ceramide. These results indicate that NO, working through cGMP, increases the ability of human DC to internalise antigens and thus modulate the initial steps leading to antigen-specific immune-responses.

In this CRADA effort, LLNL's core strengths in optics were leveraged to develop a second generation prototype system that would be characterized by improved real-time endoscopic visualization. The visualization enhancements were targeted at image features which were critical elements of the surgical decision making process and were the surgeon's only visual and major sensory link during endoscopic surgical tool positioning. Endoscopic "minimally-invasive" procedures are being performed more frequently in medicine. Many of the more routine laparoscopic and arthroscopic procedures have had their technical difficulties overcome. The demand for "minimally invasive" surgical procedures motivated new procedures to be attempted in smaller surgical fields. During these new procedures, new visualization challenges were present for which solutions were needed. The objective was to develop a system that used optics to minimize glare while preserving color integrity.

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1-specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32-base pair deletion in the C-C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5 Delta 32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C-C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell-tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development.

In spite of repeated exposures to HIV, some individuals remain seronegative and apparently uninfected. A variety of mechanisms potentially able to confer resistance to HIV infection, including cell-mediated and (unconventional) humoral immune responses, as well as mutations affecting receptors for virus entry have been considered and analysed. In this article, we want to discuss recent reports on specific immune responses and genetic factors potentially involved in mechanisms of protection, and to present some of our data relative to a cohort of people sexually exposed to HIV-1, but persistently seronegative. These EU (exposed uninfected) individuals can be distinguished from "normal" unexposed controls on the basis of significantly increased frequencies of a number of immunological parameters that might be considered "unconventional" correlates of HIV infection/protection. However, EU individuals are highly heterogeneous since the various unconventional immune responses considered can be present in all possible combinations. Aim of future research will be to ascertain the role of such immune responses in the maintenance of the protection state, or their secondary nature as signals of a particular kind of infection.

Dual-energy X-ray absorptiometry (DXA) is now a commonly used method for the determination of bone mineral status and body composition in humans. The purposes of this study were to compare fat mass by in vitro neutron activation analysis (FMIVNA) with that by DXA (FMDXA) in an anthropometrically heterogeneous sample of healthy adult men (n = 33) and women (n = 36) (19 < or = BMI < or = 39), and to determine whether differences in fat mass estimates between the two methods (delta FM) were attributable to subject anthropometry as defined by several circumference (waist, iliac crest, thigh) and skinfold thickness (umbilical, suprailiac, abdominal) measurements. No significant differences between FMDXA and FMIVNA were observed in men (p = 0.46) or women (p = 0.09). The two methods were very highly correlated in both sexes (women r2 = 0.97, p < 0.001, men r2 = 0.91, p < 0.001), although the regression line for men was significantly different from the line of identity (p = 0.043). These results suggest modest trends toward underestimation of FMDXA in men when FMIVNA < 18 kg, and overestimation in men when FMIVNA > 18 kg. delta FM (IVNA-DXA) was not significantly related to any combination of skinfold thickness and circumferences in either gender. Age explained 27% of the variance in delta FM for the men (p = 0.008). Furthermore, delta FM was not significantly related to inter-method disparity in total-body bone mineral measurements in men or women (p < 0.05). The present study demonstrates strong correlation in fat measurements between IVNA and DXA in men and women ranging from normal to markedly obese. Correction for subject anthropometry does not significantly improve this relationship.

A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study. Twelve of the HR partners were studied for in vitro stimulation of peripheral blood mononuclear cells (PBMC) by HIV envelope-derived peptides. All 12 responded overwhelmingly to a peptide containing the fifth conserved region of gp120. By generating and cloning T cell lines specific for this peptide, we concluded that in these individuals the T cell response to the envelope is mainly focused on the carboxy-terminus region of gp120 and is characterized by an oligoclonal expansion of CD4+ T cells expressing the same TCR. Eighteen HR partners and 37 HIV-1 seropositive subjects were tested for the presence of anti-CD4 antibodies (anti-CD4 Abs) using a recombinant CD4-based enzyme-linked immunosorbent assay (ELISA). Anti-CD4 Abs were detected in eight of the HR partners (six confirmed by Western blot) and in nine of the HIV-1 seropositive subjects (eight confirmed by Western blot). Results from binding competition assays with a panel of monoclonal anti-CD4 Abs suggested that the anti-CD4 Abs detected in the HR partners are directed toward epitopes that are induced by gp120 binding. Twenty-seven of the HR partners were tested for the presence of antibodies that cross-react with HLA class I and gp120 (anti-HLA Abs). Anti-HLA Abs were detected in 16 of the HR partner sera and in 494 sera from a control population of normal healthy blood donors. Taken together, the results suggest that in some individuals with a history of long-term exposure to HIV, specific immunity may develop in the absence of overt infection. The common trigger for these responses is gp120.

The inability to precisely estimate body composition with simple, inexpensive, and easily applied techniques is an impediment to clinical investigations in nutrition. In this study, predictive equations for body cell mass (BCM), fat-free mass (FFM), and total body water (TBW) were derived from direct measurements through use of single-frequency bioelectrical impedance analysis (BIA) in 332 subjects, including white, black, and Hispanic men and women, who were both healthy control subjects and patients infected with the human immunodeficiency virus (HIV). Preliminary studies showed more accurate predictions of BCM when parallel-transformed values of reactance were used rather than the values reported by the bioelectrical impedance analyzer. Modeling equations derived after logarithmic transformation of height, reactance, and impedance were more accurate predictors than equations using height2/resistance, and the use of sex-specific equations further improved accuracy. The effect of adding weight to the modeling equation was less important than the BIA measurements. The resulting equations were validated internally, and race and disease (HIV infection) were shown not to affect the predictions. The equation for FFM was validated externally against results derived from hydrodensitometry in 440 healthy individuals; the SEE was < 5%. These results indicate that body composition can be estimated with simple and easily applied techniques, and that the estimates are sufficiently precise for use in clinical investigation and practice.