CD34+ hemopoietic precursor and stem cells traffic in peripheral blood of celiac patients is significantly increased but not directly related to epithelial damage severity.

Celiac disease (CD) is a chronic inflammatory enteropathy of the small bowel resulting from a local TH1-mediated reaction to wheat gliadins and barley, rye and oat prolamins with the development of auto-antibodies to transglutaminases. As well as for other chronic inflammatory diseases, genetic background and environmental factors participate to pathogenesis. An increased traffic of CD34+ hemopoietic precursor and stem cells (HPC) has been reported in peripheral blood (PB) of subjects with allergic diseases that share in their pathogenesis immuno-mediated reactions, genetic and environmental factors. The aim of the present work was to investigate the CD34+ cell traffic and H2/H1 polarization of lymphoid T-cell lineage, in the peripheral blood of subjects with CD, by means of flow-cytometric techniques. Group A of control was of 20 healthy subjects, aged 5 to 58 years. Study population (Group B) was of twenty-eight patients, all females aged 13 to 70, receiving firstly a CD diagnosis at the SS Annunziata Hospital Digestive Physiopathology Out-standings' by means of clinical, serologic and small intestinal biopsy findings. Peripheral CD34+ HPCs were significantly increased in Group B (median value 0.16) when compared with Group A (median value 0.03) (p 0.0001) but did not correlate either with anti-transglutaminase (tTG) antibody levels (IgA: p 0.226; IgG: p 0.810) or with histological damage severity (p 0.41) that, on the contrary, was significantly related with anti-tTG IgA antibodies (p 0.027). Celiac circulating CD3+CD4+ lymphocytes expressed a chemokine-receptor pattern Th2-skewed in all but three patients investigated. Concluding, the CD34+ HPC highly increased peripheral traffic observed in celiac disease appears more related to a basic and emerging as common defect shared by chronic inflammatory diseases than to the gliadin-specific Th1 local reactions. Data are consistent with a potential NFkappaB deficiency and consequent prevalence of apoptotic versus survival programs leading to excessive cell-death; to replace lost cells a supplementary bone-marrow derived precursors supply, further to that physiologically provided by the gut stem cell "niches" that are cryptopatches, could be required.

Pharmacoeconomics of subcutaneous allergen immunotherapy.

The current burden of allergic diseases, estimated by both direct and indirect costs, is very relevant. In fact the cost estimation for rhinitis amount globally to 4-10 billion dollars/year in the U.S. and to an average annual cost of 1089 euros per child/adolescent and 1543 euros per adult in Europe. The estimated annual costs in Northern America for asthma amounted to 14 billion dollars. Consequently, preventive strategies aimed at reducing the clinical severity of allergy are potentially able to reduce its costs. Among them, specific immunotherapy (SIT) joins to the preventive capacity the carryover effect once treatment is discontinued. A number of studies, mainly conducted in the US and Germany demonstrated a favourable cost-benefit balance. In the nineties, most surveys on patients with allergic rhinitis and asthma reported significant reductions of the direct and indirect costs in subjects treated with SIT compared to those treated with symptomatic drugs. This is fully confirmed in recent studies conducted in European countries: in Denmark the direct cost per patient/year of the standard care was more than halved following SIT; in Italy a study on Parietaria allergic patients demonstrated a significant difference in favor of SIT plus drug treatment for three years versus drug treatment alone, with a cost reduction starting from the 2nd year and increasing to 48% at the 3rd year, with a highly statistical significance which was maintained up to the 6th year, i.e. 3 years after stopping immunotherapy, corresponding to a net saving for each patient at the final evaluation of 623 euros per year; in France a cost/efficacy analysis comparing SIT and current symptomatic treatment in adults and children with dust mite and pollen allergy showed remarkable savings with SIT for both allergies in adults and children.

Economic evaluation of sublingual immunotherapy: an analysis of literature.

Allergic rhinitis and asthma constitute a global health problem because of their very high prevalence and the consequent burden of disease, concerning medical and economical issues. Among the treatments of allergy, specific immunotherapy has the capacity to favourably alter the natural history of the disease both during and after its performance and thus to reduce the direct and indirect costs of allergic rhinitis and asthma. A number of studies reported such cost reduction for traditional, subcutaneous immunotherapy and recent data demonstrate that also sublingual immunotherapy (SLIT) is associated to economic advantages and/or monetary savings, specifically in terms of reduction of disease economic burden. Only few formal economic assessments of SLIT have been carried out so far, this article will present and discuss the published studies addressed to this issue. The data obtained, although the number of studies is still limited, provide preliminary evidence supporting a SLIT effect on sparing costs for respiratory allergy.

Methodology and potential pitfalls in allergic diseases study designs: measurements for the assessment of the overall severity of atopic dermatitis--the four step severity score (FSSS), SCORAD-related, electronic system, for the simple and rapid evaluation of the skin and mucosal allergic inflammation.

Medical statistics may contribute to ameliorate research by improving the design of studies and identifying the optimal method for the analysis of results. Sometimes, nevertheless, it could be misemployed flawing the benefit potential. Allergic diseases pathogenesis is recognized to be systemic but global initiatives such as GINA and ARIA documents define allergic asthma and rhinitis as organ diseases; such an asymmetrical view raises a set of known and unknown confounding that could influence the quality of the process of evidence-based decision-making (topic symptomatic therapeutic interventions versus systemic pathogenetic interventions). This article shows the first scoring system for the assessment of atopic dermatitis lesions developed in the allergy-area. A four-step severity score (FSSS) was chosen in agreement with those developed for asthma and rhinitis in global initiatives, to avoid any further differences in evaluating the severity of allergic diseases. FSSS relates each step with the objective signs of the SCORAD and rates the disease course as intermittent or persistent. A devoted electronic program has been also framed to allow a quick and simple contemporary evaluation of the SCORAD Index (Section I) and of the FSSS (Section II); the program furthermore foresees a third section named ESAS (Extra Skin Allergic Signs) (Section III) in which it is possible to check whether organs other than the skin are involved by the allergic inflammation. The limitations potential generated by a misemployment of medical statistics for clinical trials designed to establish benefits rising from specific immunotherapy for allergic diseases have been also discussed extensively.

Increased CD34+ cell peripheral traffic appears to be an unique feature of the allergic inflammation.

Evidence has been cumulated during the last years concerning the immaturity of the cells involved in the local and systemic aspects of allergic inflammation. Hematopoietic precursors (HPC) are mobilized from the bone matrix as multipotent cells or, more often, as progenitors that, after the initial white-lineage commitment reach through the peripheral blood (PB) their final destinations constituted by the target organs of allergy. Although several studies have investigated the CD34+ cells traffic and location at the level of the inflamed peripheral mucosae in allergic populations, limited information is available on their behaviour on the time-course of infectious diseases. The current study thus was designed to asses the peripheral traffic of CD34+ HPC during the infectious inflammation. To this end CD34+ HPCs have been enumerated, by flow-cytometric techniques, in PB of 24 adult healthy beings (Group A), 24 adult subjects with symptomatic extrinsic allergy (Group B) and in PB of 24 adult patients hospitalised for febrile infectious pathology (Group C). CD34+ cell values ranged 0.01-0.08% with a median of 0.03 in Group A. In Group B values ranged 0.17-0.75% with a median of 0.28 and in Group C values ranged 0.00-0.12% with a median of 0.07. Variance analysis test among the three groups was statistically significant (p<0.001) supporting the conclusion that CD34+ HPC mobilizing and increased peripheral traffic is an unique feature of the allergic inflammation.

Immunotherapy in atopic dermatitis.

Atopic dermatitis (AD) is a common inflammatory disease involving the skin and often other organs and systems, mainly respiratory. A definitive general consensus on the AD pathogenesis has not yet been established, however several lines of evidence suggest that T-cells play a crucial role in priming AD early-stage lesions. Main topics involved in the disease pathogenesis have been reviewed, which considered the concept of local and systemic haemopoietic events as important contributors to allergic inflammation, a concept now achieving great acceptance. The recently recognised atopic nature of the skin inflammation in AD has raised increasing interest for treatment with allergen-specific immunotherapy. However, we only found eight studies using specific immunotherapy (SIT) in AD, two double-blind, placebo-controlled (DBPC) and six observational. One controlled and five observational reported favourable outcomes. The one unique study providing negative results was flawed by the ineffective oral route of extract administration. Despite being encouraging, the reported results do not allow definitive conclusions based on meta-analytic techniques because the amount and quality of information in the literature is not sufficient. The highly promising sub-lingual immunotherapy (SLIT) is discussed with its potential capability of controlling not only the skin lesion severity but also its capability of preventing the development of atopic dermatitis into asthma.

Hemopoietic progenitor cells in atopic dermatitis skin lesions.

We have recently shown the expression of lymphoid early developmental markers, including CD104, Thy 1, CD1a, Pgp-1 and TdT, by the cells constituting atopic dermatitis skin infiltrates. To further characterize the cellular phenotypes we used an indirect immunoperoxidase assay to analyze sections from two atopic dermatitis lesion skin biopsies using the following as first step monoclonal antibodies (MAB): anti-CD34, CD2, CD5 and CD7. CD34+ mononuclear cells and endothelial cells were identified. A strong immunoreaction was observed for the T-lineage marker CD2 and CD5, but a poor reaction, if any, was seen for the CD7. Since CD34+ marrow and blood cells are currently believed to be the major source of the hemopoietic precursors, our data provide further substantial evidence supporting the hypothesis that the atopic dermatitis skin cell infiltrate represents an ongoing T-lineage in situ differentiation process regulated by the skin epithelial microenvironment. The observed defective expression of the CD7 antigen requires further investigation for its confirmation as a possible constant feature in atopic dermatitis.

T-cell receptor Vbeta repertoire in mite-allergic subjects after sublingual immunotherapy.

Sublingual immunotherapy has been recognized as an alternative to injected immunotherapy for the treatment of allergic diseases. Even if compelling clinical evidence supports such a view, few studies are available on its mechanisms of action. This study was carried out to investigate the peripheral lymphocyte Vbeta repertoire of subjects with mite-allergic respiratory allergy who were either not treated or treated for 2 years with mite-specific sublingual immunotherapy. The T-cell receptor Vbeta distribution was studied by flow-cytometric techniques in three subject groups. Group A (untreated) included 19 subjects with symptomatic, mite-allergic, low to moderate asthma and/or rhinitis. Group B (treated) was made up of 10 asymptomatic subjects treated for 2 years with mite-specific sublingual-swallow immunotherapy for low to moderate asthma and/or rhinitis. Group C (controls) included 10 healthy subjects. The Vbeta usage was investigated with monoclonal antibodies specific to the diverse beta segments V3, V5a, V5b, V5c, V6a, V8a, V8b and V12a. The comparison between the group A and group C repertoires showed a lower expression (p < 0.05) of the beta V8b+ T-cell subset. The group B repertoire, when compared with group A, showed a significantly greater usage of the beta V5a (p <0.05), 8a (p <0.05) and 12a (p <0.01) segments. The significantly lower expression of beta V8b observed in the symptomatic untreated group was not present in the group that was asymptomatic after treatment. The oligoclonal expansion observed in the treated group was consistent with the development of suppressor T-cell and/or of Th1 clones but not with deletion mechanisms of induced tolerance.

Expression of T-lineage early developmental markers by cells establishing atopic dermatitis skin infiltrates.

The association between the atopic dermatitis, eczema and T-cell immunodeficiency disorders are well known, thus suggesting that bone marrow T-precursors could use the micro-environment of the skin as an extrathymic site for compensatory ontogenesis. In keeping with this hypothesis, we analyzed the atopic dermatitis skin lymphocytic infiltrate phenotypes to establish their ontogenetic stage of development. Cryostatic sections (4 microns) obtained from acute lesional skin biopsies of six patients with extrinsic atopic dermatitis were processed with indirect immunoperoxidase, using a panel of first-step monoclonal antibodies (mAb) specific to CD104 (integrin beta 4 chain), CD90w (Thy 1 antigen), CD44 (phagocytic glycoprotein-1; Pgp-1), CD1a and the DNA polymerase terminal deoxynucleotidyl transferase (TdT). Within the lymphocytic dermal infiltrate different levels of immunoreactivity were observed with respect to CD104, CD90w and CD1a. A strong, spread staining was also detected for mAb specific to Pgp-1 and TdT. Together, the reported features indicate that the atopic dermatitis skin-homing lymphocytes express immunophenotypes which are distinctive of the early T-ontogeny.

Probiotics reduce the CD34+ hemopoietic precursor cell increased traffic in allergic subjects.

An increased traffic of circulating CD34+ Hemopoietic Precursors Cells (HPC) is an important feature of systemic allergic inflammation. Bacteria and bacterial products are capable of stimulating the transcription of the maturational cytokines IL12 and IFNs through the activation of Toll-Like-Receptor and the subsequent nuclear translocation of the NF-kappaB factor. In this study the probiotics differentiation/maturational effect potential on CD34+ HPC has been investigated. Fourteen consecutive subjects, 9M and 5F, aged 6-48, with clinical symptoms of asthma and /or conjunctivitis, rhinitis, urticaria, atopic dermatitis, food allergy and irritable bowel syndrome were enrolled. Allergen-specific serum IgE were found in twelve patients. Flow-cytometric measurement of peripheral blood CD34dim/bright HPC values were assessed before and after 30 days of therapy, consisting in the oral administration of one sachet a day of ENDOLAC (UCB Pharma, Turin, Italy). Each sachet contained a mixture of Lactobacillus acidophilus, L. delbrueckii and Streptococcus thermophilus for a total of 1 x 10(9) live bacteria. Circulating CD34+ cell values significantly (p < 0.001) reduced after the treatment. ENDOLAC, thus, may improve the efficacy of the standard treatments of allergic diseases.

Quantitative expression of the major homing integrins alphaL and alpha4 on human cord blood hematopoietic progenitor and stem cells.

An increased traffic of hematopoietic progenitor cells (HPC) between bone marrow and peripheral organs is a peculiar feature of the allergic inflammation. It has been recently reported that the sublingual form of specific immunotherapy (SLIT) is capable of reducing such an increased HPC traffic. The House Dust Mite major antigen Der p1 has been proved to up-regulate the expression of the ICAM-1 and VCAM-1 endothelial addressins, supporting the view of an inflammatory cell recruiting at the site of allergen extract administration. In the present work we have investigated, by flow-cytometric techniques, the expression of the two major integrins CD11a (LFA-1) and CD49d (VLA-4) that are the homing receptor cognate for ICAM-1 and VCAM-1 on human cord blood CD34 hematopoietic progenitor and stem cells. Even if both the investigated molecules resulted detectable on CD34+ HPC surfaces, being the system redundant, the density of the cellular expression was significantly higher for CD49d (median value: 158) than CD11a (median value: 20.5), suggesting a preferential usage of the homing axis VLA-4/VCAM-1. Results consistency with outcomes of clinical trials that relate SLIT efficacy to allergen dosage is discussed.

Measurement of nasal IgE in an epidemiological study: assessment of its diagnostic value in respiratory allergy.

BACKGROUND: Specific questionnaire and skin prick test (SPT) are the most used methods in epidemiological studies on respiratory allergy. SPT, however, can be positive in many subjects without evidence of any allergic disease. Nasal IgE determination has been suggested by some authors as a valuable diagnostic method, which may overcome this lack of specificity. OBJECTIVE: The aim of this study was to evaluate sensitivity and specificity of nasal specific IgE for the seven most common inhalant allergens in order to verify its reliability as a screening test. METHODS: 126 children, involved in an epidemiological study on prevalence of respiratory allergic disease, were evaluated. All children were assessed with a specific questionnaire, SPT and nasal specific IgE. Nasal specific IgE were determined with a previously described method modified for screening purposes, in order to test seven allergens at the same time. When discordant results were obtained between questionnaire, SPT and nasal IgE, an allergen specific nasal challenge (ASNC) was performed and nasal tryptase was also determined before and after challenge. RESULTS: The questionnaire was positive for respiratory allergy in 28/126 children. SPT was positive in 21 of the 28 children, but also in 5/10 children with atopic dermatitis (AD), and in 12/88 children without allergic symptoms. Nasal IgE were positive in 22/28 and also in 2/10 with AD. Nasal challenge and tryptase confirmed the negativity of nasal IgE in 12/17 children with positive SPT but totally negative for allergic respiratory disease. Moreover nasal IgE was found to be positive to dermatophagoides in one of seven children with negative SPT despite a clinical history suggestive for mite respiratory allergy. In this patient and in 2 of the 5 children with AD the positive nasal IgE to mites was confirmed by a positive ASNC and tryptase. CONCLUSIONS: Nasal IgE have shown a specificity significantly higher than SPT (98% vs. 83%) and a good sensibility. This screening test may also be useful to detect the beginning of upper airways sensitization in patients with AD.

Specific sublingual immunotherapy in atopic dermatitis. Results of a 6-year follow-up of 35 consecutive patients.

BACKGROUND: allergen-specific immunotherapy has proved to be effective in selected patients with IgE-mediated respiratory allergic diseases, and alternative routes of administration are being studied. Atopic Dermatitis (AD) is currently regarded as an allergic inflammatory disease. METHODS: we conducted a cohort study to evaluate the safety and effectiveness of sublingual-swallow immunotherapy (SLIT) in selected patients with allergic (extrinsic) AD. Thirty-five patients, 16 suffering from AD without respiratory allergic symptoms (Group A) and 19 with AD associated to mild asthma and/or rhinitis (Group B), were enrolled in the study. The severity of the skin lesions (eczema) was scored on a 0 to 4 scale (and subsequently related to the more recent SCORAD Index), where 0 indicated complete healing of the eczema and 4 indicated maximal spread of the lesions. Only patients with an eczema score of 1 to 3 were started on allergen-specific SLIT for 36 months. Eczema scores, symptoms and side effects were recorded every two months during the first 2 years and then after 36 months. After SLIT was completed, all patients attended 3 yearly follow-up visits to evaluate the long-term effects of the treatment. All patients followed a set of rules designed to control for identified confounding variables. All patients received ketotifen during the first 3 months of SLIT. RESULTS: only the complete disappearance of skin lesions (score 0) was considered to indicate effectiveness. In Group A this was observed in 12.6% of the patients after 6 months of SLIT, in 31,2% after 12 months and 68.8% after 24 months. In Group B, eczema disappeared in 0% after 6 months, in 36.8% after 12 months and 73.7% after 24 months. No patients in Group A developed asthma during SLIT, and 1 patient developed asthma 3 years after immunotherapy had ended. Three focal reactions consisting of 2 cases of mild eczema and one case of diarrhoea were recorded. One case of urticaria, due to violation of the administration schedule was the only systemic reaction observed. No life-threatening reactions appeared at any time of the study. CONCLUSIONS: the outcomes obtained, taken into account the limitations of the study design, suggest that sublingual allergen-specific immunotherapy for the treatment of the extrinsic form of Atopic Dermatitis is safe and well tolerated by patients, and may favourably affect the natural course of the disease.

Evaluation of T cell subsets in Behçet's syndrome using anti-T cell monoclonal antibodies.

Several immunological abnormalities have been described in Behçet's syndrome, the multisystem disease characterized by the triad of relapsing iridocyclitis with recurrent oral and genital ulcerations. In the present study we have evaluated T cell subsets in the peripheral blood of patients suffering from Behçet's syndrome, using a panel of anti-T cell monoclonal antibodies. When compared with normal subjects, patients with Behçet's syndrome show a discrepancy between the number of T3+ cells and those forming rosettes with sheep erythrocytes, a significantly higher number of T4+ and T8+ double labelled cells, as well as of T6+ lymphocytes in the peripheral blood. The percentage of T8 T lymphocytes is increased, thus lowering the T4+/T8+ cell ratio. The results of this study indicate that a complex imbalance of T cell subsets is present in Behçet's syndrome and give a rationale for possible treatment of these patients with immunomodulators.

CD34+ cells in peripheral blood of healthy human beings and allergic subjects: clue to acute and minimal persistent inflammation.

BACKGROUND: There is compelling evidence that hemopoietic precursor cells (HPC) play a crucial role in establishing cellular inflammation in allergic diseases. Increased levels of circulating CD34+ HPC committed to the myeloid lineage have been extensively reported in allergic rhinitis, asthma and eczema, whereas CD34+ cells have been identified within the cellular infiltrates of tissues, at peripheral sites of inflammation. METHOD: We conducted a pilot study to evaluate CD34+ traffic in the peripheral blood of 22 consecutive patients (13 men and nine women; mean age 28.9 years), independently of treatment. The patients presented rhinitis, asthma, eczema, urticaria and adverse food reactions of suspected allergic origin. Allergic reactions were extrinsic in 18 patients and intrinsic in four. In 12 patients who underwent sublingual specific immunotherapy, CD34+ cells were quantified at enrollment (T0), one year later (T1) and two years later (T2). The severity of symptoms was graded on a five-point scale (0 = absence of symptoms and 4 = severe symptoms). Twenty healthy human subjects (10 men and 10 women; mean age 24.5 years) were evaluated as controls. To obtain information about the total amount of circulating HPC, independently of the lineage commitment (Lin+/-) and the degree of differentiation (CD34bright/dim), we used a modification of the Milan protocol of peripheral blood CD34+ cell estimation. The cells were analyzed using a BD FACScan or FACSCalibur and the results were expressed as the percentage of positive cells. RESULTS: CD34+ cell traffic in the control group was very low since all values were < 0.10 (median value: 0.03 %). Values in the patient group were increased in both extrinsic and intrinsic forms with a median value of 0.25 % (interquartile range: 0.13- 0.33 %). The relationship between CD34+ traffic and the severity score was highly significant (Spearman's rho = 0.954; test of Ho: CD34; independent score: Pr > t = 0.000). CONCLUSIONS: The data reported herein suggest that the method employed is effective in assessing acute allergic inflammation, as well as minimal persistent inflammation underlying an asymptomatic clinical condition. Evaluation of CD34bright/dim peripheral traffic, if confirmed by the outcomes of a multicenter study currently being planned together with traditional study of circulating IgE, could be a reliable non-invasive laboratory tool for monitoring allergic inflammation.

Mite antigens enhance ICAM-1 and induce VCAM-1 expression on human umbilical vein endothelium.

Although sublingual allergen-specific immunotherapy has been proved to be effective in the treatment of allergic diseases, controversy surrounds the means by which such a local therapy can induce systemic immunological changes. Adhesion molecules are critical in the regulation of leukocyte traffic. It has been hypothesized that allergenic extract, administered locally, may induce an up-regulation of the mucosal vessel vascular adhesion molecules (CAMs) resulting in local recruitment of circulating inflammatory cells. In the present study we investigated whether the mite antigens, Der p1 and Der p2, can modulate CAM expression of human endothelial cells (HEC). To do this, slices of whole human umbilical cord vein underwent short-term (8 hours) cultures in the presence or absence of mite antigen (baseline, unstimulated controls). Cryostatic sections of the specimens were then evaluated immunohistochemically for expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) molecules. The results revealed that while Der p1 is capable of significantly up-regulating ICAM-1 and VCAM-1 on HEC, Der p2 antigen moderately up-regulates ICAM-1 expression but is ineffective in modulating VCAM-1. Although preliminary, these results clearly support the hypothesis that at least some of the effects of sublingual immunotherapy may derive from inflammatory cell recruitment at the site of allergen release.

IgE responses to Dermatophagoides pteronyssinus native major allergens Der p 1 and Der p 2 during long-term specific immunotherapy.

We investigated by ELISA the IgE response to whole extract of the house-dust mite Dermatophagoides pteronyssinus (Dp) and to the native major allergens, Der p 1 and Der p 2, in sera from 18 adult patients (group A) with Dp-allergic asthma before (t0) and 1, 2, 3, and 4 (t1-t4) years after subcutaneous specific immunotherapy (SIT). A qualitative reduction (P = 0.05) of the IgE responses to Dp and Der p 2 was observed from t1 to t4, but a highly statistical significant decrease appeared at t3 (P < 0.01). With regard to Der p 1 IgE values, the immunotherapy induced a significant decrease (P < 0.01) at t3, but not before. In group A, the IgE responses to Der p 1 and Der p 2 were not correlated at t0 (rs = 0.31; P = 0.21) but were correlated at t3 (rs = 0.78; P = 0.001). We also examined sera from 14 adult patients (group B, same SIT schedule as group A) who were without respiratory symptoms at the end of the third year (t3) of Dp SIT. At this time (t3), there were no significant differences in Der p 1 and Der p 2 IgE levels between group A and group B.