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.

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.

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.

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 (AU)

Aunque se ha demostrado que la inmunoterapia sublingual con alergeno específico (ITSA) es eficaz en el tratamiento de enfermedades alérgicas, se discute cómo un tratamiento local puede inducir modificaciones inmunitarias sistémicas. Las moléculas de adhesión son esenciales en la regulación leucocitaria, y se ha planteado la hipótesis de que el extracto alergénico, administrado localmente, puede inducir una estimulación de las moléculas de adhesión vascular (CAM) de los vasos de la mucosa, que se traduce en un reclutamiento local de células inflamatorias circulantes. En el presente trabajo hemos investigado si los antígenos de ácaros Der p1 y Der p2 pueden regular la expresión de células endoteliales humanas (CEH) por CAM. Para ello, se sometió a cortes de vena de cordón umbilical humano completo a cultivos a corto plazo (8 horas) en presencia o en ausencia del antígeno de ácaro (controles no estimulados basales). Después se evaluó inmunohistoquímicamente la expresión de moléculas ICAM-1 y VCAM-1 en cortes criostáticos de las muestras. Este análisis reveló que Der p1 es capaz de regular significativamente ICAM-1 y VCAM-1 sobre CEH, mientras que el antígeno Der p2 regula moderadamente la expresión de ICAM-1 pero es ineficaz para regular VCAM-1. Un que preliminares, estos resultados apoyan conclaridad la hipótesis de que al menos parte de los efectos de la inmunoterapia sublingual pueden deberse al reclutamiento de células inflamatorias en el lugar de liberación del alergeno (AU)

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.

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 (AU)

Antecedentes: Existen datos convincentes de que las células precursoras hematopoyéticas (HPC) desempeñan una función esencial en el establecimiento de la inflamación celular en las enfermedades alérgicas. Se han descrito a menudo mayores concentraciones de CD34+ HPC circulantes, comprometidas con la estirpe mieloide, en la rinitis alérgica, el asma y el eccema, mientras que se han identificado células CD34+ en los infiltrados celulares de tejidos, en zonas periféricas de inflamación. Método: Realizamos un estudio preliminar para evaluar el tráfico de CD34+ en la sangre periférica de 22 pacientes consecutivos (13 varones y 9 mujeres; promedio de edad de 28,9 años), con independencia del tratamiento. Los pacientes padecían rinitis, asma, eccema, urticaria y reacciones alimentarias adversas de presunto origen alérgico. Las reacciones alérgicas eran extrínsecas en 18 pacientes e intrínsecas en cuatro. En el caso de 12 pacientes que recibieron inmunoterapia específica por vía sublingual se cuantificaron las células CD 34 + en el momento de inscripción (T0), un año después (T1) y dos años más tarde (T2). La intensidad de los síntomas se graduó con una escala de cinco puntos (0 = ausencia de síntomas y 4 = síntomas graves). Se evaluó como testigos a 20 personas sanas (10 varones y 10 mujeres; promedio de edad de 24,5 años).Para obtener información sobre la cantidad de HPC circulantes, con independencia del compromiso de estirpe (Lin ñ ) y el grado de diferenciación (CD34bright/dim: no divididas/divididas), aplicamos una modificación del protocolo de Milán para calcular las células CD34+ en sangre periférica. Las células se analizaron empleando un BD FACScan o un FACSCalibur y los resultados se expresaron como porcentaje de células positivas. Resultados: El tráfico de células CD 34 + en el grupo de control fue muy escaso, pues todos los valores fueron t = 0,000].Conclusiones: Los datos descritos indican que el método empleado es eficaz para evaluar la inflamación alérgica aguda, así como la inflamación persistente mínima que subyace a una enfermedad clínica asintomática. La evaluación del tráfico periférico de CD34bright/dim, si lo confirman los resultados de un estudio multicéntrico que se está planificando junto con un estudio tradicional de la IgE circulante, podría ser un método de laboratorio incruento fidedigno para vigilar la inflamación alérgica. (AU)

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.

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.

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 (AU)

La inmunoterapia específica ha demostrado ser efectiva en pacientes con enfermedades respiratorias mediadas por IgE y se están estudiando nuevas rutas de administración alternativas. La Dermatitis atópica (DA) está en la actualidad considerada como una enfermedad alérgica inflamatoria. Métodos: hemos llevado a cabo un estudio de cohorte para evaluar la seguridad y eficacia de la inmunoterapia por vía sublingual (SLIT) en un grupo de pacientes seleccionados que padecían DA extrínseca. Se han incluido 35 pacientes, 16 de ellos con DA sin síntomas respiratorios (grupo A) y 19 con DA asociada a asma leve y/o rinitis (grupo B). La intensidad de las lesiones cutáneas (eczema) se ha graduado en una escala de 0-4, en relación al más reciente Índice SCORAD, donde 0 correspondía a la completa curación del eczema y 4 correspondía a la extensión máxima de las lesiones. Solamente los pacientes con eczema entre 1 y 3 recibieron SLIT durante 36 meses.La graduación del eczema, los síntomas y los efectos adversos se registraron durante visitas de control realizadas cada dos meses durante los dos primeros años y luego a los 36 meses. Después de la interrupción de la SLIT, a todos los pacientes se les hacía un seguimiento anual durante tres años para evaluar los efectos a largo plazo del tratamiento. Todos los pacientes siguieron una serie de normas de conducta con el fin de controlar las variables externas que pudiesen confundir los resultados. Todos los pacientes recibieron ketotifeno durante los tres primeros meses de SLIT. Resultados: para la evaluación de la eficacia, solamente se ha considerado la completa desaparición de las lesiones de piel (score 0). Esta evolución fue observada en el grupo A en el 12,6 por ciento de los pacientes después de seis meses de tratamiento, en el 31,7 a los 12 meses y en el 68,8 por ciento después de 24 meses.En el grupo B, el eczema desapareció en el 0 por ciento después de seis meses, en el 36,8 por ciento después de 12 meses y en el 73,7 por ciento tras 24 meses. Ningún paciente del grupo A desarrolló asma durante el tratamiento, mientras que un paciente padeció asma tres años después de haber cesado la SLIT. Se detectaron tres reacciones locales que consistieron en dos eczemas leves y diarrea. La única reacción sistémica observada fue un caso de urticaria, debido a la violación del esquema de administración. No hubo ninguna reacción que amenazara la vida. Conclusiones: los resultados obtenidos, teniendo en cuenta los límites del diseño del estudio, sugieren que la SLIT en el tratamiento de la forma extrínseca de la DA es segura y bien tolerada por los pacientes y podría alterar favorablemente al curso natural de la enfermedad. (AU)

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.