Immunomodulation of allergic immune response during specific immunotherapy

Atopic, IgE-mediated allergies are one of the major public health problems in Finland and other Western countries. These diseases are characterized by type 2 T helper (Th2) cell predominated immune responses (interleukin-4 (IL-4), IL-5) against ubiquitous environmental allergens. Despite of adequate pharmacological treatment, more than 20% of the patients with allergic rhinitis develop asthma. Allergen specific immunotherapy (SIT) is the only treatment currently available to affect to the natural course of allergic diseases. This treatment involves repeated administration of allergens to the patients either via sublingual route (sublingual immunotherapy, SLIT) or by subcutaneous injections (subcutaneous immunotherapy, SCIT). Successful treatment with SCIT or SLIT has been shown to provide long-term remission in symptoms, and prevent disease progression to asthma, but the immunological mechanisms behind these beneficial effects are not yet completely understood. Increased knowledge of such mechanisms could not only help to improve SIT efficacy, but also provide tools to monitor the development of clinical response to SIT in individual patients, and possibly also, predict the ultimate therapeutic outcome. The aim of this work was to clarify the immunological mechanisms associated with SIT by investigating the specific allergen-induced immune responses in peripheral blood mononuclear cells (PBMC) of allergic rhinitis patients during the course of SLIT and SCIT. The results of this work demonstrate that both therapies induced increases in the protective, Th2-balancing Th1 type immune responses in PBMC, e.g. by up-regulating signaling lymphocytic activation molecule (SLAM) and interferon gamma (IFN-γ) expression, and augmented tolerogenic T regulatory (Treg) cell type responses against the specific allergens, e.g. by increasing IL-10 or Forkhead box P3 (FOXP3) expression. The induction of allergen-specific Th1 and Treg type responses during SLIT were dependent on the treatment dose, favoring high allergen dose SLIT. During SCIT, the early decrease in Th2 type cytokine production - in particular of IL-4 mRNA and IL-4/IFN-γ expression ratio - was associated with the development of good therapeutic outcome. Conversely, increases in both Th2 (IL-5) and Th1 (IFN-γ, SLAM) type responses and IL-10 mRNA production were seen in the patients with less effective outcome. In addition, increase in Th17 type cytokine (IL-17) mRNA production was found in the PBMC of patients with less effective outcome during both SLIT and SCIT. These data strengthen the current hypothesis that immunomodulation of allergen-specific immune responses from the prevailing Th2-biased responses towards a more Th1 type, and induction of tolerogenic Treg cells producing IL-10 represent the two key mechanisms behind the beneficial effects of SIT. The data also give novel insight into the mechanisms why SIT may fail to be effective in some patients by demonstrating a positive correlation between the proinflammatory IL-17 responses, Th2 type IL-5 production and clinical symptoms. Taken together, these data indicate that the analysis of Th1, Th2, Treg ja Th17-associated immune markers such as IL-10, SLAM, IL-4, IL-5 and IL-17 could provide tools to monitor the development of clinical response to SIT, and thereby, predict the ultimate clinical outcome already in the early course of the treatment.

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs

Oral tolerance attenuates changes in in vitro lung tissue mechanics and extracellular matrix remodeling induced by chronic allergic inflammation in guinea pigs. J Appl Physiol 104: 1778-1785, 2008. First published April 3, 2008; doi:10.1152/japplphysiol.00830.2007.-Recent studies emphasize the presence of alveolar tissue inflammation in asthma. Immunotherapy has been considered a possible therapeutic strategy for asthma, and its effect on lung tissue had not been previously investigated. Measurements of lung tissue resistance and elastance were obtained before and after both ovalbumin and acetylcholine challenges. Using morphometry, we assessed eosinophil and smooth muscle cell density, as well as collagen and elastic fiber content, in lung tissue from guinea pigs with chronic pulmonary allergic inflammation. Animals received seven inhalations of ovalbumin (1-5 mg/ml; OVA group) or saline (SAL group) during 4 wk. Oral tolerance (OT) was induced by offering ad libitum ovalbumin 2% in sterile drinking water starting with the 1st inhalation (OT1 group) or after the 4th (OT2 group). The ovalbumin-exposed animals presented an increase in baseline and in postchallenge resistance and elastance related to baseline, eosinophil density, and collagen and elastic fiber content in lung tissue compared with controls. Baseline and post-ovalbumin and acetylcholine elastance and resistance, eosinophil density, and collagen and elastic fiber content were attenuated in OT1 and OT2 groups compared with the OVA group. Our results show that inducing oral tolerance attenuates lung tissue mechanics, as well as eosinophilic inflammation and extracellular matrix remodeling induced by chronic inflammation.

Polissensibilização alimentar : um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Histometry of the sublingual gland in male and female mice (Mus musculus) infected with the RAL strain of the Chagas parasite, Trypanosoma cruzi

Trypanosoma cruzi. The aim of this work was to analyze histologically and histometrically the sublingual gland of mice infected with the RAL strain of T cruzi, according to the sex. Swiss mice (Mus musculus) were inoculated with 2 x 10(4) blood trypomastigotes of the RAL strain of T cruzi. In the peak of the parasitemia (12th day) the mice were sacrificed, and the sublingual glands were fixed in ALFAC. HE-stained histological sections were evaluated histometrically. The parasitemia was higher in females. Histopatologically, acini of the infected animals were smaller, with scanty production of secretion, and smaller striated ducts. The nuclei of the demilunes were smaller and showed amastigote nests in the cytoplasm. Karyometrically, nuclei of the acini, demilunes and striated ducts were smaller in the infected mice. Stereologically, it was observed that relative volumes of acini and ducts were smaller and, inversely, relative volumen were greater for the conjunctive tissue in the infected males. The surface densities of acini and ducts were bigger and the diameter and thickness of the wall were smaller in this group. On the other hand, relative volume of acini was smaller and those of the ducts and conjunctive tissue were bigger in the infected females. The diameter and thickness of the wall of acini were smaller, and those of the striated ducts were bigger in this group. The RAL strain of T cruzi caused general atrophy in the sublingual gland, with numerous nests of parasites in the glandular parenchyma.

The prospects for immunotherapy in smoking cessation

Context Smoking is a major preventable cause of death and disability that is maintained by dependence on nicotine. Smoking cessation reduces mortality and morbidity. Although existing pharmacological aids to smoking cessation and relapse prevention (nicotine replacement therapy and bupropion) improve on unassisted quitting and behavioural methods, they are only modestly effective. More effective pharmacological methods are required that improve compliance, reduce side-effects, and can be used in combination with existing cessation methods. Starting point A nicotine vaccine is a promising immunotherapeutic approach to smoking cessation and relapse prevention. Such a vaccine would induce the immune system to form specific antibodies to nicotine to prevent it from crossing the blood-brain barrier to act on receptor sites in the central nervous system. Recent studies in rats provide proof of principle by showing that nicotine-specific antibodies can prevent the reinstatement of nicotine self-administration (N Lindblom et al, Respiration 2002; 69: 254–60) and block dopamine release in the shell of the nucleus accumbens (Sde Villiers et al, Respiration 2002; 69: 247–53). A phase 1 trial of a human cocaine vaccine has also recently been successfully completed (T Kosten et al, Vaccine 2002; 20: 1196–204). A safe and effective human nicotine vaccine would potentially have fewer side-effects and better compliance than existing smoking-cessation pharmacotherapies. It could also be used in combination with some of them (eg, bupropion). Where next? The most promising clinical application of a human nicotine vaccine is likely to be in relapse prevention in abstinent smokers. A vaccine may also have a role in preparing smokers to quit. Clinical trials of safety and efficacy in human smokers and ex-smokers are warranted. If a nicotine vaccine proves to be safe and effective, the health-care system will need to ensure that it is registered for clinical use and that the poorer members of the community (among whom smoking prevalence is now highest in developed countries) have access to the vaccine. The community will need to be appropriately informed about the role of a nicotine vaccine to ensure that it is not prematurely used for preventive purposes in children and adolescents.

Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

Considering that mycobacterial heat-shock protein 65 (hsp65) gene transfer can elicit a profound antitumoral effect, this study aimed to establish the safety, maximum-tolerated dose (MTD) and preliminary efficacy of DNA-hsp65 immunotherapy in patients with advanced head and neck squamous cell carcinoma (HNSCC). For this purpose, 21 patients with unresectable and recurrent HNSCC were studied. Each patient received three ultrasound-guided injections at 21-day intervals of: 150, 600 or 400 mu g of DNA-hsp65. Toxicity was graded according to CTCAE directions. Tumor volume was measured before and after treatment using computed tomography scan. The evaluation included tumor mass variation, delayed-type hypersensitivity response and spontaneous peripheral blood mononuclear cell proliferation before and after treatment. The MTD was 400 mg per dose. DNA-hsp65 immunotherapy was well tolerated with moderate pain, edema and infections as the most frequent adverse effects. None of the patients showed clinical or laboratory alterations compatible with autoimmune reactions. Partial response was observed in 4 out of 14 patients who completed treatment, 2 of which are still alive more than 3 years after the completion of the trial. Therefore, DNA-hsp65 immunotherapy is a feasible and safe approach at the dose of 400 mg per injection in patients with HNSCC refractory to standard treatment. Further studies in a larger number of patients are needed to confirm the efficacy of this novel strategy.

Submandibular and sublingual glands involvement in advanced acquired immunodeficiency syndrome (AIDS): an autopsy-based study

Objective. To assess the histopathological, immunohistochemical (IHC), and in situ hybridization (ISH) features found in the submandibular (SM) and sublingual (SL) glands of 105 acquired immunodeficiency syndrome (AIDS) patients at autopsy. Study design. Gender, age, CD4 cell level, and clinical histories were obtained from clinical charts (SM: n = 103; SL: n = 92). Histologic analysis of hematoxylin and eosin, Gomori-Grocott, and Ziehl-Neelsen stained tissues, IHC to detect infectious agents and characterize inflammatory cells in sialadenitis, and ISH for EBER-1/2 were performed. Results. The mean age of the patients and CD4 cell count were 36 years and 76 cells/mu L, respectively. Fifty-eight cases (SM: n = 51 [49%]; SL: n = 54 [59%]) were considered to be microscopically normal. The most common infectious conditions were mycobacteriosis (SM: n = 11 [10%]; SL: n = 7 [7%]), followed by cytomegalovirus (CMV) (SM: n = 14 [13%]; SL: n = 2 [2%]), and cryptococcosis (SM: n = 3 [3%]; SL: n = 4 [4%]). Human immunodeficiency virus (HIV) p24 (SM: n = 2 [2%]; SL: n = 1 [1%]) and EBER-1/2 (SM: n = 9 [39%]; SL: n = 4 [20%]) were seen only in macrophages and lymphocytes, respectively. The most prevalent cells seen in chronic nonspecific sialadenitis (SM: n = 25; SL: n = 25) were CD8+ T lymphocytes, whereas CD68+ macrophages were predominant in the mycobacteriosis-associated granulomatous and nonspecific diffuse macrophagic sialadenitis. Concomitant infections occurred in 5 cases (SM: n = 4; SL: n = 1) and non-Hodgkin lymphoma in 1 case. Conclusions. Infectious diseases and chronic nonspecific sialadenitis were the main alterations found in the SM and SL glands. These alterations were greater in the SM than in the SL glands. CD8+ T lymphocytes and CD68+ macrophages might be relevant to the pathogenesis of the sialadenitis. Clinicians should consider these diseases when assessing the major salivary glands in advanced AIDS patients and follow biosafety procedures to avoid contamination by HIV, CMV, mycobacteriosis, and cryptococcosis. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108: 216-226)

Comparison of oral versus sublingual piroxicam during postoperative pain management after lower third molar extraction

In this study, 53 patients received piroxicam, administered orally or sublingually, after undergoing removal of symmetrically positioned lower third molars, during two separate appointments. This study used a randomized, blind, cross-over protocol. Objective and subjective parameters were recorded for comparison of postoperative results for 7 days after surgery. Patients treated with oral or sublingual piroxicam reported low postoperative pain scores. The patients who received piroxicam orally took a similar average amount of analgesic rescue medication compared with patients who received piroxicam sublingually (p > 0.05). Patients exhibited similar values for mouth opening measured just before surgery and immediately following suture removal 7 days later (p > 0.05), and showed no significant differences between routes of piroxicam administration for swelling control during the second or seventh postoperative days (p > 0.05). In summary, pain, trismus and swelling after lower third molar extraction, independent of surgical difficulty, could be controlled by piroxicam 20 mg administered orally or sublingually and no significant differences were observed between the route of delivery used in this study.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Metastatic lesions in the joint associated with acute inflammatory arthritis after dendritic cell immunotherapy for metastatic melanoma

A 47 year old man undergoing immunotherapy for metastatic melanoma with autologous dendritic cells pulsed with autologous tumour peptide and hepatitis a surface antigen developed acute left ankle arthritis. Gout and acute infection were excluded, and an autoimmune aetiology or occult metastasis were considered. The arthritis initially subsided with indomethacin, but the symptoms recurred 2 months later, and magnetic resonance imaging demonstrated metastatic melanoma of the left talus. Immunohistochemical staining of a cerebral metastatic deposit biopsied 1 week after the onset of arthritis demonstrated T-cell and macrophage infiltration of the tumour. In addition, the patient developed melanoma-specific delayed type hypersensitivity and cytotoxic T-cell responses after vaccination. Thus, the monoarthritis represented an 'appropriate' inflammatory response directed against metastatic melanoma. (C) 2001 Lippincott Williams & Wilkins.

Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune disease

Dendritic cells (DC) have a key role in controlling the immune response, by determining the outcome of antigen presentation to T cells. Through costimulatory molecules and other factors, DC are involved in the maintenance of peripheral tolerance through modulation of the immune response. This modulation occurs both constitutively, and in inflammation, in order to prevent autoimmunity and to control established immune responses. Dendritic cell control of immune responses may be mediated through cytokine or cell-contact dependent mechanisms. The molecular and cellular basis of these controls is being understood at an increasingly more complex level. This understanding is reaching a level at which DC-based therapies for the induction of immune regulation in autoimmunity can be tested in vivo. This review outlines the current state of knowledge of DC in immune tolerance, and proposes how DC might control both T cell responses, and themselves, to prevent autoimmunity and maintain peripheral tolerance.

Monitoring and isolation of blood dendritic cells from apheresis products in healthy individuals: a platform for cancer immunotherapy

The fundamental role of dendritic cells (DC in initiating and directing the primary immune response is well established. Furthermore, it is now accepted that DC may be useful in new vaccination strategies for preventing certain malignant and infectious diseases. As blood DC (BDC physiology differs from that of the DC homologues generated in vitro from monocyte precursors, it is becoming more relevant to consider BDC for therapeutic interventions. Until recently, protocols for the isolation of BDC were laborious and inefficient; therefore, their use for investigative cancer immunotherapy is not widespread. In this study, we carefully documented BDC counts, yields and subsets during apheresis (Cobe Spectra), the initial and essential procedure in creating a BDC isolation platform for cancer immunotherapy. We established that an automated software package (Version 6,0 AutoPBPC) provides an operator-independent reliable source of motionuclear cells (MNC for BDC preparation. Further, we observed that BDC might be recovered in high yields, often greater than 100% relative to the number of circulating BDC predicted by blood volume. An average of 66 million (range, 17-179) BDC per 10-1 procedure were obtained, largely satisfying the needs for immunization. Higher yields were possible on total processed blood volumes of 151. BDC were not activated by the isolation procedure and, more importantly, both BDC subsets (CD11c(+)CD123(low) and CD11c(-)CD123(high)) were equally represented. Finally, we established that the apheresis product could be used for antibody-based BDC immunoselection and demonstrated that fully functional BDC can be obtained by this procedure. (C) 2002 Published by Elsevier Science B.V.

Donor-derived b2a2-specific T cells for immunotherapy of patients with chronic myeloid leukemia

The BCR-ABL fusion proteins, b2a2 and b3a2, are potential targets for a beneficial graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation for chronic myeloid leukemia (CML). This study demonstrates that CD4(+) T cells specific to the b2a2 peptide can be generated from a normal allogeneic stem cell transplant donor after stimulation with monocyte-derived dendritic cells (Mo-DC) using culture conditions applicable to clinical use. Stimulation of donor T-cell enriched mononuclear cells (MNC) with b2a2-pulsed Mo-DC produced approximately 3 x 10(9) b2a2-specific CD4(+) T cells. The CD4(+) T cells were HLA-DR7 restricted. These results confirm that the generation of donor derived b2a2-specific T cells for clinical use is feasible and warrants clinical testing after stem cell transplantation.