Secrets and lies of host-microbial interactions: MHC restriction and trans-regulation of T cell trafficking conceal the role of microbial agents on the edge between health and multifactorial/complex diseases.

Here we critically discuss data supporting the view that microbial agents (pathogens, pathobionts or commensals alike) play a relevant role in the pathogenesis of multifactorial diseases, but their role is concealed by the rules presiding over T cell antigen recognition and trafficking. These rules make it difficult to associate univocally infectious agents to diseases' pathogenesis using the paradigm developed for canonical infectious diseases. (Cross-)recognition of a variable repertoire of epitopes leads to the possibility that distinct infectious agents can determine the same disease(s). There can be the need for sequential infection/colonization by two or more microorganisms to develop a given disease. Altered spreading of infectious agents can determine an unwanted activation of T cells towards a pro-inflammatory and trafficking phenotype, due to differences in the local microenvironment. Finally, trans-regulation of T cell trafficking allows infectious agents unrelated to the specificity of T cell to modify their homing to target organs, thereby driving flares of disease. The relevant role of microbial agents in largely prevalent diseases provides a conceptual basis for the evaluation of more specific therapeutic approaches, targeted to prevent (vaccine) or cure (antibiotics and/or Biologic Response Modifiers) multifactorial diseases.

House dust mite allergy and shrimp allergy: a complex interaction.

Summary: Background and Objective. Sensitization and allergy to shrimp among Italian house dust mite allergic patients are not well defined and were investigated in a large multicenter study. Methods. Shrimp sensitization and allergy were assessed in 526 house dust mite (HDM)-allergic patients submitted to the detection of IgE to Der p 10 and 100 atopic control not sensitized to HDM. Results. Shrimp allergy occurred in 9% of patients (vs 0% of 100 atopic controls not sensitized to HDM; p minor 0.001). Shrimp-allergic patients were less frequently hypersensitive to airborne allergens other than HDM than crustacean-tolerant subjects (35% vs 58.8%; p minor 0.005). Only 51% of tropomyosin-sensitized patients had shrimp allergy, and these showed significantly higher Der p 10 IgE levels than shrimp-tolerant ones (mean 22.2 KU/l vs 6.2 KU/l; p minor 0.05). Altogether 53% of shrimp-allergic patients did not react against tropomyosin. Conclusions. Shrimp allergy seems to occur uniquely in association with hypersensitivity to HDM allergens and tropomyosin is the main shrimp allergen but not a major one, at least in Italy. Along with tropomyosin-specific IgE levels, monosensitization to HDM seems to represent a risk factor for the development of shrimp allergy among HDM allergic patients.

Prosthetic Aortic Valve Thrombosis: Surgery or Thrombolysis.

Mechanical prosthetic valve thrombosis is a serious complication which necessitates immediate intervention. The presenting signs and symptoms of this illness are somewhat variable, but physical examination and trans-esophageal-echocardiography enable rapid diagnosis. Valve replacement or thrombolysis in the correct hospital setting must be performed to avoid life-threatening complication without delay. But it is not proven entirely which therapy is superior. For any given patient, the risks of thrombolytic therapy, including bleeding, systemic embolism and failure to restore valvular function, must be weighed against the risks of surgical intervention. In spite of aggressive therapy, morbidity and mortality from prosthetic valve thrombosis and its treatment are not less indeed. This report describes the case of a woman with aortic prosthetic valves who presents with heart failure and evidence of severe prosthetic aortic valve dysfunction after a period of suboptimal anticoagulation.

TLR2: a crossroads between infections and autoimmunity?

Environment has both pathogenic and protective roles in the determination of autoimmune disease development, possibly through infectious agents. TLR2 has the capability to recognize the widest range of PAMPs, and it is important for the recognition of mycobacteria and gram-positive bacteria. Here we review recent information showing that TLR2 ligands, its signaling machinery and the effects of its engagement on T cell polarization and differentiation, all play a decisive role in experimental models of autoimmunity. Thus, we propose that engagement of TLR2 is an important crossroad between encounter with bacteria and development of self-reactive diseases.

Skewed T-cell receptor repertoire: more than a marker of malignancy, a tool to dissect the immunopathology of inflammatory diseases.

The highly diverse heterodimeric surface T cell receptor (TCR) gives the T lymphocyte its specificity for MHC-bound peptides needed to initiate antigen-recognition. In normal peripheral blood, spleen and lymph nodes, the TCR repertoire of the T lymphocytes is usually polyclonal. However, in malignancies such as leukemias, as well as in lymphoproliferative diseases of mature T cells, the TCR is a reflection of the clonality of the malignant cells and is therefore monoclonal. Several clinical conditions (mainly solid tumors and autoimmune diseases) have been described where the TCR repertoire is restricted. The ability to demonstrate clonal TCR usage provides a useful tool to dissect the immunopathology of inflammatory diseases. In this review we discuss these findings and propose to sub-divide diseases with restricted TCR repertoire into a group of conditions in which there is a known TCR ligand, as opposed to diseases in which the restricted TCR repertoire is the result of impaired T-cell development. This classification sheds light on the pathogenesis of several inflammatory diseases.

Study of the effects of Lemna minor extracts on human immune cell populations.

OBJECTIVE: Lemna minor is a plant with a huge repertoire of secondary metabolites. The literature indicates that extracts of Lemna minor have antioxidant, antiradical, immunomodulatory and anti-inflammatory properties. The objective of the present study was to find a suitable technique to extract active compounds from this plant and verify whether these extracts have immunomodulatory activity. MATERIALS AND METHODS: We grew L. minor on a standard medium with Gamborg B5 and vitamins. We extracted compounds from the plant by maceration and decoction. The phytochemical profile of the extracts was characterized by chromatography, spectrophotometry, and spectroscopy. The extracts were tested on cultures of mononuclear cells from four human subjects. These cells were pulsed with carboxyfluorescein succinimidyl ester, grown in triplicate in standard culture medium without (control) and with increasing concentrations of Lemna extracts. Flow cytometry was used to evaluate cell death and proliferation of the total mononuclear cell population and of CD4+, CD8+, B cell and monocyte populations. RESULTS: The Lemna extracts were not cytotoxic and did not cause cell necrosis or apoptosis in immune cells. At low concentrations, they induced very limited proliferation of CD4+ cells within 48 hours. At high concentrations, they induced proliferation of CD8+ cells and B lymphocytes within 48 hours. CONCLUSIONS: Unfortunately, we failed to confirm any immunomodulatory activity of Lemna extracts. Growth and death rates of human immune cells were not significantly affected by adding Lemna extracts to the culture medium.

Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant.

We have analyzed the relative contribution of dendritic cells (DC) and B cells in the presentation of peptide-class II complexes in an inflammatory situation in vivo. Draining lymph node cells from mice immunized subcutaneously with hen egg-white lysozyme (HEL) in adjuvant display HEL peptide-major histocompatibility complex class II complexes able to stimulate, in the absence of any further antigen addition, specific T hybridoma cells. The antigen-presenting capacity of three different antigen-presenting cell (APC) populations recruited in lymph nodes, DC (N418+, class II+, B220-, low buoyant density), large B cells (B220+, low buoyant density), and small B cells (B220+, high buoyant density), was analyzed. After immunization with HEL in adjuvant, DC are the only lymph node APC population expressing detectable HEL peptide-class II complexes. These results indicate that lymph node DC and not B cells are the APC initiating the immune response in vivo after administration of antigen in adjuvant.

Awareness of medical radiation exposure among patients: A patient survey as a first step for effective communication of ionizing radiation risks.

INTRODUCTION: The European Directive 2013/59/EURATOM requires patient radiation dose information to be included in the medical report of radiological procedures. To provide effective communication to the patient, it is necessary to first assess the patient's level of knowledge regarding medical exposure. The goal of this work is to survey patients' current knowledge level of both medical exposure to ionizing radiation and professional disciplines and communication means used by patients to garner information. MATERIAL AND METHODS: A questionnaire was designed comprised of thirteen questions: 737 patients participated in the survey. The data were analysed based on population age, education, and number of radiological procedures received in the three years prior to survey. RESULTS: A majority of respondents (56.4%) did not know which modality uses ionizing radiation. 74.7% had never discussed with healthcare professionals the risk concerning their medical radiological procedures. 70.1% were not aware of the professionals that have expertise to discuss the use of ionizing radiation for medical purposes, and 84.7% believe it is important to have the radiation dose information stated in the medical report. CONCLUSION: Patients agree with new regulations that it is important to know the radiation level related to the medical exposure, but there is little awareness in terms of which modalities use X-Rays and the professionals and channels that can help them to better understand the exposure information. To plan effective communication, it is essential to devise methods and adequate resources for key professionals (medical physicists, radiologists, referring physicians) to convey correct and effective information.

Antibodies elicited by naked DNA vaccination against the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain idiotypic determinants of B-lymphoproliferative disorders specifically react with patients' tumor cells.

Several reports have suggested that the mechanism of protection induced by antiidiotypic vaccination against low-grade lymphoproliferative disorders is likely to be antibody mediated. Here we test the hypothesis that DNA vaccination with the short peptide encompassing the complementary-determining region 3 hypervariable region of immunoglobulin heavy chain (VH-CDR3) may elicit a specific antibody immune response able to recognize the native antigens in the form required for therapy. As a test system, we used the VH-CDR3 sequences derived from two patients with non-Hodgkin's B lymphomas (PA, AS) and one patient with hairy cell leukemia (BA) to immunize outbred Swiss mice. This experimental model could mimic a clinical setting in which different patients present distinct HLA haplotypes. Individual tumor-specific VH-CDR3 sequences were amplified by a two-step procedure and directly cloned into multigenic plasmid vectors (pRC100 and derived) with and without mouse interleukin 2 (mIL-2). Each tumor-specific sequence was characterized by sequencing. Female Swiss mice were vaccinated i.m. with plasmids expressing the tumor-specific VH-CDR3 sequence alone (pRC101-PA), mIL-2 plus the VH-CDR3 sequence (pRC111-PA), or a different unrelated antigen (NS3 of hepatitis C virus; pRC112), the sole mIL-2 (pRC110), and the empty plasmid (pRC100). Boost injections were performed at 3 and 16 weeks from the first vaccination, and sera were drawn before each vaccination and at 6, 9, and 19 weeks. Induction of anti-VH-CDR3s antibodies in the sera and their ability to recognize native antigens on patients' tumor cells were evaluated by FACS analysis. Up to 56% (n = 25) of mice vaccinated with pRC111-PA plasmid and 20% (n = 15) of mice vaccinated with pRC101-PA developed a specific immune response that was maintained throughout 19 weeks of observation in 40% of pRC111-PA-vaccinated mice. No response was detected in sera obtained from mice vaccinated with the other plasmids (n = 45). pRC111-PA injection s.c. was less effective (13%, n = 15) than i.m. injection (53%, n = 15). Indeed, we demonstrated that antibodies elicited by naked DNA vaccination against three different patient-derived VH-CDR3 peptides (pRC111-PA or BA or AS) readily reacted with binding epitopes on the idiotypic proteins expressed on the surface of tumor cells derived from each patient; 60, 40, and 40% of, respectively, PA-, BA-, and AS-vaccinated mice developed specific antibodies. No cross-reactivity was detected among the three different CDR3s against tumor cells derived from the other two patients. The outbred mouse strategy confirmed the significant matching potential of three different VH-CDR3 peptides to be efficaciously presented through different MHCs. We conclude that individual VH-CDR3 DNA vaccination can result in a potentially effective specific immune response against non-Hodgkin's B lymphoma cells by a rapid and low-cost therapeutic approach.

Molecular characterization of the T cell repertoire using immunoscope analysis and its possible implementation in clinical practice.

T lymphocytes play a central role in the pathogenesis of a large number of human conditions including autoimmunity and graft rejection. Although T cells are key players in mounting immune responses, the assessment of T cell repertoires has yet to find an important role in clinical decision making. In this review, we discuss the "immunoscope" technique and its potential diagnostic role in a variety of clinical scenarios. This is an RT-PCR based approach that subdivides a bulk T cell population (i. e. from blood, lymph, spleen, or tissue) into approximately 2800 groups based upon rearranged variable beta (Vbeta)/joining beta (Jbeta) gene segments and the resulting length of the T cell receptor's (TCR's) third complementarity determining region (CDR-3). This extensive subdivision, or focusing, allows clonal expansions to be directly observed. Such a fine-tuned analysis has revealed previously unappreciated aspects of the T cell repertoire. For instance, an antigen-specific immune response can be divided into both public and non-public components. The non-public repertoire contains the majority of the expanding T cells which are unique to the individual (private), or shared by only some (semi-private), while "public" T cells can be found responding to the antigenic determinant in every individual. Although they are often a minority of the response, the public T cell repertoire seems to play a more important role in defining, as well as driving, the overall immune phenotype in the animal. Immunoscope analysis has identified public and non-public responses in human pathologies, such as multiple sclerosis. The ability to characterize the driver T cells dictating the state of immunity/autoimmunity in individual patients will be an important step towards understanding autoimmunity and designing effective treatment for a variety of conditions including rheumatoid arthritis and multiple sclerosis. We review the current literature involving public and non-public repertoires and discuss the prospect that immunoscope analysis may play a central role in the study and perhaps the management of human autoimmune diseases, and cancer.

Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

[Essential mixed cryoglobulinemia with liver involvement: a still open problem]. / Crioglobulinemia mista essenziale con coinvolgimento epatico: un problema ancora aperto.

Essential mixed cryoglobulinemia (EMC) is a syndrome characterized by cryoglobulinemia and clinical features including purpura, arthralgia, asthenia (Meltzer-Franklin syndrome) without evidence of any systemic disease Liver involvement in the course of EMC is described in 50-84% of patients. It consists of mild silent hepatosplenomegaly and slightly rise of serum amino transferase. Eleven patients with clinical and laboratory findings suggestive for EMC (five type II and six type III) underwent percutaneous liver biopsy to evaluate the degree of liver involvement. Two liver cirrhosis, two chronic active hepatitis, one chronic persistent hepatitis and a case of hepatic steatosis were found. A type III cryoglobulinemia was present in four of the six patients with liver involvement. All the patients were Hbs Ag negative but three of them were Hbs Ab positive. The pathogenesis of liver involvement in the course of EMC is still now uncertain. The authors believe that a previous HBV infection plays no role in the pathogenesis of EMC syndrome. This syndrome must be considered different from mixed cryoglobulinemia secondary to chronic liver disease. They suggest that liver biopsy is mandatory during the course of EMC even when clinical and laboratory data are silent.

Preparation of a monoclonal antibody against rat MnSOD, using a COOH-terminal peptide.

In the present paper we report the production of a monoclonal antibody against rat MnSOD, a supposed tumor-suppressor protein, using a purified synthetic peptide encompassing amino acids 184-198 to immunize mice, without conjugation to a carrier. The resulting antibody is able to recognize the native form of the protein, since it can immunoprecipitate the MnSOD activity in rat liver homogenate. In Western blot studies, the antibody recognizes a protein of 24 KD M(r), whose concentration varies according to the MnSOD activity and it apparently recognizes also human and mouse MnSODs. The protocol of immunization gives high yield of secreting lines. This monoclonal antibody will allow the detection of structural and functional alterations of MnSOD.

Failure of presented, non-dominant self epitope to induce tolerance: implications for autoimmune diseases.

It has been observed that a hierarchy exists among epitopes such that fewer epitopes are actually involved in the induction of T cell response and tolerance than there are epitopes available in a given antigen. Some epitopes which are "cryptic" for immune activity within the protein, are nevertheless able to elicit a response if administered alone and can also be used to by-pass tolerance. We report that tolerance to a self protein shows the same phenomenon seen for non self proteins. In fact, we elicit a proliferative response toward a predicted minor cryptic epitope, to which animals are clearly not self-tolerant. The minor epitope escapes the induction of tolerance to self proteins more easily than the major epitopes, since we cannot elicit proliferative response to the major epitope. A striking feature of our results however is that lack of self tolerance to the minor epitope appears as not being due to the failure of presentation of this epitope in normal, healthy animals.

Regulation of T-cell responses by CNS antigen-presenting cells: different roles for microglia and astrocytes.

Analysis of the mechanisms underlying CNS immune surveillance and immunopathology have provided new insights into the intracerebral regulation of immune responses. Here, Francesca Aloisi, Francesco Ria and Luciano Adorini review the role of CNS antigen presenting cells and focus on the control of Th1 and Th2 responses by microglia and astrocytes.

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells.

Dendritic cells are the most relevant antigen-presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen-specific T cells, dendritic cells (DC) expressing appropriate peptide-MHC class II complexes secrete IL-12, a cytokine that drives Th1 cell development. To analyze the T cell-mediated regulation of IL-12 secretion by DC, we have examined their capacity to secrete IL-12 in response to stimulation by antigen-specific Th1 and Th2 DO11.10 TCR-transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen-specific Th1, but not Th2 cells, induces IL-12 p40 and p75 secretion by DC. The induction of IL-12 production by Th1 cells does not depend on their IFN-gamma secretion, but requires direct cell-cell contact mediated by peptide/MHC class II-TCR and CD40-CD40L interactions. Th2 cells not only fail to induce IL-12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL-12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2-derived cytokines, IL-10, but not IL-4 inhibit Th1-driven IL-12 secretion. IL-10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1 -induced IL-12 secretion, but it does not account for the failure of Th2 cells to induce IL-12 production by DC. Collectively, these results demonstrate that Th1 cells up-regulate IL-12 production by DC via IFN-gamma-independent cognate interaction, whereas this is inhibited by Th2-derived IL-10. The inhibition of Th1 -induced IL-12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.

Chromatographic and electrophoretic studies of immune complexes in non-A, non-B hepatitis.

Immune complexes isolated from two patients with chronic non-A, non-B hepatitis, one patient with acute non-A, non-B hepatitis and one patient with juvenile rheumatoid arthritis were examined by means of a combined chromatographic and electrophoretic method. Both analyses showed the presence of complexes consisting of IgG, IgM, complement c1q factor and albumin; no antigen constituents were detected. The IgG-to-IgM ratio varied from 1:1 to 4:1, suggesting that one could be dealing with complexes of both IgG-IgM and IgG-IgG types. Moreover, the detectable presence of c1q factor might indicate that such complexes were capable of activating complement.