Immunological Effects of Probiotic Bacteria in Prevention and Treatment of Allergic Diseases in Children

Epidemiological and experimental studies suggest that changes in gut microbial balance are associated with increases in the prevalence of allergic diseases. Probiotics are proposed to provide beneficial immunoregulatory signals which aid in oral tolerance achievement and alleviation of symptoms of allergic diseases. The present study evaluates both the immunological mechanisms of probiotics in infants with allergic diseases and their preventive aspect among infants prone to allergy. Furthermore, the purpose of the study was to characterise the immunological features of cord blood mononuclear cells (CBMCs) in infants at high genetic risk for allergy. GATA-3 expression (p = 0.03), interleukin (IL) -2(p = 0.026), and IL-5 (p = 0.013) secretion of stimulated CBMCs were higher in IgE-sensitized infants at age 2 than in non-allergic, non-sensitized infants. Lactobacillus GG (LGG) treatment increased secretion of IFN-γ by PBMCs in vitro in infants with cow s milk allergy (CMA) (p = 0.006) and in infants with IgE-associated eczema (p = 0.017), when compared to levels in the placebo group. A probiotic mixture, increased secretion of IL-4 by PBMCs in vitro in infants with CMA (p = 0.028), when compared with placebo-group levels. The LGG treatment induced higher plasma C-reactive protein (CRP) (p = 0.021) and IL-6 (p = 0.036) levels in infants with IgE-associated eczema than in the placebo group. The probiotic mixture induced higher plasma IL-10 levels in infants with eczema (p = 0.016). In the prevention study of allergic dis-eases, the infants receiving the probiotic mixture had higher plasma levels of CRP (p = 0.008), total IgA (p = 0.016), total IgE (p = 0.047), and IL-10 (p = 0.002) than did infants in the placebo group. Increased CRP level at age 6 months was associated with a decreased risk for eczema at age 2 not only in the infants who received probiotics but also in the placebo group (p = 0.034). In conclusion, the priming of the GATA-3 and IL-5 pathway can occur in utero, and a primary feature of T-cells predisposing to IgE-sensitization seems to directly favour Th2 deviation. LGG treatment induced increased plasma levels of CRP and IL-6 in infants with IgE-associated eczema, suggesting an activation of innate immu-nity. The probiotic mixture, when given to allergy-prone infants, induced inflammation, detected as increased plasma CRP levels, which at age 6 months was associated with decreased risk for eczema at age 2.The probiotic-induced response in allergy prone infants was characterized by their higher plasma IL-10, total IgE, and CRP levels, without induction of an allergen-specific IgE response. In this respect, the probiotics in infancy appear to induce protective immune profiles that are characteristic for chronic low-grade inflammation, a response resembling that of helminth-like infections.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M.tuberculosis and, in particular, the proline-glutamic acid-polymorphicguanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Thymidine phosphorylase in cancer; Enemy or friend?

Thymidine phosphorylase (TP) is a nucleoside metabolism enzyme that plays an important role in the pyrimidine pathway.TP catalyzes the conversion of thymidine to thymine and 2-deoxy-α-D-ribose-1-phosphate (dRib-1-P). Although this reaction is reversible, the main metabolic function of TP is catabolic. TP is identical to the angiogenic factor platelet-derived endothelial-cell growth factor (PD-ECGF). TP is overexpressed in several human cancers in response to cellular stressful conditions like hypoxia, acidosis, chemotherapy and radiotherapy. TP has been shown to promote tumor angiogenesis, invasion, metastasis, evasion of the immune-response and resistance to apoptosis. Some of the biological effects of TP are dependent on its enzymatic activity, while others are mediated through cytokines like interleukin 10 (IL-10), basic fibroblast growth factor (bFGF) and tumour necrosis factor α (TNFα). Interestingly, TP also plays a role in cancer treatment through its role in the conversion of the oral fluoropyrimidine capecitabine into its active form 5-FU. TP is a predictive marker for fluoropyrimidine response. Given its various biological functions in cancer progression, TP is a promising target in cancer treatment. Further translational research is required in this area.

Stimulating immune responses to fight cancer: Basic biology and mechanisms

Chronic inflammation is now recognized as a major cause of malignant disease. In concert with various mechanisms (including DNA instability), hypoxia and activation of inflammatory bioactive lipid pathways and pro-inflammatory cytokines open the doorway to malignant transformation and proliferation, angiogenesis, and metastasis in many cancers. A balance between stimulatory and inhibitory signals regulates the immune response to cancer. These include inhibitory checkpoints that modulate the extent and duration of the immune response and may be activated by tumor cells. This contributes to immune resistance, especially against tumor antigen-specific T-cells. Targeting these checkpoints is an evolving approach to cancer immunotherapy, designed to foster an immune response. The current focus of these trials is on the programmed cell death protein 1 (PD-1) receptor and its ligands (PD-L1, PD-L2) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Researchers have developed anti-PD-1 and anti-PDL-1 antibodies that interfere with the ligands and receptor and allow the tumor cell to be recognized and attacked by tumor-infiltrating T-cells. These are currently being studied in lung cancer. Likewise, CTLA-4 inhibitors, which have had success treating advanced melanoma, are being studied in lung cancer with encouraging results.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosismand host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid-polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Autoimmune Regulator (AIRE) in the Maintenance of Immunological Tolerance

Autoimmune regulator (AIRE) is the gene mutated in the human polyglandular autoimmune disease called Autoimmune polyendocrinopathy, candidiasis, and ectodermal dystrophy (APECED) that belongs to the Finnish disease heritage. Murine Aire has been shown to be important in the generation of the T cell central tolerance in the thymus by promoting the expression of ectopic tissue-specific antigens in the thymic medulla. Aire is also involved in the thymus tissue organization during organogenesis. In addition to the thymus, AIRE/Aire is expressed in the secondary lymphoid organs. Accordingly, a role for AIRE/Aire in the maintenance of peripheral tolerance has been suggested. Peripheral tolerance involves mechanisms that suppress immune responses in secondary lymphoid organs. Regulatory T cells (Tregs) are an important suppressive T cell population mediating the peripheral tolerance. Tregs are generated in the thymus but also in the peripheral immune system T cells can acquire the Treg-phenotype. The aim of this study was to characterize Tregs in APECED patients and in the APECED mouse model (Aire-deficient mice). In the mouse model, it was possible to separate Aire expression in the thymus and in the secondary lymphoid organs. The relative importance of thymic and peripheral Aire expression in the maintenance of immunological tolerance was studied in an experimental model that was strongly biased towards autoimmunity, i.e. lymphopenia-induced proliferation (LIP) of lymphocytes. This experimental model was also utilised to study the behaviour of T cells with dual-specific T cell receptors (TCR) during the proliferation. The Treg phenotype was studied by flow cytometry and relative gene expression with real-time polymerase chain reaction. TCR repertoires of the Tregs isolated from APECED patients and healthy controls were also compared. The dual-specific TCRs were studied with the TCR repertoire analysis that was followed with sequencing of the chosen TCR genes in order to estimate changes in the dual-specific TCR diversity. The Treg function was tested with an in vitro suppression assay. The APECED patients had normal numbers of Tregs but the phenotype and suppressive functions of the Tregs were impaired. In order to separate Aire functions in the thymus from its yet unknown role in the secondary lymphoid organs, the phenomenon of LIP was utilised. In this setting, the lymphocytes that are adoptively transferred to a lymphopenic recipient proliferate to stimuli from self-originating antigens. This proliferation can result in autoimmunity if peripheral tolerance is not fully functional. When lymphocytes that had matured without Aire in the thymus were transferred to lymphopenic Aire-sufficient recipients, no clinical autoimmunity followed. The Aire-deficient donor-originating lymphocytes hyperproliferated, and other signs of immune dysregulation were also found in the recipients. Overt autoimmunity, however, was prevented by the Aire-deficient donor-originating Tregs that hyperproliferated in the recipients. Aire-deficient lymphopenic mice were used to study whether peripheral loss of Aire had an impact on the maintenance of peripheral tolerance. When normal lymphocytes were transferred to these Aire-deficient lymphopenic recipients, the majority of recipients developed a clinically symptomatic colitis. The colitis was confirmed also by histological analysis of the colon tissue sections. In the Aire-deficient lymphopenic recipients Tregs were proliferating significantly less than in the control group s recipients that had normal Aire expression in their secondary lymphoid organs. This study shows that Aire is not only important in the central tolerance but is also has a significant role in the maintenance of the peripheral tolerance both in mice and men. Aire expressed in the secondary lymphoid organs is involved in the functions of Tregs during an immune response. This peripheral expression appears to be relatively more important in some situations since only those lymphopenic recipients that had lost peripheral expression of Aire developed a symptomatic autoimmune disease. This AIRE-related Treg defect could be clinically important in understanding the pathogenesis of APECED.

Glycodelin A suppresses the cytolytic activity of CD8(+) T lymphocytes

Maternal tolerance to the semi-allogenic fetus is brought about by several mechanisms in humans Glycodelin A (GdA) secreted by the uterine mucosa and decidua is induced to high levels by progesterone between 12 and 16 weeks of pregnancy The glycoprotein an immunomodulator has been shown to be inhibitory to the survival and functions of almost all the immune cells CD8(+) T cells which predominate the T lymphocyte population in the decidua are relatively less studied We attempted to find out the possible mechanism if any of regulation of the cytolytic function of CD8(+) T cells during pregnancy Alloactivated CD8(+) T cells harbouring specific cytolytic activity against target cells exhibited compromised activity upon treatment with high concentrations of GdA Interestingly unlike the CD4(+) T cells CD8(+) T cells were resistant to GdA-induced apoptosis The inhibition of cytotoxic T lymphocyte activity was brought about by the downregulation of transcription of the cytolytic effector molecules granzyme B and perform and the degranulation of cytolytic vesicles These results suggest a protective role played by GdA during pregnancy by regulating the cytolytic activity of CD8(+) T cells (C) 2010 Elsevier Ltd All rights reserved

Characterization and immune response of a protein produced by a cDNA clone of foot and mouth disease virus, type Asia 1 63/72

A 0.9 kb double stranded cDNA of foot and mouth disease virus (FMDV) Type Asia 1, 63/72 was cloned in an expression vector, pUR222. A protein of 38 kd was produced by the clone which reacted with the antibodies raised against the virus. A 20 kd protein which may be derived from the 38 kd protein contained the antigenic epitopes of the protein VP1 of the virus. Injection of 10-20 micrograms of the partially purified 38 and 20 kd proteins or a lysate of cells containing 240 micrograms of the proteins elicited high titers of FMDV specific antibodies in guinea pigs and cattle respectively. Also, at these concentrations, the proteins protected 5 of 8 guinea pigs and 3 of 8 cattle when challenged with a virulent virus.

The use of murine polyclonal anti-idiotypic antibodies as surrogate allergens in the diagnosis of Parthenium hypersensitivity

Background: Anti-idiotypic antibodies (Ab-2), which are the mirror images of idiotypic antibodies (Ab-1), may be useful as diagnostic reagents and for use as immunogen to induce antigen-specific immune responses. Methods and Results: To explore the biologic potential of Ab-2 as diagnostic reagents in allergic diseases, murine mouse (m) Ab-2 were raised by immunizing Balb/c mice with affinity purified rabbit (r) Ab-1 specific for the pollen of Parthenium hysterophorus, an allergenic weed that grows wild on the Indian subcontinent and in Australia, Mexico, and the southern United States. Affinity purified Parthenium-specific human (h)AB-1 could successfully inhibit the binding of mAb-2 to immobilized rAb-1. Further, Balb/c mice immunized with mAb-2 induced Parthenium-specific anti-anti-idiotypic IgE and IgG antibodies. Specificity of the Ab-2 was confirmed by the ability of Parthenium pollen extracts to inhibit the binding of allergen-specific IgE and IgG Ab-1 in the sera of patients with rhinitis to immobilized mAb-2. Parthenium-sensitive patients with rhinitis who had positive results on skin prick tests to Parthenium pollen extracts also responded with a positive skin reaction to mAb-2. Conclusion: Our data demonstrate that Parthenium-specific mAb-2 may be of value as surrogate allergens in allergen standardization and for in vitro diagnosis.

Needleless Vaccine Delivery Using Micro-Shock Waves

Shock waves are one of the most efficient mechanisms of energy dissipation observed in nature. In this study, utilizing the instantaneous mechanical impulse generated behind a micro-shock wave during a controlled explosion, a novel nonintrusive needleless vaccine delivery system has been developed. It is well-known that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle-free devices for epidermal delivery have inherent problems from the perspective of the safety and comfort of the patient. The penetration depth of less than 100 mu m in the skin can elicit higher immune response without any pain. Here we show the efficient utilization of our needleless device (that uses micro-shock waves) for vaccination. The production of liquid jet was confirmed by high-speed microscopy, and the penetration in acrylamide gel and mouse skin was observed by confocal microscopy. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model, and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. Vaccination using our device elicits better protection and an IgG response even at a lower vaccine dose (10-fold less) compared to other routes of vaccination. We anticipate that our novel method can be utilized for effective, cheap, and safe vaccination in the near future.