Wirla Maria da Silva Cunha Tamashiro

Inflammatory Bowel Disease: An Overview of Immune Mechanisms and Biological Treatments

Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the intestinal tract associated with an imbalance of the intestinal microbiota. Crohns disease (CD) and ulcerative colitis (UC) are the most widely known types of IBD and have been the focus of attention due to their increasing incidence. Recent studies have pointed out genes associated with IBD susceptibility that, together with environment factors, may contribute to the outcome of the disease. In ulcerative colitis, there are several therapies available, depending on the stage of the disease. Aminosalicylates, corticosteroids, and cyclosporine are used to treat mild, moderate, and severe disease, respectively. In Crohns disease, drug choices are dependent on both location and behavior of the disease. Nowadays, advances in treatments for IBD have included biological therapies, based mainly on monoclonal antibodies or fusion proteins, such as anti-TNF drugs. Notwithstanding the high cost involved, these biological therapies show a high index of remission, enabling a significant reduction in cases of surgery and hospitalization. Furthermore, migration inhibitors and new cytokine blockers are also a promising alternative for treating patients with IBD. In this review, an analysis of literature data on biological treatments for IBD is approached, with the main focus on therapies based on emerging recombinant biomolecules.

Yacon (Smallanthus sonchifolius): A Functional Food

Yacon (Smallanthus sonchifolius) is an Andean tuberous root that is regarded as a functional food given that it contains fructooligosaccharides (FOS), inulin and phenolic compounds. The consumption of FOS and inulin improves the growth of bifidobacteria in the colon, enhances mineral absorption and gastrointestinal metabolism and plays a role in the regulation of serum cholesterol. Furthermore, the literature reports that the consumption of these prebiotics promotes a positive modulation of the immune system, improving resistance to infections and allergic reactions. Certain studies have demonstrated the potential of yacon as an alternative food source for those patients with conditions that require dietary changes. This review intends to describe the potential of yacon as a prebiotic and its cultivation and industrial processing for human consumption.

Aging alters the production of iNOS, arginase and cytokines in murine macrophages

The limited amount of information on the primary age-related deficiencies in the innate immune system led us to study the production of inducible nitric oxide synthase (iNOS), arginase, and cytokines in macrophages of young (8 weeks old) and old (72 weeks old) female BALB/c mice. We first evaluated iNOS and arginase inducers on peritoneal (PMΦ) and bone marrow-derived (BMMΦ) macrophages of young BALB/c and C57BL/6 mice, and then investigated their effects on macrophages of old mice. Upon stimulation with lipopolysaccharide (LPS), resident and thioglycolate-elicited PMΦ from young mice presented higher iNOS activity than those from old mice (54.4%). However, LPS-stimulated BMMΦ from old mice showed the highest NO levels (50.1%). Identical NO levels were produced by PMΦ and BMMΦ of both young and old mice stimulated with interferon-γ. Arginase activity was higher in resident and elicited PMΦ of young mice stimulated with LPS (48.8 and 32.7%, respectively) and in resident PMΦ stimulated with interleukin (IL)-4 (64%). BMMΦ of old mice, however, showed higher arginase activity after treatment with IL-4 (46.5%). In response to LPS, PMΦ from old mice showed the highest levels of IL-1α (772.3 ± 51.9 pg/mL), whereas, those from young mice produced the highest amounts of tumor necrosis factor (TNF)-α (937.2 ± 132.1 pg/mL). Only TNF-α was expressed in LPS-treated BMMΦ, and cells from old mice showed the highest levels of this cytokine (994.1 ± 49.42 pg/mL). Overall, these results suggest that macrophages from young and old mice respond differently to inflammatory stimuli, depending on the source and maturity of the cell donors.

Induction of systemic tolerance in normal but not in transgenic mice through continuous feeding of ovalbumin.

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)‐2 and interferon (IFN)‐γ] and Th2 (IL‐4 and IL‐10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T‐cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)‐β secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF‐β as a regulatory cytokine in oral tolerance in the murine model.

NK1.1+ cells and T-cell activation in euthymic and thymectomized C57BL/6 mice during acute Trypanosoma cruzi infection

Natural killer (NK) cells may provide the basis for resistance to Trypanosoma cruzi infection, because the depletion of NK1.1 cells causes high levels of parasitemia in young C57Bl/6 mice infected with T. cruzi. Indeed, NK1.1 cells have been implicated in the early production of large amounts of interferon (IFN)‐γ, an important cytokine in host resistance. The NK1.1 marker is also expressed on special subpopulations of T cells. Most NK1.1+ T cells are of thymic origin, and their constant generation may be prevented by thymectomy. This procedure, by itself, decreased parasitemia and increased resistance in young mice. However, the depletion of NK1.1+ cells by the chronic administration of a monoclonal antibody (MoAb) (PK‐136) did not increase the parasitemia or mortality in thymectomized C57Bl/6 mice infected with T. cruzi (Tulahuen strain). To study the cross‐talk between NK1.1+ cells and conventional T cells in this model, we examined the expression of activation/memory markers (CD45RB) on splenic CD4+ and CD8+ T cells from young euthymic or thymectomized mice with or without depletion of NK1.1+ cells and also in aged mice during acute infection. Resistance to infection correlated with the amount of CD4+ T cells that are already activated at the moment of infection, as judged by the number of splenic CD4+ T cells expressing CD45RB−. In addition, the specific antibody response to T. cruzi antigens was precocious and an accumulation of immunoglobulin (Ig)M with little isotype switch occurred in euthymic mice depleted of NK1.1+ cells. The data presented here suggest that NK1.1+ cells have important regulatory functions in euthymic, but not in thymectomized mice infected with T. cruzi. These regulatory functions include a helper activity in the generation of effector or activated/memory T cells.

Aging reduces the primary humoral response and the in vitro cytokine production in mice

Aging is accompanied by a decrease in several physiological functions that make older individuals less responsive to environmental challenges. In the present study, we analyzed the immune response of female BALB/c mice (N = 6) of different ages (from 2 to 96 weeks) and identified significant age-related alterations. Immunization with hapten-protein (trinitrophenyl-bovine serum albumin) conjugates resulted in lower antibody levels in the primary and secondary responses of old mice (72 weeks old). Moreover, young mice (2, 16, and 32 weeks old) maintained specific antibodies in their sera for longer periods after primary immunization than did old mice. However, a secondary challenge efficiently induced memory in old mice, as shown by the increased antibody levels in their sera. The number of CD4+ and CD8+ T cells in the spleen increased until 8 weeks of age but there was no change in the CD4+/CD8+ ratio with aging. Splenic T cells from old mice that had or had not been immunized were less responsive to concanavalin-A and showed reduced cytokine production compared to young mice (IL-2: 57-127 vs 367-1104 pg/mL, IFN-gamma: 2344-12,836 vs 752-23,106 pg/mL and IL-10: 393-2172 vs 105-2869 pg/mL in old and young mice, respectively). These data suggest that there are significant changes in the organization of the immune system throughout life. However, the relevance of these alterations for the functioning of the immune system is unknown.

Oral tolerance and OVA-induced tolerogenic dendritic cells reduce the severity of collagen/ovalbumin-induced arthritis in mice

Dietary proteins play an important role in the regulation of systemic immune response, in a phenomenon known as oral tolerance (OT). To evaluate the effects of OT on a murine model of type II collagen (CII) plus ovalbumin (OVA)-induced arthritis (CIA), mice were fed with OVA either before or after CIA induction. OT significantly reduced the paw edema and synovial inflammation, as well as serum levels of anti-CII, the ex vivo proliferation and inflammatory cytokine production by spleen cells from CIA mice. The frequencies of Foxp3(+) and IL-10(+) cells were higher, whereas IFNγ(+) cells and IL-17(+) cells were lower, among gated CD4(+) spleen T cells from tolerized CIA mice than in those from non-tolerized CIA mice. Adoptive transfer of tolerogenic dendritic cells (DCs) before CIA induction mimics the effects observed in the OT. We demonstrate here that bystander suppression induced by OT can modify the course of CIA and tolerogenic DCs play a role this phenomenon.

Role of the Mac-1 and VLA-4 integrins, and concomitant Th2-cytokine production, in nitric oxide modulated eosinophil migration from bone marrow to lungs in allergic mice

Although numerous studies demonstrate the participation of nitric oxide (NO) in various inflammatory diseases, the precise function of NO in allergic asthma remains unclear. We investigated whether iNOS inhibition could interfere with the kinetics of VLA-4 and Mac-1 expression and adhesion properties of bone marrow and peripheral blood eosinophils of sensitized mice after antigen exposure. Treatment of allergic mice with 1400 W (iNOS inhibitor) increased the adhesion of bone marrow eosinophils to ICAM-1, but not blood eosinophils, at 24h and 48 h after OVA-challenge. Conversely, adhesion of blood eosinophils from 1400 W-treated mice to VCAM-1 diminished at 24h and was almost completely blocked at 48 h. 1400 W did not induce any change in the adhesion of bone marrow eosinophils to VCAM-1, at 24h, but cells collected 48 h after challenge showed significantly lower adherence. Flow cytometry demonstrated that 1400 W resulted in a significantly increased Mac-1 expression on bone marrow eosinophils at 24h, as compared to control mice. However, at 24h, 1400 W significantly decreased Mac-1 and VLA-4 expressions on blood eosinophils. At 48 h, the expressions of both Mac-1 and VLA-4 returned to previous levels. Results show a temporal effect of iNOS upon Mac-1 expression and function, the chief adhesion molecule involved in the eosinophil efflux from the bone marrow at 24h. In contrast, Mac-1 and VLA-4 were involved in eosinophil mobilization from blood to lungs at 48 h after antigen challenge. Data suggest an important role of the Mac-1 and VLA-4 in the iNOS-modulated migration of eosinophils to the lungs of allergic mice.

Tolerogenic Dendritic Cells on Transplantation: Immunotherapy Based on Second Signal Blockage

Dendritic cells (DCs), the most important professional antigen-presenting cells (APC), play crucial role in both immunity and tolerance. It is well known that DCs are able to mount immune responses against foreign antigens and simultaneously tolerate self-antigens. Since DCs can be modulated depending on the surrounding microenvironment, they can act as a bridge between innate and adaptive immunity. However, the mechanisms that support this dual role are not entirely clear. Recent studies have shown that DCs can be manipulated ex vivo in order to trigger their tolerogenic profile, what can be a tool to be used in clinical trials aiming the treatment of various diseases and the prevention of transplant rejection. In this sense, the blockage of costimulatory molecules on DC, in the attempt of inhibiting the second signal in the immunological synapse, can be considered as one of the main strategies under development. This review brings an update on current therapies using tolerogenic dendritic cells modulated with costimulatory blockers with the aim of reducing transplant rejection. However, although there are current clinical trials using tolerogenic DC to treat allograft rejection, the actual challenge is to modulate these cells in order to maintain a permanent tolerogenic profile.

Evaluation of in vitro anti-inflammatory effects of crude ginger and rosemary extracts obtained through supercritical CO2 extraction on macrophage and tumor cell line: the influence of vehicle type.

BackgroundNumerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line.MethodsGinger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts.ResultsGinger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-α reduction than ginger extract in primary macrophages.ConclusionsAmongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.