Recent insights into the mucosal reactions associated with Giardia lamblia infections.

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.

Efficacy of a Nasal Spray from Citrus limon and Cydonia oblonga for the Treatment of Hay Fever Symptoms - A Randomized, Placebo Controlled Cross-Over Study

Nasal spray from lemon and quince (LQNS) is used to treat hay fever symptoms and has been shown to inhibit histamine release from mast cells in vitro. Forty-three patients with grass pollen allergy (GPA) were randomized to be treated either with placebo or LQNS for one week, respectively, in a cross-over study. At baseline and after the respective treatments patients were provoked with grass pollen allergen. Outcome parameters were nasal flow measured with rhinomanometry (primary), a nasal symptom score, histamine in the nasal mucus and tolerability. In the per protocol population absolute inspiratory nasal flow 10 and 20 min after provocation was higher with LQNS compared to placebo (-37 ± 87 mL/s; p = 0.027 and -44 ± 85 mL/s; p = 0.022). The nasal symptom score showed a trend (3.3 ± 1.8 in the placebo and 2.8 ± 1.5 in the LQNS group; p = 0.070) in favor of LQNS; the histamine concentration was not significantly different between the groups. Tolerability of both, LQNS and placebo, was rated as very good. LQNS seems to have an anti-allergic effect in patients with GPA. Copyright © 2016 John Wiley & Sons, Ltd.

Cardiac chymase: pathophysiological role and therapeutic potential of chymase inhibitors

On release from cardiac mast cells, alpha-chymase converts angiotensin I (Ang I) to Ang II. In addition to Ang II formation, alpha-chymase is capable of activating TGF-beta 1 and IL-1 beta, forming endothelins consisting of 31 amino acids, degrading endothelin-1, altering lipid metabolism, and degrading the extracellular matrix. Under physiological conditions the role of chymase in the mast cells of the heart is uncertain. In pathological situations, chymase may be secreted and have important effects on the heart. Thus, in animal models of cardiomyopathy, pressure overload, and myocardial infarction, there are increases in both chymase mRNA levels and chymase activity in the heart. In human diseased heart homogenates, alterations in chymase activity have also been reported. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of cardiac disease. The selective chymase inhibitors developed to date include TY-51076, SUN-C8257, BCEAB, NK320, and TEI-E548. These have yet to be tested in humans, but promising results have been obtained in animal models of myocardial infarction, cardiomyopathy, and tachycardia-induced heart failure. It seems likely that orally active inhibitors of chymase could have a place in the treatment of cardiac diseases where injury-induced mast cell degranulation contributes to the pathology.

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: Relationship to cystatins and the tumor suppressor TIG1

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 Angstrom resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

A review of non-pharmacological and pharmacological management of seasonal and perennial allergic conjunctivitis

Allergic eye disease encompasses a group of hypersensitivity disorders which primarily affect the conjunctiva and its prevalence is increasing. It is estimated to affect 8% of patients attending optometric practice but is poorly managed and rarely involves ophthalmic assessment. Seasonal allergic conjunctivitis (SAC) is the most common form of allergic eye disease (90%), followed by perennial allergic conjunctivitis (PAC; 5%). Both are type 1 IgE mediated hypersensitivity reactions where mast cells play an important role in pathophysiology. The signs and symptoms are similar but SAC occurs periodically whereas PAC occurs year round. Despite being a relatively mild condition, the effects on the quality of life can be profound and therefore they demand attention. Primary management of SAC and PAC involves avoidance strategies depending on the responsible allergen(s) to prevent the hypersensitivity reaction. Cooled tear supplements and cold compresses may help bring relief. Pharmacological agents may become necessary as it is not possible to completely avoid the allergen(s). There are a wide range of anti-allergic medications available, such as mast cell stabilisers, antihistamines and dual-action agents. Severe cases refractory to conventional treatment require anti-inflammatories, immunomodulators or immunotherapy. Additional qualifications are required to gain access to these medications, but entry-level optometrists must offer advice and supportive therapy. Based on current evidence, the efficacy of anti-allergic medications appears equivocal so prescribing should relate to patient preference, dosing and cost. More studies with standardised methodologies are necessary elicit the most effective anti-allergic medications but those with dual-actions are likely to be first line agents.

A review of non-pharmacological and pharmacological management of seasonal and perennial allergic conjunctivitis

Allergic eye disease encompasses a group of hypersensitivity disorders which primarily affect the conjunctiva and its prevalence is increasing. It is estimated to affect 8% of patients attending optometric practice but is poorly managed and rarely involves ophthalmic assessment. Seasonal allergic conjunctivitis (SAC) is the most common form of allergic eye disease (90%), followed by perennial allergic conjunctivitis (PAC; 5%). Both are type 1 IgE mediated hypersensitivity reactions where mast cells play an important role in pathophysiology. The signs and symptoms are similar but SAC occurs periodically whereas PAC occurs year round. Despite being a relatively mild condition, the effects on the quality of life can be profound and therefore they demand attention. Primary management of SAC and PAC involves avoidance strategies depending on the responsible allergen(s) to prevent the hypersensitivity reaction. Cooled tear supplements and cold compresses may help bring relief. Pharmacological agents may become necessary as it is not possible to completely avoid the allergen(s). There are a wide range of anti-allergic medications available, such as mast cell stabilisers, antihistamines and dual-action agents. Severe cases refractory to conventional treatment require anti-inflammatories, immunomodulators or immunotherapy. Additional qualifications are required to gain access to these medications, but entry-level optometrists must offer advice and supportive therapy. Based on current evidence, the efficacy of anti-allergic medications appears equivocal so prescribing should relate to patient preference, dosing and cost. More studies with standardised methodologies are necessary elicit the most effective anti-allergic medications but those with dual-actions are likely to be first line agents. © 2011 British Contact Lens Association.

Investigating ocular allergy:can we determine a better measurement?

Objective: Ocular allergy is a broad group of allergic conditions involving inflammation of the conjunctiva and the most common forms are seasonal allergic conjunctivitis (SAC; 90% of cases) and perennial allergic conjunctivitis (PAC; 5% of cases). The main symptom is ocular itching caused by mast cell degranulation leading to the release of histamine and other mediators such as tryptase. Tryptase is a neutral protease that is selectively concentrated in the secretory granules of human mast cells and has been shown to be a sensitive and specific marker of type I hypersensitivity reaction. The objective was to ascertain the best assay method for determining the tryptase levels in tear samples and whether this can be used to determine the efficacy of non-pharmacological treatments compared to no treatment or their combined effect with anti-allergic medication for SAC and PAC. Method: Thirty patients with a history of SAC were recruited into a randomised blind study during winter months when all the patients were asymptomatic. Suitability was determined by skin prick and conjunctival provocation tests. Patients were randomly assigned to either a non-pharmacological or a pharmacological Intervention group and received each test condition assigned to their group in a randomly assigned order. Symptoms were provoked by exposure to pollen in an environmental test chamber where the temperature, humidity and grass pollen levels were set to a high pollen count day. Tear samples were taken set intervals during the visit and then processed by enzyme linked immunosorbent assay (ELISA) for the detection of tryptase levels. Preliminary results: Results are still being analysed but the preliminary optimisation experiments tested four different ELISA systems; two indirect assays and two capture ‹sandwich› assays. The results suggest that in both sandwich assay systems non-specific binding occurred which could not be easily overcome. The indirect assay systems both showed specific reactions, and the sensitivity achieved was greater with the monoclonal than the polyclonal antibody. Using these findings the indirect assay system was optimised to provide a standardised system for measuring tryptase. Initial trials using human tear samples displayed tryptase levels between 23.1 and 175.1 ng/ml; levels which fall within the anticipated range for patients with SAC. Further statistical work is needed to determine whether tryptase levels vary between the treatments 75.

Análise da imunoexpressão da podoplanina e da triptase em carcinoma epidermóide de língua e sua relação com parâmetros clinicopatológicos

Oral tongue squamous cell carcinoma (OTSCC) has an aggressive biological behavior, with a high propensity for the development of lymph node metastases. In this context, lymphangiogenesis is considered an important phenomenon for the spread of tumor cells and may be influenced by microenvironmental stimuli. Mast cells have been implicated in tumor progression, although their influence in the formation of lymphatic vessels is not well established. The aim of this study was to analyze, in a case series of OTSCC (n=50), possible correlations between lymphatic vessel density (LVD), mast cell count and clinicopathological features, including tumor-node-metastasis (TNM) stage, histological grade of malignancy (Bryne, 1998), and nodal metastasis. LVD was established as the mean number of lymphatic vessels immunostained by anti-podoplanin (D2-40) antibody, identified in five microscopic fields (200x). For the analysis of mast cells, tryptase-immunoreactive cells were quantified in five fields (400x). Both immunostainings were analyzed in the tumor center and invasion front. Intratumoral lymphatic density (ILD) was higher in cases in advanced clinical stages (III-IV), compared to those in initial stages (I-II), as well as in metastatic cases in respect of non-metastatic (p<0,05). There were no statistically significant differences between low-grade and high-grade malignancy cases with respect to ILD (p>0,05). Peritumoral lymphatic density (PLD) and mast cell counts showed no significant relations with any of the clinicopathological parameters evaluated (p>0,05). Also there were no significant correlations between LVD and mast cell counts, whether in intratumoral (r = -0,004; p=0,977) or peritumoral region (r = -0,154; p=0,285). The results of the present study suggest that intratumoral lymphatic vessels may contribute in part to the progression of OTSCC, although PLD may be insufficient to justify differences in biological behavior. This supports the hypothesis of involvement of other mechanisms in metastatic spread of malignant cells, which could complement the effects of lymphangiogenesis. Although mast cells perform several pro- and antitumoral functions, they do not appear to directly influence aggressiveness of OTSCC. In addition, the quantity of these cells may not be essential for lymphatic vessel formation.

Efeito da oxigenação hiperbárica no reparo ósseo de ratos diabéticos

The chronic state of hyperglycemia due to diabetes mellitus affects multiples organs impairing life quality. In bone, diabetes alters strength and mineral density and also suppresses the osteoblast activity, leading to an unbalanced bone healing process. Hyperbaric oxygen therapy (HBO) is suggested as an adjuvant treatment to accelerate bone repair. This study evaluated the effects of HBO in the number of mast cells and in new bone formation at the initial stage of bone repair in normoglycemic and diabetic rats. It was hypothesized that HBO treatment may improve bone repair in diabetic bone. The rats were equally divided in four groups: Control (C); Control + HBO (CH); Diabetes (D) and Diabetes + HBO (DH). Diabetes was induced by streptozotocin (65mg/kg) and femoral bone defects were created thirty days after diabetes induction in all groups. HBO initiated immediately after surgery procedure and was performed daily, for 7 days, in the CH e DH groups. Seven days after surgery, all animals were euthanized. The femur diaphyses were removed, fixated, decalcified and processed for paraffin embedding. The semi-serial histological sections obtained were stained with Hematoxylin-Eosin (HE), Mallory Trichrome and Toluidine Blue. The qualitative analysis was conducted in the histology slides stained with HE, where it was evaluated the morphological aspects of bone repair in the lesion area, observing the presence of clot, inflammatory cells, granulation tissue, type of bone tissue, morphology of bone cells, and thickness and organization of bone trabeculae. In the slides stained with Mallory Trichrome and Toluidine Blue were evaluated the percentage of new bone formation and number of mast cells, respectively. The qualitative analysis showed that the CH group presented a more advanced stage of bone repair compared to the C group, showing thicker trabeculae and greater bone filling of the lesion area. In D and DH group, the lesion area was partially filled with new bone formation tissue and presented thinner trabeculae and fewer areas associated to osteoclasts compared to control group. The histomorphometric analysis showed a significant improvement in new bone formation (p<0.001) comparing CH (38.08 ± 4.05) and C (32.05 ± 5.51); C and D (24.62 ± 2.28 and CH and DH (27.14 ± 4.21) groups. In the normoglycemic rats there was a significant increasing in the number of mast cells (p<0.05) comparing C (8.06 ± 5.15) and CH (21.06 ± 4.91) groups. In conclusion, this study showed that diabetes impaired bone repair and HBO was only able to increase new bone formation and the number of mast cells in the normoglycemic animals.

On the Origin of Natural Antibody

Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.

In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.

In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.

In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.

The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.

Why Serological Responses during Cystitis are Limited.

The high frequency of urinary tract infections (UTIs), some of which appear to be endogenous relapses rather than reinfections by new isolates, point to defects in the host's memory immune response. It has been known for many decades that, whereas kidney infections evoked an antibody response to the infecting bacteria, infections limited to the bladder failed to do so. We have identified the existence of a broadly immunosuppressive transcriptional program associated with the bladder, but not the kidneys, during infection of the urinary tract that is dependent on bladder mast cells. This involves the localized secretion of IL-10 and results in the suppression of humoral immune responses in the bladder. Mast cell-mediated immune suppression could suggest a role for these cells in critically balancing the needs to clear infections with the imperative to prevent harmful immune reactions in the host.

Characterization of three human sec14p-like proteins: a-Tocopherol

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled α-tocopherol to mitochondria in the same order of magnitude as the human α-tocopherol transfer protein (α-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.