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Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 254521, 12 pages
http://dx.doi.org/10.1155/2010/254521
The Role of Lipopeptidophosphoglycan in the Immune Response to Entamoeba histolytica
Isabel Wong-Baeza,1,3 Marcela Alcántara-Hernández,1,3 Ismael Mancilla-Herrera,1,4 Itzmel Ramírez-Saldívar,1,5 Lourdes Arriaga-Pizano,1 Eduardo Ferat-Osorio,1,2 Constantino López-Macías,1 and Armando Isibasi1
1Medical Research Unit on Immunochemistry, Specialties Hospital, National Medical Centre "Siglo XXI", Mexican Institute for Social Security (IMSS), 06720 Mexico City, Mexico
2Gastrointestinal Surgery Department, Specialties Hospital, National Medical Centre "Siglo XXI", Mexican Institute for Social Security (IMSS), 06720 Mexico City, Mexico
3Graduate Program on Immunology, National School of Biological Sciences, National Polytechnic Institute, 11350 Mexico City, Mexico
Graduate Program on Molecular Biomedicine and Biotechnology, National School of Biological Sciences, National Polytechnic Institute, 11350 Mexico City, Mexico
Graduate Program on Chemical and Biological Sciences, National School of Biological Sciences, National Polytechnic Institute, 11350 Mexico City, Mexico
Received 30 June 2009; Accepted 12 October 2009
Academic Editor: Abhay R. Satoskar
Copyright © 2010 Isabel Wong-Baeza et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The sensing of Pathogen Associated Molecular Patterns (PAMPs) by innate immune receptors, such as Toll-like receptors (TLRs), is the first step in the inflammatory response to pathogens. Entamoeba histolytica, the etiological agent of amebiasis, has a surface molecule with the characteristics of a PAMP. This molecule, which was termed lipopeptidophosphoglycan (LPPG), is recognized through TLR2 and TLR4 and leads to the release of cytokines from human monocytes, macrophages, and dendritic cells; LPPG-activated dendritic cells have increased expression of costimulatory molecules. LPPG activates NKT cells in a CD1d-dependent manner, and this interaction limits amebic liver abscess development. LPPG also induces antibody production, and anti-LPPG antibodies prevent disease development in animal models of amebiasis. Because LPPG is recognized by both the innate and the adaptive immune system (it is a "Pamptigen"), it may be a good candidate to develop a vaccine against E. histolytica infection and an effective adjuvant.
Amebiasis is a disease caused by Entamoeba histolytica, a parasite protozoan that infects humans and is responsible for 40,000 to 110,000 deaths per year [1]. Ten percent of infected persons exhibit clinical symptoms; 80% to 98% of these are intestinal, and 2% to 20% are extraintestinal. The clinical symptoms can range from a mild and nonspecific presentation (constipation alternated with diarrhea, pain in the lower abdomen, mild nausea during or after meals, and mild abdominal distension with pain in the right iliac fossa) to dysentery, fulminating colitis, and toxic megacolon. Less frequently, amebiasis can cause appendicitis and ameboma. Amebic liver abscess is the most frequent presentation of invasive extraintestinal amebiasis, but the lungs, heart, brain, skin, and genitals can also be affected [2].
Approximately 500 million people in the world are currently infected with E. histolytica [1]. The incidence of amebiasis has decreased significantly in recent years because of increased sanitation in many countries and the use of effective therapeutic agents. The World Health Organization and the Pan-American Health Organization recommend the treatment of all patients with confirmed E. histolytica infection, regardless of the presence of symptoms. The treatments of choice for asymptomatic intestinal amebiasis are the luminal amebicides paromomycin sulfate and diloxanide furoate. Symptomatic intestinal or extraintestinal infection is treated with metronidazole in combination with a luminal amebicide. Nitazoxanide is an effective luminal amebicide, and it is also effective for invasive amebiasis. Gastrointestinal complications, such as perforation, intestinal obstruction, and toxic megacolon, are treated with surgery. Most hepatic abscesses respond to metronidazole, but if they do not, they can be aspirated by puncture or treated with open surgery [3–5].
In spite of the effective therapeutic agents that are available for the treatment of amebiasis, it still constitutes a global health problem [6]. The prevalence of amebiasis varies from 1% in industrialized countries to 50%–80% in tropical countries [7–10].
2. Identification of Lipopeptidophosphoglycan
In the 1970s, amebiasis was the fourth most frequent infectious disease in Mexico, with an incidence of 118.9 per 10,000 inhabitants (almost 1500 times higher than the incidence in the United States in the same year) [11]. This situation prompted many researchers to study several aspects of this parasitic disease, including comparative studies of drugs for the treatment of acute amebic liver abscess [12] and various studies of seroepidemiology of amebiasis in adults [13–17].
Several genes from E. histolytica were cloned, sequenced and expressed in an effort to identify new drug targets for this parasite, including the alcohol dehydrogenase gene (Ehadh3) [18], the ferredoxin oxidoreductase gene [19], the EhDEAD1 RNA helicase gene [20], and the Ehvma2 gene (which encodes the B subunit of the vacuolar ATPase) [21]. Mechanisms of drug resistance in E. histolytica were also studied, and it was determined that the multidrug-resistant phenotype is regulated at the transcriptional level by the P-glycoprotein-like genes (EhPgp) 1 and 5 [22]. A protein complex (EhCPADH) was identified on the surface of E. histolytica. This complex is formed by a cysteine proteinase that digests gelatin, collagen type I, fibronectin and hemoglobin (EhCP112), and an adhesin (EhADH112), and is involved in adherence, phagocytosis, and cytolysis [23, 24]. Polypeptides derived from this complex were assessed as vaccine candidates, and it was demonstrated that they confer partial protection from amebic liver abscess in hamsters (Mesocricetus auratus) [25].
The role of the immune response in the pathogenesis of amebiasis was also studied; the early approaches demonstrated that serum from infected patients could neutralize the virulence of E. histolytica cultures [26] and that this serum could confer antiameba passive immunity in hamsters [27]. The importance of cellular immunity in the control of amebiasis was addressed in several studies, which demonstrated the ability of activated eosinophils to kill the parasite in vitro [28] and to protect from amebic liver abscess in vivo [29], and the killing of trophozoites by peritoneal macrophages in hamsters [30] and by activated T lymphocytes and macrophages in humans [31]. It was shown that patients cured from amebic liver abscess had specific T lymphocytes that killed trophozoites in vitro [31]. It was also demonstrated that molecules from E. histolytica were able modulate the host immune response. The supernatant fluid of axenically grown E. histolytica could inhibit chemotaxis and random mobility of human monocytes, without affecting the locomotion of neutrophils [32]. The effect was attributed to a monocyte locomotion inhibitory factor (MLIF), and physicochemical analysis revealed that MLIF is a heat-stable pentapeptide (Met-Gln-Cys-Asn-Ser) that inhibits locomotion of monocytes, respiratory burst of monocytes and neutrophils, and delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs (Cavia porcellus) [33]. MLIF decreased the expression of macrophage inflammatory protein- (MIP-) 1alpha, MIP-1beta, and chemokine receptor CCR1 in a phorbol myristate acetate- (PMA-) stimulated human monocyte cell line, which suggests that the inhibition of monocyte locomotion could be attributed to downregulation of chemokines and chemokine receptors [34]. MLIF also decreased interleukin- (IL-) 1beta and increased IL-10 production by PMA-stimulated human CD4 T lymphocytes [35]. Immunization with a tetramer of MLIF around a lysine core completely protected gerbils (Meriones unguiculatus) against amebic liver abscess [36].
In 1969, Galanos et al. developed a new method for the extraction of bacterial lipopolysaccharide (LPS, Figure 1(a)) [37] and, in the following years, many of the chemical, biological, and immunological properties of the so-called endotoxin were determined [38–44]. LPS is a major structural component of the outer membrane of Gram-negative bacteria; it activates many cell types, induces inflammation, and produces fever and shock. We decided to determine if E. histolytica had a surface molecule with chemical and immunological properties similar to those of bacterial LPS. The use of a modified phenol-water extraction procedure on E. histolytica trophozoites yielded a molecule with 85% carbohydrate, 8% peptide, 2.5% lipid, and 1% phosphate, which was termed lipopeptidophosphoglycan (LPPG, Figure 1(c)) [45, 46]. The isolation and structural characterization of microbial molecules can lead to the identification of new drug targets and new antigens that are recognized by the immune system; some antigens are good candidates for vaccine development. LPPG was first identified as an antigen; antiameba IgG antibodies were detected in rats after intracecal inoculation of trophozoites [47], anti-LPPG IgA antibodies were found in colostrum of healthy volunteers [48], and antiameba plasma cells were found in peripheral blood of patients with amebic liver abscess [49]. Monoclonal antiproteophosphoglycan antibodies were described by several groups [50–53]. However, as research in immunology progressed, LPPG was studied as a molecule that could be sensed not only by the adaptive immune system but also by the innate immune system.
Figure 1: (a) Lipopolysaccharide (LPS) from the Gram-negative bacterium Escherichia coli is the most potent activator of TLR4 [54]. (b) Alpha-galactosyl ceramide from the marine sponge Agelas mauritanius is presented via CD1d and activates NKT cells [55]. (c) Partial structure of lipopeptidophosphoglycan (LPPG) from Entamoeba histolytica of the HM1 : IMSS strain, which was originally isolated from a patient with liver abscess [56, 57]. The structure of the active phosphoinositol moiety of LPPG was characterized in [58].
3. Sensing of Parasites by the Immune System
The relevance of adaptive immunity (whose main effectors are T and B lymphocytes) in protection against infections was well recognized in the last decades of the past century, while the role of neutrophils, monocytes, macrophages, and other cells of the innate immune system was seen as a that of a "first line of defense'' that contained infections until adaptive immunity was fully activated. It was known that LPS, a component of the outer membrane of Gram-negative bacteria, caused fever and shock in animal models and that it induced the secretion of proinflammatory cytokines by monocytes, macrophages, and epithelial cells. However, the receptor that sensed LPS remained elusive. Many phagocytic receptors on macrophages had been described, but none of these was responsible for the biological properties of LPS [59].
Two strains of mice, C3H/HeJ and C57BL/10ScCr, which were resistant to endotoxic shock, were identified [60, 61]. In 1998, the positional cloning of the affected locus in C3H/HeJ mice showed a point mutation in Toll-like receptor (TLR) 4, a previously orphan receptor, and C57BL/10ScCr mouse were found to lack TLR4 [62]. TLR4 is a member of a family of proteins that share a signaling domain (TIR) with IL-1 receptor and are related to the Toll proteins of the fruit fly, Drosophila. Toll was described in 1988 as a transmembrane protein that is required for establishing the embryonic dorsal-ventral pattern in flies [63]. In 1996, Toll was shown to be critical for the antifungal response in Drosophila [64], and it was suspected that the human homologues of Toll, which were described in 1998 [65], would be relevant for the immune response in humans. Indeed, it was found that a constitutively active mutant of human TLR4, transfected into a monocytic cell line, could induce the activation of NF-kappaB and the expression of the proinflammatory cytokines IL-1, IL-6, and IL-8 as well as the expression of the costimulatory molecule CD80 (B7.1), which is required for the activation of naïve T lymphocytes [66]. After the establishment of TLR4 as the main sensor for LPS in mice, it was immediately suggested that other members of the Toll family in mammals could also serve as sensors for microbial molecules. Many molecules of bacteria, viruses, and fungi, and others that are found during viral replication, have been identified as agonists of mammalian TLRs (Table 1) [67, 68]. The study of TLRs and other innate receptors has established innate immunity not only as a first line of defense against infections but also as a critical component of the immune system that induces and regulates the adaptive response [69].
Table 1: Some TLR agonists from bacteria, viruses, and fungi (modified from [67, 68]).
The molecules that are sensed by TLRs are widely distributed among groups of microorganisms, and they are essential for the metabolism or the structural integrity of the microbe, so they are highly conserved in evolution. These molecules were termed pathogen-associated molecular patterns (PAMPs) [70], although their expression is not restricted to pathogens, and this term is used widely to this day. Several molecules from protozoan and helminth parasites were also identified as PAMPs [71] (Table 2). In protozoan parasites, many surface molecules are linked to glycosylphosphatidylinositol (GPI), which is inserted in the plasma membrane. GPI-anchored molecules include lipophosphoglycan (LPG) and LPPG; they are essential for survival and virulence of the parasite, and they are likely the major macromolecules on the trophozoite surface [72]. In Leishmania, LPG is involved in intestinal adhesion and resistance to insect hydrolases; LPG-deficient strains are unable to survive in their vector. Leishmania LPG gains phosphosaccharide domains as procyclic promastigotes in the vector midgut differentiate to infectious metacyclic promastigotes; this structural change in LPG mediates detachment from vector midgut and acquisition of complement resistance [73]. LPG also induces the production of nitric oxide and proinflammatory cytokines by macrophages in the host [74]. LPG from metacyclic promastigotes is a more effective activator of TLR2 in NK cells than LPG from procyclic promastigotes [75].
Table 2: TLR agonists from protozoan and helminth parasites.
Plasmodium GPI-anchored molecules are required for the induction of proinflammatory responses, which promote pathogenesis [76]. However, activation of innate and adaptive immune responses is necessary to control parasite growth and frequent tlr4 polymorphisms predispose African children to severe malaria [77]. Therefore, it is proposed that overactivation or deregulation of the inflammatory response is the cause of the pathological condition [78, 79]. Several mucin-like GPI-anchored glycoproteins have been isolated from the Trypanosoma cruzi surface. A T. cruzi trans-sialidase adds sialic acid residues to these molecules, which are required for survival and infectivity [72, 80]. GPI-anchored molecules purified from T. cruzi trypomastigotes signal through TLR2 and induce the production of IL-12, tumor necrosis factor (TNF)-alpha and nitric oxide by murine macrophages [81]; signaling through TLR2 synergizes with TLR9 and is crucial to control the infection [82]. Tc52 is a soluble molecule that is released by T. cruzi during parasitemia, and it activates macrophages and dendritic cells via TLR2 [83]. GPI-anchored molecules isolated from Toxoplasma activate TLR4, while glycan cores and phospholipid moieties from these molecules activate both TLR2 and TLR4 [84]. Lysophosphatidylserine-containing lipids from Schistosoma mansoni induce the maturation of dendritic cells that prime Th2 and regulatory T cell responses, which favor the establishment of chronic infections with little tissue damage [87].
4. Function of LPPG as a PAMP and Role of Inflammation in the Pathogenesis of Amebiasis
The similarities in chemical structure between LPS and LPPG (Figures 1(a) and 1(c)), and the presence of a GPI anchor in LPPG, suggested that LPPG might be a PAMP. This would explain how the innate immune system senses the presence of E. histolytica, an event that is necessary for the orchestration of the inflammatory response in amebiasis. Studies from our laboratory demonstrated that LPPG is recognized through TLR2 and TLR4. Human embryonic kidney- (HEK-) 293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Coexpression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF-kappaB and release of IL-8, IL-10, IL-12p40, and TNF-alpha from human monocytes [85, 96]. Human macrophages and dendritic cells internalize LPPG. As shown by colocalization of LPPG with late endosomes marked with fluorescein isothiocyanate–dextran and LAMP-1, the internalization process involves intracellular traffic from the cell membrane to late endosomes. LPPG-activated dendritic cells have increased expression of costimulatory molecules CD80, CD86, and CD40 and produce TNF-alpha, IL-8, and IL-12 [97]. These results show that LPPG activates antigen-presenting cells and reaches intracellular compartments that are involved in antigen presentation. Responsiveness of mouse macrophages lacking TLR2 expression (TLR2−/−) or functional TLR4 (C3H/HeJ) to LPPG challenge was impaired, while macrophages from C3H/HeJ/TLR2−/− mice were unresponsive. In contrast to wild-type and TLR2−/− mice, which succumbed to LPPG-induced shock, C3H/HeJ mice were resistant [85]. All these results clearly establish that LPPG is a PAMP from E. histolytica that induces the activation of innate immunity.
In humans, the pathogenesis of E. histolytica requires adhesion of trophozoites to the host cells, phagocytosis of host cells and bacteria by trophozoites, and tissue destruction by amebic enzymes and by enzymes released from lysed neutrophils. The adhesion of trophozoites to host cells is required for tissue invasion; this adhesion is mediated, in part, by a galactose/N-acetylgalactosamine- (Gal/GalNAc-) binding lectin, which is also cytotoxic and confers protection from complement. Other important adhesins are a 220 kDa cell surface protein, a 112 kDa adhesin (EhADH112), and a surface LPG [23, 98–100]. Phagocytosis is regulated by adhesins and by signaling pathways that control cytoskeleton structure and vesicular traffic. A phagosome-associated transmembrane kinase (PATMK) binds to phosphatidylserine on host cells and initiates their phagocytosis by trophozoites [101]. Trophozoites cause damage of host cells and extracellular matrix through the action of amebapore, amebic phospholipases, and proteolytic enzymes (cysteine endopeptidases, cysteine proteinase, acid and neutral proteinases, collagenases, histolysin, amebapain, cathepsin B) [102–108]. Neutrophils are the first cells that infiltrate the necrotic lesions caused by E. histolytica in the intestine and liver [109–113], where they are killed by trophozoites. The enzymes and reactive oxygen species released from these neutrophils increase tissue damage, and in this context, LPPG could be seen as a virulence factor that promotes tissue invasion by causing inflammatory damage to host cells. However, the role of inflammation in amebiasis is still controversial [114]: in susceptible animals (hamsters and gerbils) inflammation is related to host cell lysis and facilitates the spreading of trophozoites [115], while in resistant animals (mice, guinea pigs), inflammatory cells protect the host by killing trophozoites [116, 117].
E. histolytica genomic DNA is recognized by TLR9 and induces the production of TNF-alpha by a macrophage cell line [94], and Gal/GalNAc-binding lectin activates NF-kappaB and mitogen-activated protein (MAP) kinases in macrophages. These transcription factors increase the expression of several genes, including TLR2 [118]. It is likely that genomic DNA and Gal/GalNAc-binding lectin, along with LPPG, contribute to the initiation of inflammation in response to E. histolytica.
Silencing of the expression of E. histolytica GPI-anchored molecules by antisense RNA-mediated inhibition of their biosynthetic pathways suppresses endocytosis, adhesion, and proliferation of the trophozoites [119]. Specific blockade of LPG and LPPG by monoclonal antibody EH5 reduces intestinal inflammation and tissue damage in a severe combined immunodeficient (SCID) mouse model of intestinal amebiasis with human intestine xenograft [120]. EH5 also prevents liver abscess development in an SCID mouse model [52] and E. histolytica adhesion and cytotoxicity to a hamster cell line [101]. These results suggest that LPPG is a virulence factor of E. histolytica. Mirelman and colleagues found that a nonvirulent E. histolytica strain had reduced expression of LPG and LPPG; they also found no LPG and a modified LPPG (with a higher negative charge and different lengths of oligosaccharide chains) in the low-virulence strain Rahman and in the nonpathogenic Entamoeba dispar [121].
Recently, a role for LPPG in protection against invasive amebiasis was shown. The chemical structure of LPPG has some similarities with alpha-galactosyl ceramide, a known activator of NKT cells [55] (Figures 1(b) and 1(c)). NKT cells share many surface receptors with natural killer (NK) cells and, like conventional T cells, express T cell receptors that are generated by somatic DNA rearrangement. However, most NKT cells express semi-invariant T cell receptors, consisting of V 14-J 18/V 8.2 chains in mouse and V 24-J 18/V 11 chains in humans [122]. This limited repertoire, conserved between individuals and presumably selected by evolution, is more closely related to the pattern-recognition receptors of innate immunity than to the highly diverse receptors of adaptive immunity. NKT cells recognize glycolipid antigens presented by nonpolymorphic CD1d molecules; these glycolipids can be endogenous, like lysosomal isoglobotrihexosyl ceramide [123], and exogenous, like glycosyl ceramides from Gram-negative, LPS-negative Sphingomonas capsulata [124]. In a mouse model, it was demonstrated that NKT cells play a central role in the control of amebic liver abscess caused by E. histolytica. Specific activation of NKT cells by alpha-galactosyl ceramide or LPPG induced significant protection, while CD1d−/− mice suffered from severe abscess development [58]. The phosphoinositol moiety of LPPG was shown to induce interferon- (IFN-) gamma but not IL-4 secretion in NKT cells. NKT cell activation was dependent on the presence of CD1d and simultaneous TLR receptor signaling, as indicated by the absence of IFN-gamma secretion in antigen-presenting cells from TLR2- or TLR6-deficient mice [58]. These results suggest that NKT cell activation by LPPG is important to limit amebic liver abscess development and may help to explain why the vast majority of E. histolytica-infected individuals do not develop invasive amebiasis.
5. LPPG as a Molecule That Is Sensed by Both the Innate and the Adaptive Immune Systems
Molecules that are recognized by receptors of both innate and adaptive immune systems are, in general, highly immunogenic; we have referred to these molecules as "Pamptigens'' [125, 126]. Some examples of molecules that present this dual recognition include porins, profilin, polysaccharide A, yellow fever vaccine, and respiratory syncytial virus vaccine. Salmonella typhi porins are recognized by TLR2 and TLR4 [126], and they induce high antibody titers that persist during the whole lifetime of mice [127]. Toxoplasma gondii profilin, a TLR11 agonist, is an immunodominant antigen in the CD4+ T cell response to the pathogen [128]. Bacteroides fragilis polysaccharide A activates CD4+ T cells by a mechanism that depends on TLR2 signaling and antigen presentation by the MHCII pathway [129]. Live attenuated yellow fever vaccine 17D, one of the most effective vaccines available, activates TLR2, 7, 8, and 9 and induces antigen-specific CD8+ T cells [130]. Poor TLR signaling by a formalin-inactivated respiratory syncytial virus vaccine led to the induction of low-affinity antibodies and to the failure of the vaccine to protect immunized children [131].
Molecules that are recognized by innate and adaptive receptors of the immune system are also effective adjuvants. Innate immunity participates in the induction and regulation of adaptive responses; without adjuvants, molecules that are recognized by adaptive receptors but not by innate receptors fail to elicit antibody or T cell responses. Antigen recognition alone is not sufficient to activate adaptive immune responses, and innate signals are required to indicate the microbial origin of the antigen; adjuvants provide this signal by activating innate immune receptors [132, 133].
LPPG signals through TLR2 and TLR4, and it induces the production of IFN-gamma (a cytokine that activates macrophages and increases cytotoxic T cell responses) by NKT cells. LPPG is also an antigen, because anti-LPPG antibodies have been detected in animal models and in patients with amebiasis. The mechanism that leads to the production of these antibodies has not been determined, but it is probably influenced by the innate signaling of LPPG (Figure 2). The intrinsic immunogenicity of LPPG and the fact that it is a virulence factor of E. histolytica make LPPG an attractive candidate for vaccine development. Its properties as an adjuvant also deserve further study.
Figure 2: The role of lipopeptidophosphoglycan (LPPG) in the immune response to Entamoeba histolytica. During E. histolytica infection, amebic enzymes and enzymes and reactive oxygen species from neutrophils cause tissue damage. LPPG released from lysed trophozoites is recognized through TLR2 and TLR4/CD14 and induces the production of IL-8, IL-10, IL-12p40, and TNF-alpha by monocytes [85, 96]. Macrophages and dendritic cells internalize LPPG into LAMP-1+ endosomes, and LPPG-activated dendritic cells have increased expression of costimulatory molecules CD80, CD86, and CD40 and produce TNF-alpha, IL-8, and IL-12 [97]. NKT cells are also activated by LPPG, and this depends on the presence of CD1d on dendritic cells and simultaneous TLR2 and TLR6 signaling [58]. Anti-LPPG antibodies have been described in humans and in animal models [47–53]. The mechanism that leads to the production of these antibodies has not been determined, but it is probably influenced by the innate signaling of LPPG on dendritic cells and B cells.
6. Concluding Remarks
The study of E. histolytica was initially motivated by the high morbidity and mortality of amebiasis, and in our group, this research led to the identification of LPPG, one of the first PAMPs described in parasites, and a promising vaccine candidate and potential adjuvant. The incidence and severity of amebiasis has declined, because of improved sanitation and effective treatments, but this disease is still a health problem in many parts of the world. The development of a vaccine that effectively protects against E. histolytica infection would have a positive impact on global health.
Gal/GalNAc: Galactose/N-acetylgalactosamine
GPI: Glycosylphosphatidylinositol
IFN: Interferon
IL: Interleukin
MAP kinases: Mitogen-activated protein kinases
LAMP: Lysosome-associated membrane glycoprotein
LPG: Lipophosphoglycan
LPPG: Lipopeptidophosphoglycan
LPS: Lipopolysaccharide
MHCII: Major histocompatibility complex class II
MIP: Macrophage inflammatory protein
MLIF: Monocyte locomotion inhibitory factor
NK: Natural killer
PAMP: Pathogen associated molecular pattern
PATMK: Phagosome-associated transmembrane kinase
PMA: Phorbol myristate acetate
SCID: Severe combined immunodeficiency
TLR: Toll-like receptor
TNF: Tumor necrosis factor.
I. Wong-Baeza, M. Alcántara-Hernández, I. Mancilla-Herrera, and I. Ramírez-Saldívar received scholarships from Consejo Nacional de Ciencia y Tecnología (CONACyT) and IMSS. The authors thank Omar Agni García-Hernández for assistance in the preparation of Figure 2.
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I am so excited to reveal our nursery and the beautiful Shutterfly Home Decor pieces that brought it all together! When planning my son's nursery, I wanted it to be light, natural and cozy, with a nod to the outdoors. Having a place we can be comfortable and find peace in is very important with kids, so I also wanted to keep his room fairly simple with a clean color palette of black, white, grey and natural.
I started searching Pinterest for ideas and came across the Ulm crib from Spot on Square made of sustainably harvested solid birch and knew it was the perfect centerpiece to plan my nursery around.
Once that was chosen, I thought canvas wall art would be the perfect complement for this focal point and turned to the Design-A-Wall tool from Shutterfly to create three coordinating framed canvases to hang above the crib with the picture window square layout.
I knew I wanted to incorporate more black and white and some of Everett's newborn photos in the nursery, and loved the natural media options Shutterfly offers like canvas, metal and wood. I had an idea of creating a custom magnet board, so decided to use metal prints above the dresser. I selected three square metal wall art prints to hang above the gallery panoramic metal print I chose as the magnet board by using the Design-A-Wall tool again, and am so glad I did because hanging them was a breeze!
I started with the accompanying template, stuck the level on, and positioned it center above the dresser. Each piece also comes with an index card of where the top of the artwork is and where the hardware will be, so I attached this card in the coordinating spot for the magnet board, marked the wall for all four pieces, hammered, hung and stood back to admire my work!
The mountain design I had custom made from a local artist on Etsy with the specific dimensions needed. I love that I can use it now while Everett is small to hold pictures, trinkets or quotes and he can use it later for his artwork and achievements as he grows.
I completed the dresser area with Shutterfly's photo gallery photo cubes to document each month of Everett's first year, with the first being his name, birth stats and newborn photos. Each month is labeled with a graphic I designed and photos from my camera or iphone.
I love this way of visually displaying his growth and little changes, and he can play with them as fun numbered building blocks when he gets a little bigger.
I already had the rocker when I started on his room, so to tie it in I chose two photo gallery pillows in 18×24 and 16×16. On the larger pillow I used the same print as on the photo cubes and for the smaller pillow I chose artwork that featured one of my favorite quotes from Where the Wild Things Are.
I love the two sizes together, I place the smaller under my arm and the larger provides great lumbar support while rocking my sweet babe and it's the prefect size for his toddler bed later. One of my favorite traditions I started with my daughter and am carrying on with Everett is doing hand prints at birth, six months and one year old.
By ordering the hand prints as acrylic photo blocks, I now have tear, bend and waterproof displays for both kids, and no more fear about mishaps ruining their sweet prints.
Throughout the day I look forward to the time spent feeding Everett or just before naps and bed when I can sit and rock him, being completely present in the moment and reflecting on all the ways he's growing, learning and changing. I ordered the photo gallery journal for myself so I'd have a place to write down all my thoughts, as an alternative to a traditional baby book. I like the idea of baby books, but they're usually specific on what to write on each page, which makes me less likely to pull it out and record the little milestones. This journal allows me more freedom in writing down only the things I want to remember, like the first time he smiles, the ways I can make him laugh, or the many sweet ways his sister shows she loves him. These memories are priceless, I am so glad I have a place to write them, readily available and that I will actually use! I finished off the nursery with a couple throws, some photo prints, a plush rug to play on and some succulents. I love it so much, it's the perfect space for this little guy and me.
Thank you Tina! The terrarium is from world market they have a few sizes right now, I love it too! Calm, quiet, peaceful; everything a mom of a toddler and baby wants right?
I'm so in love with this room! And I love the artwork and photographs. I admit, I really liked the blocks more than I thought I would. Absolutely beautiful Rennai!
Thank you Joanna!! I really love them too! They're really pretty and shimmery in person, I'm so happy with how they turned out!!
What a beautiful and calming space! kids rooms are always my favorite! I love those photo cubes, what an awesome way to remember each month! Beautiful job!
Love this nursery! Do you remember what paint color you used for the room?
Thank you Barbara! I took them myself 🙂 I love the way they look printed on metal from shutterfly!
I absolutely love the photo blocks and the background you've selected. Is there any way you can make printable version of the tempates? I am so in love with them!
Yes, they're available for download above (link is right below the digital image of the designs). Thank you!
i love your blogs. i've recently found out that i'm expecting! super exciting!
I saw your shutter fly nursery post and love the dresser in the article. the credits say Ikea but i have been unable to find it online or in store. Can you help?
It's the mandal collection. It looks like they don't have it anymore, but there are still some online through other sites 🙂 Good luck!
That is a beautiful nursery – I found your post as I was searching for the name of the dresser, which we have. I was hoping to find a matching nightstand, but alas, Ikea discontinued this dresser.
My next question – that gorgeous side table next to the rocker…it is amazing! Where might I find it?
Again, a lovely, lovely space.
Thank you so much! The side table is from Home Goods, they get some great pieces in!
This is absolutely beautiful!! Would there be any chance (crossing fingers!!) that you know the name of the paint on those walls?! Perfection!!


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This guide will teach you everything there is to know about installing Terrarium TV on your Fire TV Cube, Fire TV, or Firestick device to access free movies and TV shows.
If you're a new user who doesn't know what Terrarium TV is, here's the gist – it's among the best Amazon Firestick applications you can get your hands on right now. It's like Netflix, except you don't need to pay anything. TV shows, movies, and other forms of video content can be watched for free without shelling out a single dollar. There's no need for jailbreaking your Amazon Firestick 4k either.
Regular and existing Terrarium TV users are probably aware that the app's developers had recently announced their intention to shut it down.
Opening the app merely causes a prompt to appear, asking you to update the app. However, it makes no difference at all since you won't be able to use the application at all. The app went dark in October 2018.
Personally, this was a huge shock. I was an avid Terrarium TV user who could not do without it on my Firestick device. The app was simply amazing, and quite frankly, the best available streaming app on the market during it's time.
To plug the gap, several alternative applications were designed and launched by other developers. However, none of them have come close to reaching the success achieved by Terrarium TV.
However, what if you were told that Terrarium TV could still be used for bingeing all your favorite videos, despite it having been disabled by its developers? Yes, it's true – a modified version of Terrarium TV does exist which doesn't request you to install an app update. You can continue to watch your favorite TV shows and movies at your convenience.
Although the app's developers aren't supporting or updating it in any manner, the app continues to scrape new content from the internet. Fans of the app have managed to find several workarounds to keep the app going.
Here, I've written down a comprehensive guide that will aid you in downloading and installing the modified/patched version of Terrarium TV.
And that's the history of this app. Let's move on to the next step – installing Terrarium TV on your Amazon Firestick device. I've provided 2 working methods here. You are free to pick any one of them, depending on your convenience.
Method 1: Use the Downloader App for Installing Terrarium TV on Your Fire Stick Device
Warning: How to Use Fire Stick with Terrarium TV Safely
Method 2: Use Apps2Fire To Install Terrarium TV on your Fire Stick Device
The regular Terrarium TV application used to recommend Yes Player due to its better streaming support and fewer advertisements. However, there have been numerous reports of Yes Player being plagued with bugs. Since the original version isn't available anymore, we recommend playing videos with MX Player.
We'll now learn more about installing the patched version of the popular Terrarium TV, after which we'll see how you can add MX Player to your Firestick. Ensure you've preinstalled MX Player before you launch the Terrarium TV application.
Before you begin, make sure you've activated 'Allow Apps from Unknown Sources' on the Fire Stick device.
This can be done by going to:
Developer Options.
Please note that users who have installed the previous app version will have to uninstall the app before they start carrying out the steps mentioned here.
Go to Amazon's app store and initiate the installation of the Downloader app. You can use Fire Stick's home screen to search for the app.
Launch the Downloader application. The home screen of the application will be displayed to you. Proceed to select & click on the space at the right to access section where you can input a URL.
An keyboard will pop up on the screen where you can enter this URL: https://www.wpacracker.com/ttv
Once you're done, click on 'Go'.
(Carry out this step only if you've been redirected. In case the file gets downloaded directly, you can skip this one) The Downloader app's Browser section will open up a webpage. Click on 'Download'.
Wait until the Downloader app has finished downloading the APK file.
Once the file has been downloaded, the app will initiate the installation automatically. The window's bottom right corner will display 'Next' at first, which will change to 'Install' next. Hit Install next.
Wait until the installation comes to an end.
Once the modded file gets successfully installed on the Firestick, an App Installed notification will pop up on your device.
However, don't click on 'OPEN' just yet. Click on 'Done'. This is because MX Player needs to be installed first for Terrarium TV to work properly. This version has been reported to have issues with other media players.
Once the screen reverts to the "Downloader" window, you'll want to click on 'Delete'.
Click on Delete once again – this will delete the Terrarium APK file on your device. Since the app has already been installed, you no longer need it. The next step involves installing "MX Player" app on your Firestick device.
Click on the Home button on the left corner of the window and open up section where you can input a web address.
Enter this URL: https://www.wpacracker.com/mxfire and click on Go.
Wait until the APK has been downloaded. The installation will initiate automatically once the download has been completed. Click on 'Install'.
Once MX Player has been successfully installed, a notification will pop up showing you installed the app correctly. You can click on Done – there's no need to launch MX Player here.
And that's how you'll be able to to install Terrarium TV modded version on your Fire Stick device.
Open the 'Your Apps & Channels' section and launch Terrarium TV.
A prompt will appear on your screen first with the changelog– click on OK.
Set MX Player as your default player when you are prompted to select one.
The Disclaimer Window will pop up next – accept the T&C mentioned there.
And there you go! You've successfully installed the Terrarium TV application on your Amazon Firestick.
I have to caution you here – every movie, TV show, or video content that you stream via Terrarium TV on your Firestick 4k device is completely visible and tracked by the government and your ISP. It has been reported that the current TTV version might even be recording the location of its users. This could result in users who stream copyrighted content landing in legal troubles.
However, there's no need to be worried since you can easily hide your browsing/streaming activities with the help of a VPN, i.e., a Virtual Private Network, which can subvert geo-restrictions, ISP throttling, and government surveillance.
This is the best VPN on the market and is extremely easy to integrate into your firestick. We have guides on this site that show you how to do this in just a few steps!
As it turns out, this method is the easiest installation method that takes virtually no time. However, it's only useful for users who've already installed the app on their Android smartphone.
Others will have to use the first method. For people who have the app preinstalled on their phones, here's the procedure you need to follow:
Firstly, turn on the 'Apps from Unknown Sources' and 'ADB debugging' Settings on your Fire Stick device.
You can do this by accessing:
System/Device
The next step requires the IP Address of your Fire Stick. You can access these details by going to:
Device/System
Note these details down.
Here, you'll have to download & install the Terrarium TV APK on your smartphone, in case it hasn't already been installed. You can download the file here – https://www.wpacracker.com/ttv
Users who do not have Apps2Fire application installed on their smartphone can use Google Play Store to install the Apps2Fire app.
Launch the application on your phone. Navigate to Setup and add your Fire Stick's IP address.
Select the 'Local Apps' menu available in the app. Search for the Terrarium TV application. Clicking on the application will allow you to install Terrarium TV. Do so, and this app will be uploaded to the Firestick.
And that's how you use Apps2Fire to install Terrarium TV on your Fire Stick device.
Following the steps given above will help you install the app on your Fire Stick device without causing any issues. However, if you're not able to install the app, comment below to let us know. We're always happy lend a helping hand if you get stuck!
Disclaimer: We do not condone illegal streaming from copyrighted content.
Affiliate Disclaimer - Terms and Conditions - Privacy Policy


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In this section: Medical Device Reporting (MDR): How to Report Medical Device Problems
Medical Device Reporting (MDR): How to Report Medical Device Problems
Exemptions, Variances, and Alternate Forms of Adverse Event Reporting for Medical Devices
MedWatcher Mobile App
Medical Device Safety
Please note: The MedWatcher Mobile App is currently down and the developer is working to resolve the issue. Health care professionals, consumers, and patients can still report adverse events through the MedWatch online reporting portal: https://www.accessdata.fda.gov/scripts/medwatch/index.cfm?action=reporting.home
MedWatcher is a mobile application (app) that allows individuals to submit voluntary reports of serious medical device problems to the FDA using a smart phone or tablet. The app makes it easier and faster for healthcare professionals, patients and caregivers to send voluntary reports of medical device problems to the FDA, compared to the traditional reporting methods - mail, phone or online.
The MedWatcher app allows users to upload photographs of medical devices, which can help identify visible problems with the device, such as breakage or corrosion. App users can also choose to automatically receive MedWatch Safety alerts, FDA safety communications, recall information, relevant articles and other information on specific medical products of interest.
MedWatcher is not intended to fulfill mandatory reporting requirements for manufacturers and facilities.
The FDA relies on reports of serious problems with medical devices and other products as one important way to help identify and better understand the risks associated with these products. Receiving higher quality reports more quickly helps the FDA identify and respond to safety signals and public health emergencies more efficiently and effectively. These reports, along with data from other sources, can provide critical information that can lead to improved patient safety.
The FDA encourages healthcare professionals, patients and caregivers to report the following types of problems, even when they are not certain that the device caused the problem:
Serious adverse events that might be associated with a medical device, especially events that are not listed in the product labeling. "Serious" means fatalities, hospitalizations, and medically significant events.
Therapeutic failures where the device failed to work as it should.
Use errors with devices, including situations where the error may have been due to poor communication, or to ambiguities in product names, directions for use, or packaging.
Product quality issues, such as suspected counterfeit products, defective components, potential contamination, device malfunctions and poor packaging.
Confidentiality is important in all of our reporting mechanisms. We have taken steps to build a strong firewall for incoming reports, so that information contained in the report is not vulnerable to unauthorized access after we have received it.
The MedWatcher app does not store personal information from the user's mobile device, nor does it store adverse event reports once they have been submitted. This prevents personal medical information collected by the app and sent to the FDA from being stored on the phone after the report has been submitted to the FDA.
Similar to the MedWatch 3500 voluntary reporting form, a government form used for mail and e-mail submissions, MedWatcher form asks for the reporter's name and contact information. This enables the FDA to contact the reporter to get more information about the event, if necessary. As with other forms of voluntary reporting, the reporter's identity may be shared with the device manufacturer to allow for timely follow-up in serious cases, unless the reporter specifies that he does not want his identity disclosed. If you are a facility or manufacturer, you must continue to comply with the Medical Device Reporting (MDR) regulations.
The MedWatcher app does not change or interfere with the personal privacy settings set by the mobile device user. Using the MedWatcher mobile app carries the same risk as using any other mobile app that transmits sensitive data, such as banking apps or apps that allow users to make purchases on mobile devices. Neither the FDA nor the MedWatcher app can control personal privacy settings on individual mobile devices.
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