RESUMEN
The lack of disease models adequately resembling human tissue has hindered our understanding of amoebic brain infection. Three-dimensional structured organoids provide a microenvironment similar to human tissue. This study demonstrates the use of cerebral organoids to model a rare brain infection caused by the highly lethal amoeba Balamuthia mandrillaris. Cerebral organoids were generated from human pluripotent stem cells and infected with clinically isolated B. mandrillaris trophozoites. Histological examination showed amoebic invasion and neuron damage following coculture with the trophozoites. The transcript profile suggested an alteration in neuron growth and a proinflammatory response. The release of intracellular proteins specific to neuronal bodies and astrocytes was detected at higher levels postinfection. The amoebicidal effect of the repurposed drug nitroxoline was examined using the human cerebral organoids. Overall, the use of human cerebral organoids was important for understanding the mechanism of amoeba pathogenicity, identify biomarkers for brain injury, and in the testing of a potential amoebicidal drug in a context similar to the human brain.
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Amebiasis , Balamuthia mandrillaris , Encéfalo , Organoides , Humanos , Organoides/parasitología , Balamuthia mandrillaris/efectos de los fármacos , Encéfalo/parasitología , Encéfalo/patología , Amebiasis/parasitología , Amebiasis/tratamiento farmacológico , Trofozoítos/efectos de los fármacos , Neuronas/parasitología , Células Madre PluripotentesRESUMEN
Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-ß signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1ß, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-ß signaling pathways, and exhibiting anti-inflammatory properties.
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Diferenciación Celular , Proliferación Celular , Condrogénesis , Glucólisis , Inflamación , Sericinas , Transducción de Señal , Proteína Smad2 , Proteína smad3 , Factor de Crecimiento Transformador beta , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteína Smad2/metabolismo , Animales , Transducción de Señal/efectos de los fármacos , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Condrogénesis/efectos de los fármacos , Sericinas/farmacología , Glucólisis/efectos de los fármacos , Ratones , Inflamación/metabolismo , Inflamación/patología , Inflamación/tratamiento farmacológico , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Línea Celular , Bombyx/metabolismoRESUMEN
BACKGROUND: Endothelial cells (ECs) play a major role in malaria pathogenesis, as a point of direct contact of parasitized red blood cells to the blood vessel wall. The study of cytoskeleton structures of ECs, whose main functions are to maintain shape and provide strength to the EC membrane is important in determining the severe sequelae of Plasmodium falciparum malaria. The work investigated the cytoskeletal changes (microfilaments-actin, microtubules-tubulin and intermediate filaments-vimentin) in ECs induced by malaria sera (Plasmodium vivax, uncomplicated P. falciparum and complicated P. falciparum), in relation to the levels of pro-inflammatory cytokines. METHODS: Morphology and fluorescence intensity of EC cytoskeleton stimulated with malaria sera were evaluated using immunofluorescence technique. Levels of tumour necrosis factor (TNF) and interferon (IFN)-gamma (γ) were determined using enzyme-linked immunosorbent assay (ELISA). Control experimental groups included ECs incubated with media alone and non-malaria patient sera. Experimental groups consisted of ECs incubated with malaria sera from P. vivax, uncomplicated P. falciparum and complicated P. falciparum. Morphological scores of cytoskeletal alterations and fluorescence intensity were compared across each experiment group, and correlated with TNF and IFN-γ. RESULTS: The four morphological changes of cytoskeleton included (1) shrinkage of cytoskeleton and ECs with cortical condensation, (2) appearance of eccentric nuclei, (3) presence of "spiking pattern" of cytoskeleton and EC membrane, and (4) fragmentation and discontinuity of cytoskeleton and ECs. Significant damages were noted in actin filaments compared to tubulin and vimentin filaments in ECs stimulated with sera from complicated P. falciparum malaria. Morphological damages to cytoskeleton was positively correlated with fluorescence intensity and the levels of TNF and IFN-γ. CONCLUSIONS: ECs stimulated with sera from complicated P. falciparum malaria showed cytoskeletal alterations and increased in fluorescence intensity, which was associated with high levels of TNF and IFN-γ. Cytoskeletal changes of ECs incubated with complicated P. falciparum malaria sera can lead to EC junctional alteration and permeability changes, which is mediated through apoptotic pathway. The findings can serve as a basis to explore measures to strengthen EC cytoskeleton and alleviate severe malaria complications such as pulmonary oedema and cerebral malaria. In addition, immunofluorescence intensity of cytoskeleton could be investigated as potential prognostic indicator for malaria severity.
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Malaria Cerebral , Malaria Vivax , Humanos , Vimentina , Tubulina (Proteína) , Células Endoteliales , Citoesqueleto , Microtúbulos , Factor de Necrosis Tumoral alfa , Técnica del Anticuerpo FluorescenteRESUMEN
The noncontagious immune-mediated skin disease known as psoriasis is regarded as a chronic skin condition with a 0.09-11.4% global prevalence. The main obstacle to the eradication of the disease continues to be insufficient treatment options. Sericin, a natural biopolymer from Bombyx mori cocoons, can improve skin conditions via its immunomodulatory effect. Many external therapeutic methods are currently used to treat psoriasis, but sericin-based hydrogel is not yet used to treat plaques of eczema. Through the use of an imiquimod rat model, this study sought to identify the physical and chemical characteristics of a silk sericin-based poly(vinyl) alcohol (SS/PVA) hydrogel and assess both its therapeutic and toxic effects on psoriasis. The cytokines, chemokines, and genes involved in the pathogenesis of psoriasis were investigated, focusing on the immuno-pathological relationships. We discovered that the SS/PVA had a stable fabrication and proper release. Additionally, the anti-inflammatory, antioxidant, and anti-apoptotic properties of SS/PVA reduced the severity of psoriasis in both gross and microscopic skin lesions. This was demonstrated by a decrease in the epidermal histopathology score, upregulation of nuclear factor erythroid 2-related factor 2 and interleukin (IL)-10, and a decrease in the expression of tumor necrosis factor (TNF)-α and IL-20. Moreover, the genes S100a7a and S100a14 were downregulated. Additionally, in rats given the SS/PVA treatment, blood urea nitrogen, creatinine, and serum glutamic oxaloacetic transaminase levels were within normal limits. Our findings indicate that SS/PVA is safe and may be potentiated to treat psoriasis in a variety of forms and locations of plaque because of its physical, chemical, and biological characteristics.
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Psoriasis , Sericinas , Ratas , Animales , Sericinas/farmacología , Sericinas/uso terapéutico , Sericinas/química , Alcohol Polivinílico/química , Psoriasis/tratamiento farmacológico , Hidrogeles , VendajesRESUMEN
Mouse antibodies specific to dengue NS1 have been widely investigated for their cross-reactivity with several human biomolecules. This is the first study demonstrating the cross-reactivity of human monoclonal antibodies (HuMAbs) specific to dengue NS1 isolated from patients infected with dengue virus serotype-2 (DENV-2). Nine anti-NS1 HuMAbs, which were mainly derived from patients in convalescent-phase after secondary infection of DENV-2, were characterized. Their cross-reactivity with plasminogen, thrombin, and endothelial cells was investigated, following which plasmin-formation assays were performed. All anti-NS1 HuMAbs exhibited cross-reactivity with human plasminogen (Plg), but not with thrombin or endothelial cells. Moreover, all HuMAbs exhibiting cross-reactivity with Plg converted Plg to plasmin in the plasmin-formation assay. These results suggest the implications and drawbacks of using anti-NS1 antibodies in immunotherapy.
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Virus del Dengue , Dengue , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales , Células Endoteliales , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas no Estructurales ViralesRESUMEN
In the present study, we developed a genus-specific rGroEL1-524 IgM-ELISA assay for use in screening diagnosis of suspected leptospirosis among acute undifferentiated febrile illness patients during acute fever. The diagnostic accuracies of the rGroEL1-524 IgM-ELISA, commercial Panbio IgM-ELISA, and Virion-Serion Classic IgG-ELISA were evaluated using 133 Thai leptospirosis sera and 210 controls. Sensitivities were 91.7%, 59.6%, and 17.7% for acute infection, and the specificities were 92.6%, 90.2%, and 88.3% for the non-leptospirosis control, respectively. The rGroEL1-524 IgM-ELISA had high sensitivity, at 92.3% and 91.7%, among culture-positive and MAT-negative cases at 1-3 days post-onset of symptoms (DPO1-3), respectively. Impaired specificity on scrub typhus was found, possibly from antibody cross-reaction to ortholog GroEL. Commercial Panbio IgM-ELISA had sensitivities at DPO1-3 of 30.8% and 41.7% for culture-positive and MAT-negative cases whereas Virion-Serion IgG-ELISA showed sensitivities of 5.9% and 13.3%, respectively. The rGroEL1-524 IgM-ELISA could be useful as a screening test for early diagnosis. The performance of the commercial ELISA suggests the applicability of IgM-ELISA for diagnosis, while IgG-ELISA is useful for seroprevalence surveys. However, confirmation by reference tests is recommended.
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Chaperonina 60/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Inmunoglobulina M/inmunología , Leptospirosis/diagnóstico , Leptospirosis/epidemiología , Juego de Reactivos para Diagnóstico , Anticuerpos Antibacterianos/inmunología , Epítopos/inmunología , Humanos , Leptospira/inmunología , Tamizaje Masivo , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Tailandia/epidemiologíaRESUMEN
Clostridioides difficile infection is the most common cause of nosocomial and antibiotic-associated diarrhea. C. difficile treatment is increasingly likely to fail, and the recurrence rate is high. Antimicrobial peptides are considered an alternative treatment for many infectious diseases, including those caused by antibiotic resistant bacteria. In the present study, we identified a CM peptide, a hybrid of cecropin A and melittin, and its derivative which possesses potent antimicrobial activity against C. difficile strain 630. CM peptide exhibited antibacterial activity with minimum inhibitory concentration of 3.906 µg/ml (2.21 µM). A modified derivative of CM, CM-A, exhibited even greater activity with a minimum inhibitory concentration of 1.953 µg/ml (1.06 µM) and a minimum bactericidal concentration of 7.8125 µg/ml (4.24 µM), which indicates that CM-A peptide is more efficient than its parent peptide. A fluorescence-activated cell sorter analysis revealed that the membrane of C. difficile 630 could be an important target for CM-A. This peptide induced high levels of cell depolarization and cell permeability on C. difficile cell membrane. Moreover, electron microscopy imaging showed that CM-A interferes with the C. difficile cell membrane. Hence, the antimicrobial peptide CM-A may represent a promising novel approach for the treatment of C. difficile infections.
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Péptidos Catiónicos Antimicrobianos/química , Clostridioides difficile/efectos de los fármacos , Infecciones por Clostridium/tratamiento farmacológico , Meliteno/química , Péptidos/química , Antiinfecciosos , Péptidos Antimicrobianos/química , Células CACO-2 , Membrana Celular/efectos de los fármacos , Diseño de Fármacos , Colorantes Fluorescentes/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de ProteínaRESUMEN
OBJECTIVES: The aims of the study were two-fold: (1) antigen (Ag) preparation and evaluation of three antigens of Gnathostoma spinigerum infective larvae (GsL3), crude somatic antigen (CSAg), excretory-secretory antigen (ESAg) and partially purified antigens (namely P1Ag, P2Ag and P3Ag) to differentiate IgE, IgG, IgG1-4 and IgM for human gnathostomiasis diagnosis; and (2) application of the selected ELISA for following up stored sera of patients treated with ivermectin (IVM) and albendazole (ABZ). METHODS: Different antigens were analysed by antibodies of gnathostomiasis cases, other parasite infections and healthy controls using indirect ELISA to differentiate IgE, IgG, IgG1-4 and IgM. Then, prominent antigen and immunoglobulin were used in antibody predictions of gnathostomiasis cases treated with albendazole or ivermectin. RESULTS: Sensitivity of all evaluated ELISAs: IgM-, IgG-, IgG1- and IgG4-ELISA, was 100%. IgM-ELISA with CSAg and P3Ag exhibited the highest specificity of 99%. IgG-ELISA with P2Ag resulted in the highest specificity of 92.3%. IgG1-ELISA with P2Ag and P3Ag showed excellent results with 100% specificity. Finally, P2Ag evaluated IgG1 of the followed-up cases with ABZ and IVM. Decreasing antibody IgG1 levels were mostly found in both treatments at Month 9 and long follow-up was over 12 months. A Gnathostoma worm was extracted from each two treated patients. CONCLUSIONS: Using IgG1-ELISA against P2Ag and P3Ag gave excellent results with 100% sensitivity and specificity. These tests can be an alternative to immunoblotting for gnathostomiasis. IgG1 decreased at least 9 months in most cases, so long-term treatment should be performed over 1 year.
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Antígenos Helmínticos/inmunología , Gnathostoma/inmunología , Gnathostomiasis/sangre , Gnathostomiasis/diagnóstico , Pruebas Inmunológicas/métodos , Albendazol/uso terapéutico , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Antiparasitarios/uso terapéutico , Gnathostomiasis/tratamiento farmacológico , Gnathostomiasis/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Ivermectina/uso terapéutico , Larva/inmunología , Sensibilidad y EspecificidadRESUMEN
Allergen-specific-immunotherapy (ASIT) can cause long-term resolution of allergic diseases, reduces drug use and chances of new allergen sensitization. Nevertheless, therapeutic vaccine and data on ASIT efficacy for cockroach (CR) allergy are relatively scarce. In this study, efficacy and mechanism of a novel intranasal vaccine consisting of liposome (L)-entrapped mixture of American CR (Periplaneta americana) major allergen (Per a 9) and immunosuppressive protein of Brugia malayi nematode named transforming growth factor-beta homologue (TGH) in treatment of CR allergy were investigated along with two other vaccines (L-Per a 9 alone and L-TGH alone). All three vaccines could reduce pathogenic type 2 response and lung immunopathology in the vaccines-treated CR-allergic mice, but by different mechanisms. L-Per a 9 caused a deviation of the pathogenic type 2 to type 1 response (IFN-γ-upregulation), whereas the L-(TGH + Per a 9) and L-TGH generated regulatory immune responses including up-expression of immunosuppressive cytokine genes and increment of serum adenosine and lung indoleamine-2,3-dioxygenase-1 which are signatures of regulatory T cells (Tregs) and tolerogenic dendritic cells, respectively. The L-(TGH + Per a 9) should be further evaluated towards clinical application, as this vaccine has a propensity to induce broadly effective therapeutic effects for inhalant allergies.
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Alérgenos/inmunología , Arginina Quinasa/inmunología , Brugia Malayi/inmunología , Desensibilización Inmunológica/métodos , Hipersensibilidad/inmunología , Hipersensibilidad/prevención & control , Inmunosupresores/inmunología , Proteínas de Insectos/inmunología , Periplaneta/inmunología , Factor de Crecimiento Transformador beta/inmunología , Vacunas/inmunología , Administración Intranasal , Alérgenos/sangre , Animales , Arginina Quinasa/sangre , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Hipersensibilidad/sangre , Hipersensibilidad/parasitología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Liposomas , Masculino , Ratones , Ratones Endogámicos BALB C , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/sangre , Resultado del Tratamiento , Vacunas/administración & dosificaciónRESUMEN
(1) Background: Ebolavirus (EBOV) poses as a significant threat for human health by frequently causing epidemics of the highly contagious Ebola virus disease (EVD). EBOV glycoprotein (GP), as a sole surface glycoprotein, needs to be cleaved in endosomes to fully expose a receptor-binding domain (RBD) containing a receptor-binding site (RBS) for receptor binding and genome entry into cytoplasm for replication. RBDs are highly conserved among EBOV species, so they are an attractive target for broadly effective anti-EBOV drug development. (2) Methods: Phage display technology was used as a tool to isolate human single-chain antibodies (HuscFv) that bind to recombinant RBDs from a human scFv (HuscFv) phage display library. The RBD-bound HuscFvs were fused with cell-penetrating peptide (CPP), and cell-penetrating antibodies (transbodies) were made, produced from the phage-infected E. coli clones and characterized. (3) Results: Among the HuscFvs obtained from phage-infected E. coli clones, HuscFvs of three clones, HuscFv4, HuscFv11, and HuscFv14, the non-cell-penetrable or cell-penetrable HuscFv4 effectively neutralized cellular entry of EBOV-like particles (VLPs). While all HuscFvs were found to bind cleaved GP (GPcl), their presumptive binding sites were markedly different, as determined by molecular docking. (4) Conclusions: The HuscFv4 could be a promising therapeutic agent against EBOV infection.
RESUMEN
Previous studies have reported activation of the B cell-activating factor (BAFF)/a proliferation-inducing ligand (APRIL) system in T independent immunity against malaria infection. Plasmodium falciparum (P. falciparum) infected animal model is not feasible. Therefore, little is known about the occurrence of BAFF/APRIL system and changes in falciparum lymphoid tissues. This study aimed to investigate the expression of BAFF/APRIL system components in lymphoid tissues from P. falciparum infected patients. Spleen and lymph node samples from 14 patients were collected at autopsy. Normal spleens and bacterially infected tonsils served as controls. The protein and/or mRNA expression of BAFF/APRIL and their cognate receptors, BAFF-R, TACI and BCMA, were determined by immunohistochemistry and RT-qPCR, respectively. The spleens of the patients exhibited significantly higher BAFF-R protein expression than normal spleens. Although without appropriate control, BCMA protein was markedly observed only in the lymph nodes. BAFF and BCMA mRNA levels were also significantly elevated in the spleen tissues of the patients compared with normal spleens. The overall BAFF-R protein levels in the lymphoid tissues of the patients correlated positively with parasitaemia. These findings are the first to confirm that BAFF/APRIL system activation in lymphoid tissues and is positively correlated with the parasitaemia levels in falciparum malaria.
Asunto(s)
Factor Activador de Células B/metabolismo , Proteínas de Unión al ADN/metabolismo , Ganglios Linfáticos/metabolismo , Malaria Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Bazo/metabolismo , Factores de Transcripción/metabolismo , Receptor del Factor Activador de Células B/biosíntesis , Antígeno de Maduración de Linfocitos B/biosíntesis , Regulación de la Expresión Génica , Humanos , Ganglios Linfáticos/parasitología , Ganglios Linfáticos/patología , Malaria Falciparum/patología , Bazo/parasitología , Bazo/patología , Proteína Activadora Transmembrana y Interactiva del CAML/biosíntesisRESUMEN
Human gnathostomiasis is mainly caused by third-stage larvae of Gnathostoma spinigerum (G. spinigerum L3). Excretory-secretory products (ES) released from infective helminthic larvae are associated with larval migration and host immunity modulation. Natural killer (NK) cells have important immune functions against helminth infection. Currently, the effects of ES from G. spinigerum L3 (G. spinigerum ES) on NK cell activity are unclear. This study investigated whether G. spinigerum ES affected human NK cells. Human normal peripheral blood mononuclear cell (PBMC) cultures were used to mimic immune cells within the circulation. PBMC were co-cultured with G. spinigerum ES (0.01-0.05 µg/ml) for 5 or 7 days. Levels of IFN-γ in cultured supernatants were measured by enzyme-linked immunosorbent assay. The expressions of mRNA encoding NK cell receptors, especially the C type killer cell lectin-like family (KLR; NKG2A, NKG2C, and NKG2D) and IFN-γ in ES induced PBMC were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). ES induced PBMC markedly decreased the levels of IFN-γ and increased the expressions of NKG2A and NKG2D on NK cells. In conclusion, low amounts of G. spinigerum ES modulated NK cells by downregulating the transcription of IFN-γ and upregulating the expressions of KLR (NKG2A and NKG2D receptors) during the 7-day observation period. These findings indicate more in-depth studies of NK cell function are required to better understand the mechanism involved in immune evasive strategies of human gnathostomiasis.
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Gnathostoma/inmunología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Receptores Similares a Lectina de Células NK/metabolismo , Animales , Técnicas de Cocultivo , Regulación hacia Abajo , Gnathostoma/crecimiento & desarrollo , Gnathostomiasis/inmunología , Humanos , Células Asesinas Naturales/metabolismo , Larva/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.
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Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Epítopos Inmunodominantes/inmunología , Leptospira/inmunología , Leptospirosis/inmunología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Monoclonales/uso terapéutico , Proteínas de la Membrana Bacteriana Externa/química , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Persona de Mediana Edad , Péptidos/química , Péptidos/inmunología , Conejos , Pruebas Serológicas , Adulto JovenRESUMEN
Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA-indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed.
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Allergen-specific immunotherapy (AIT) facilitates long-term resolution of allergic morbidity resulting in reduced drug use and increased refractoriness to new sensitization. AIT effectiveness has been demonstrated in seasonal and perennial allergies, and insect stings. However, data and studies in AIT relative to cockroach (CR) allergy are relatively scarce. In this study, mice allergic to American CR (Periplaneta americana) were treated with a liposome (L)-entrapped vaccine made of mouse Tregitope289-Per a 9 of the CR, Tregitope167-Per a 9, or Per a 9 alone - or placebo. Allergic mice that received an individual vaccine intranasally had reduced Th2 response, reduced lung inflammation, and reduced respiratory tissue remodeling. However, only L-Tregitope289-Per a 9 and L-Tregitope167-Per a 9 induced expression of immunosuppressive cytokine genes (IL-10, TGF-ß, and IL-35 for L-Tregitope289-Per a 9, and IL-10 and TGF-ß for L-Tregitope167-Per a 9) and increment of idoleamine-2,3-dioxygenase 1 (IDO1), indicating that these vaccines caused allergic disease suppression and reversal of respiratory tissue remodeling via generation of regulatory lymphocytes. Liposome entrapped-recombinant Per a 9 (L-Per a 9) did not cause upregulation of immunosuppressive cytokine genes and IDO1 increment; rather, L-Per a 9 induced high expression of IFN-γ in lungs of treated mice, which resulted in mitigation of allergic manifestations. This study provides compelling evidence that both liposome-entrapped vaccines made of single refined major allergen alone and single refined major allergen linked with Tregitopes are effective for reducing allergen-mediated respiratory tissue inflammation and remodeling, but through different mechanisms.
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Desensibilización Inmunológica/métodos , Hipersensibilidad/inmunología , Linfocitos T Reguladores/inmunología , Vacunas/inmunología , Administración Intranasal , Alérgenos/inmunología , Animales , Proteínas de la Cápside/inmunología , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , PeriplanetaRESUMEN
This narrative review article summarizes past and current technologies for generating antibodies for passive immunization/immunotherapy. Contemporary DNA and protein technologies have facilitated the development of engineered therapeutic monoclonal antibodies in a variety of formats according to the required effector functions. Chimeric, humanized, and human monoclonal antibodies to antigenic/epitopic myriads with less immunogenicity than animal-derived antibodies in human recipients can be produced in vitro. Immunotherapy with ready-to-use antibodies has gained wide acceptance as a powerful treatment against both infectious and noninfectious diseases. Influenza, a highly contagious disease, precipitates annual epidemics and occasional pandemics, resulting in high health and economic burden worldwide. Currently available drugs are becoming less and less effective against this rapidly mutating virus. Alternative treatment strategies are needed, particularly for individuals at high risk for severe morbidity. In a setting where vaccines are not yet protective or available, human antibodies that are broadly effective against various influenza subtypes could be highly efficacious in lowering morbidity and mortality and controlling unprecedented epidemic/pandemic. Prototypes of human single-chain antibodies to several conserved proteins of influenza virus with no Fc portion (hence, no ADE effect in recipients) are available. These antibodies have high potential as a novel, safe, and effective anti-influenza agent.
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Anticuerpos Antivirales , Vacunas contra la Influenza , Gripe Humana/inmunología , Animales , Anticuerpos Monoclonales , Humanos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/terapia , Infecciones por Orthomyxoviridae , PandemiasRESUMEN
Plasmodium falciparum, the most virulent malaria parasite species, causes severe symptoms especially acute lung injury (ALI), of which characterized by alveolar epithelium and endothelium destruction and accelerated to blood-gas-barrier breakdown. Parasitized erythrocytes, endothelial cells, monocytes, and cytokines are all involved in this mechanism, but hemozoin (HZ), the parasitic waste from heme detoxification, also mainly contributes. In addition, it is not clear why type II pneumocyte proliferation, alveolar restorative stage, is rare in malaria-associated ALI. To address this, in vitro culture of A549 cells with Plasmodium HZ or with interleukin (IL)-1ß triggered by HZ and monocytes (HZ-IL-1ß) was conducted to determine their alveolar apoptotic effect using ethidium bromide/acridine orange staining, annexin-V-FITC/propidium iodide staining, and electron mircroscopic study. Caspase recruitment domain-containing protein 9 ( CARD9), the apoptotic regulator gene, and IL-1ß were quantified by reverse-transcriptase PCR. Junctional cellular defects were characterized by immunohistochemical staining of E-cadherin. The results revealed that cellular apoptosis and CARD9 expression levels were extremely high 24 h after induction by HZ-IL-1ß when compared to the HZ- and non-treated groups. E-cadherin was markedly down-regulated by HZ-IL-1ß and HZ treatments. CARD9 expression was positively correlated with IL-1ß expression and the number of apoptotic cells. Interestingly, the localization of HZ in the vesicular surfactant of apoptotic pneumocyte was also identified and submitted to be a cause of alveolar resolution abnormality. Thus, HZ triggers monocytes to produce IL-1ß and induces pneumocyte type II apoptosis through CARD9 pathway in association with down-regulated E-cadherin, which probably impairs alveolar resolution in malaria-associated ALI. Impact statement The present work shows the physical and immunomodulatory properties of hemozoin on the induction of pneumocyte apoptosis in relation to IL-1ß production through the CARD9 pathway. This occurrence may be a possible pathway for the retardation of lung resolution leading to blood-gas-barrier breakdown. Our findings lead to the understanding of the host-parasite relationship focusing on the dysfunction in ALI induced by HZ, a possible pathway of the recovering lung epithelial retardation in malaria-associated ARDS.
Asunto(s)
Lesión Pulmonar Aguda/parasitología , Células Epiteliales Alveolares/patología , Apoptosis/fisiología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Hemoproteínas/metabolismo , Interleucina-1beta/metabolismo , Alveolos Pulmonares/lesiones , Células A549 , Lesión Pulmonar Aguda/patología , Células Epiteliales Alveolares/parasitología , Antígenos CD/biosíntesis , Cadherinas/biosíntesis , Línea Celular Tumoral , Humanos , Pulmón/metabolismo , Malaria Falciparum/patología , Plasmodium falciparum/patogenicidad , Alveolos Pulmonares/citología , Alveolos Pulmonares/parasitología , Células THP-1RESUMEN
Allergen specific immunotherapy (AIT) can modulate the allergic response causing a long-term symptom subsidence/abolishment which leads to reduced drug use and prevention of new sensitization. AIT of house dust mite allergy (HDM) using the mite crude extract (CE) as the therapeutic agent is not only less effective than the AIT for many other allergens, but also frequently causes adverse effects during the treatment course. In this study, mouse model of Dermatophagoides pteronyssinus (Dp) allergy was invented for testing therapeutic efficacies of intranasally administered liposome (L) encapsulated vaccines made of single Dp major allergens (L-Der p 1, L-Der p 2), combined allergens (L-Der p 1 and Der p 2), and crude Dp extract (L-CE). The allergen sparing intranasal route was chosen as it is known that the effective cells induced at the nasal-associated lymphoid tissue can exert their activities at the lower respiratory tissue due to the common mucosal traffic. Liposome was chosen as the vaccine delivery vehicle and adjuvant as the micelles could reduce toxicity of the entrapped cargo. The Dp-CE allergic mice received eight doses of individual vaccines/placebo on alternate days. All vaccine formulations caused reduction of the Th2 response of the Dp allergic mice. However, only the vaccines made of single refined allergens induced expressions of immunosuppressive cytokines (TGF-ß, IL-35 and/or IL-10) which are the imperative signatures of successful AIT. The data emphasize the superior therapeutic efficacy of single refined major allergen vaccines than the crude allergenic extract vaccine.
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Alérgenos/inmunología , Inmunoterapia , Liposomas , Vacunas/administración & dosificación , Animales , Dermatophagoides pteronyssinus/inmunologíaRESUMEN
BACKGROUND: Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. OBJECTIVE: To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. METHODS: A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. RESULTS: Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). CONCLUSIONS: Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.
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Neoplasias de los Conductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Línea Celular Tumoral , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Humanos , Invasividad Neoplásica , Pronóstico , Proteínas de Unión a GTP rab7RESUMEN
A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials.