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BACKGROUND: Viral infection is implicated in development of autoimmunity. Parvovirus B19 (B19V) nonstructural protein, NS1, a helicase, covalently modifies self double-stranded deoxyribonucleic acid (dsDNA) and induces apoptosis. This study tested whether resulting apoptotic bodies (ApoBods) containing virally modified dsDNA could induce autoimmunity in an animal model. METHODS: BALB/c mice were inoculated with (1) pristane-induced, (2) B19V NS1-induced, or (3) staurosporine-induced ApoBods. Serum was tested for dsDNA autoantibodies by Crithidia luciliae staining and enzyme-linked immunosorbent assay. Brain, heart, liver, and kidney pathology was examined. Deposition of self-antigens in glomeruli was examined by staining with antibodies to dsDNA, histones H1 and H4, and TATA-binding protein. RESULTS: The B19V NS1-induced ApoBod inoculation induced dsDNA autoantibodies in a dose-dependent fashion. Histopathological features of immune-mediated organ damage were evident in pristane-induced and NS1-induced ApoBod groups; severity scores were higher in these groups than in staurosporine-treated groups. Tissue damage was dependent on NS1-induced ApoBod dose. Nucleosomal antigens were deposited in target tissue from pristane-induced and NS1-induced ApoBod inoculated groups, but not in the staurosporine-induced ApoBod inoculated group. CONCLUSIONS: This study demonstrated proof of principle in an animal model that virally modified dsDNA in apoptotic bodies could break tolerance to self dsDNA and induce dsDNA autoantibodies and end-organ damage.
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Anticuerpos Antinucleares/sangre , ADN/inmunología , Vesículas Extracelulares/inmunología , Proteínas no Estructurales Virales/metabolismo , Animales , Anticuerpos Antinucleares/metabolismo , Apoptosis/efectos de los fármacos , Autoinmunidad , Encéfalo/patología , Inhibidores Enzimáticos/farmacología , Femenino , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Inmunosupresores/farmacología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Hígado/patología , Ratones , Miocardio/patología , Parvovirus B19 Humano , Estaurosporina/farmacología , Terpenos/farmacologíaRESUMEN
The spirochaete bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common tick-borne infection in the northern hemisphere. There is a long-standing debate regarding the role of pleomorphic forms in Lyme disease pathogenesis, while very little is known about the characteristics of these morphological variants. Here, we present a comprehensive analysis of B. burgdorferi pleomorphic formation in different culturing conditions at physiological temperature. Interestingly, human serum induced the bacterium to change its morphology to round bodies (RBs). In addition, biofilm-like colonies in suspension were found to be part of B. burgdorferi's normal in vitro growth. Further studies provided evidence that spherical RBs had an intact and flexible cell envelope, demonstrating that they are not cell wall deficient, or degenerative as previously implied. However, the RBs displayed lower metabolic activity compared with spirochaetes. Furthermore, our results indicated that the different pleomorphic variants were distinguishable by having unique biochemical signatures. Consequently, pleomorphic B. burgdorferi should be taken into consideration as being clinically relevant and influence the development of novel diagnostics and treatment protocols.
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Borrelia burgdorferi/química , Borrelia burgdorferi/crecimiento & desarrollo , Enfermedad de Lyme/microbiología , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Pared Celular/química , Pared Celular/metabolismo , HumanosRESUMEN
Emerging organoid research is paving way for studies in infectious diseases. Described here is a technique for the generation of stem-cell derived organoids for human small intestine and lung together with methods to infect such organoids with a mock pathogen (Cryptosporidium parvum). Such systems are amenable to imaging and processing for molecular biological analyses. It is the intent of this chapter to provide a simple, routine organoid procedure so that in vitro studies with Borrelia such as cell invasion and dissemination can be conducted.
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Enfermedades Transmisibles , Criptosporidiosis , Cryptosporidium , Humanos , Organoides , Intestino DelgadoRESUMEN
Standard clinical markers can improve tick-borne infection (TBI) diagnoses. We investigated immune and other clinical biomarkers in 110 patients clinically diagnosed with TBIs before (T0) and after antibiotic treatment (T2). At T0, both the initial observation group and patients without seroconversion for tick-borne pathogens exhibited notably low percentages and counts of CD3 percentage (CD3%), CD3+ cells, CD8+ suppressors, CD4 percentage (CD4%), and CD4+ helper cells, with the latter group showing reductions in CD3%, CD3+, and CD8+ counts in approximately 15-22% of cases. Following treatment at the T2 follow-up, patients typically experienced enhancements in their previously low CD3%, CD3+ counts, CD4%, and CD4+ counts; however, there was no notable progress in their low CD8+ counts, and a higher number of patients presented with insufficient transferrin levels. Moreover, among those with negative serology for tick-borne infections, there was an improvement in low CD3% and CD3+ counts, which was more pronounced in patients with deficient transferrin amounts. Among those with CD57+ (n = 37) and CD19+ (n = 101) lymphocyte analysis, 59.46% of patients had a low CD57+ count, 14.85% had a low CD19 count, and 36.63% had a low CD19 percentage (CD19%). Similar findings were observed concerning low CD57+, CD19+, and CD19% markers for negative TBI serology patients. Overall, this study demonstrates that routine standard clinical markers could assist in a TBI diagnosis.
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The field of data analysis, preparation, and machine learning is rapidly expanding, offering numerous libraries and resources for exploration. Researchers gain knowledge through various channels, but few resources provide a comprehensive framework for building machine-learning models. We present a step-by-step framework for constructing a robust Random Forest classification model to fill this gap. Using the trained model, we predict if individuals visiting Sanoviv Medical Institute between 2020 and 2023 participated in the Lyme disease program based on age, symptoms, blood count, and chemistry results. While not exhaustive, the methods in each step provide a valuable starting point for researchers, promoting an understanding of the fundamental approach to model creation. The framework encourages researchers to explore beyond the outlined techniques, fostering innovation and experimentation.
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Aprendizaje Automático , Participación del Paciente , Humanos , Enfermedad de LymeRESUMEN
The rising prevalence of tick-borne infections (TBIs) necessitates further attention. This study retrospectively investigated the types of TBIs, symptoms, and if combination antibiotics were helpful within a patient cohort at an infectious disease clinic in Ireland. In this chart audit of 301 individuals (184 female, 117 male) tested for TBIs, 140 (46.51%) had positive antibody responses for TBIs from an ELISA (enzyme-linked immunoassay) that was based on a modified two-tiered testing protocol. A total of 93 (66.43%) patients had positive antibody responses to one TBI: 83 (59.29%) for Borrelia, 7 (5.00%) for Rickettsia, and 1 (0.71%) each for either Babesia, Bartonella, or Ehrlichia. The remaining 47 (33.57%) patients were infected with multiple TBIs. These patients were treated with combination antibiotics and monitored at two subsequent follow-ups. Only 2 of 101 patients (1.98%) had discontinued treatment by the second follow-up. In the first follow-up with 118 patients, 70 (59.32%) reported pain and 48 (40.68%) had neurological symptoms. In the next follow-up of 101 patients, 41 (40.59%) had pain while 30 (29.70%) had neurological symptoms. There were statistically significant reductions in the incidence of pain (41.43%) and neurological (37.50%) symptoms between follow-ups. Thus, our study demonstrates that combination antibiotics effectively relieve TBI symptoms with good patient tolerance.
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Like many bacterial species, Borrelia burgdorferi, the pleomorphic bacterium that causes Lyme borreliosis, produces outer membrane vesicles (OMVs). Borrelial OMVs (BbOMVs) have been identified as containing virulence factors, such as outer surface proteins (Osps) A, B, and C, as well as DNA. However, the pathogenicity of BbOMVs in disease development is still unclear. In this study, we characterized purified BbOMVs by analyzing their size and immunolabeling for known antigenic markers: OspA, OspC, p39, and peptidoglycan. In addition, BbOMVs were cocultured with human non-immune cells for cytotoxicity analysis. The results demonstrated that, on average, the vesicles were small, ranging between 11 and 108 nm in diameter. In addition, both OspA and OspC, as well as Lyme arthritis markers p39 and peptidoglycan, were detected from BbOMVs. Furthermore, BbOMVs were cocultured with non-immune cells, which did not result in cell death. Combined, these results suggested that BbOMVs could participate in the induction of infection by functioning as a decoy for the host immune system. Furthermore, BbOMVs might serve as a means for persistent antigens to remain in the host for prolonged periods of time.
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Mycotoxin exposure in humans is primarily assessed through its occurrence in external sources, such as food commodities. Herein, we have developed a direct competitive ELISA to facilitate the detection of aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin (FUM B1/B2), ochratoxin A (OTA), and zearalenone (ZEA) in human serum. The analytical validation of the assay followed practices endorsed by the international research community and the EU directive 96/23/EC in order to examine detection capability, recovery, and cross-reactivity. The assay demonstrated a lower limit of quantitation (LLOQ) for AFB1 [0.61 ng/mL (hereon ng/mL = ppb)], DON (19.53 ppb), FUM (4.88 ppb), OTA (19.53 ppb), and ZEA (0.15 ppb). Recovery from human serum for all mycotoxins spanned from 73% to 106%. Likewise, the specificity for monoclonal antibodies against cross-reactant mycotoxins ranged from 2% to 11%. This study compares the LLOQ and recovery values with commercial and emerging immuno-based methods for detecting mycotoxins in foodstuffs. The LLOQ values from the present study were among the lowest in commercial or emerging methods. Despite the differences in the extraction protocols and matrices, the recovery range in this study, commercial tests, and other procedures were similar for all mycotoxins. Overall, the assay detected AFB1, DON, FUM, OTA, and ZEA in human serum with excellent accuracy, precision, and specificity.
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Fumonisinas , Micotoxinas , Zearalenona , Humanos , Micotoxinas/análisis , Fumonisinas/análisis , Zearalenona/análisis , Aflatoxina B1/análisis , Ensayo de Inmunoadsorción Enzimática , Contaminación de Alimentos/análisisRESUMEN
Currently, there are over 602 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and 6.4 million COVID-19 disease-related deaths worldwide. With ambitious vaccine strategies, reliable and accurate serological testing is needed to monitor the dynamics of the novel coronavirus pandemic and community immunity. We set out to improve serological testing of the immune response against SARS-CoV-2. We hypothesize that by multiplexing the serological diagnostic test kit (SARSPLEX) and screening for three antibodies, an even more robust diagnostic can be developed. A total of 293 sera were analyzed for IgM, IgG, or IgA immune reactions to the subunit 1 spike glycoprotein and the nucleocapsid protein in a standardized ELISA platform. Testing IgM, IgG, and IgA demonstrated high positive and negative agreements compared to RT-PCR and serology reference tests. Comparison with the pre-2019-CoV (n = 102) samples highlighted the specificity of this test kit and indicated that no unspecific binding, even with the summer flu patients (n = 44), was detected. In addition, SARSPLEX demonstrated to be a valuable occupational surveillance tool used in a functional medicine facility. With increased and broader testing, SARSPLEX will be a valuable tool in monitoring immunity and aid in prioritizing access to the SARS-CoV-2 vaccine for high-risk patients.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Vacunas contra la COVID-19 , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Glicoproteína de la Espiga del Coronavirus , Inmunoglobulina G , Inmunoglobulina M , Inmunoglobulina A , Sensibilidad y EspecificidadRESUMEN
Parvovirus B19 is a widespread virus with diverse clinical presentations. The viral nonstructural protein, NS1, binds to and cleaves the viral genome, and induces apoptosis when transfected into nonpermissive cells, such as hepatocytes. We hypothesized that the cytotoxicity of NS1 in such cells results from chromosomal DNA damage caused by the DNA-nicking and DNA-attaching activities of NS1. Upon testing this hypothesis, we found that NS1 covalently binds to cellular DNA and is modified by PARP, an enzyme involved in repairing single-stranded DNA nicks. We furthermore discovered that the DNA nick repair pathway initiated by poly(ADPribose)polymerase and the DNA repair pathways initiated by ATM/ATR are necessary for efficient apoptosis resulting from NS1 expression.
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Apoptosis/fisiología , Daño del ADN , Parvovirus B19 Humano/fisiología , Proteínas no Estructurales Virales/fisiología , Western Blotting , Línea Celular , Reparación del ADN , Humanos , Inmunoprecipitación , Proteínas no Estructurales Virales/genéticaRESUMEN
Lyme borreliosis is a multisystemic disease caused by the pleomorphic bacteria of the Borrelia burgdorferi sensu lato complex. The exact mechanisms for the infection to progress into a prolonged sequelae of the disease are currently unknown, although immune evasion and persistence of the bacteria in the host are thought to be major contributors. The current study investigated B. burgdorferi infection processes in two human cell lines, both non-immune and non-phagocytic, to further understand the mechanisms of infection of this bacterium. By utilizing light, confocal, helium ion, and transmission electron microscopy, borrelial infection of chondrosarcoma (SW1353) and dermal fibroblast (BJ) cells were examined from an early 30-min time point to a late 9-days post-infection. Host cell invasion, viability of both the host and B. burgdorferi, as well as, co-localization with lysosomes and the presence of different borrelial pleomorphic forms were analyzed. The results demonstrated differences of infection between the cell lines starting from early entry as B. burgdorferi invaded BJ cells in coiled forms with less pronounced host cell extensions, whereas in SW1353 cells, micropodial interactions with spirochetes were always seen. Moreover, infection of BJ cells increased in a dose dependent manner throughout the examined 9 days, while the percentage of infection, although dose dependent, decreased in SW1353 cells after reaching a peak at 48 h. Furthermore, blebs, round body and damaged B. burgdorferi forms, were mostly observed from the infected SW1353 cells, while spirochetes dominated in BJ cells. Both infected host cell lines grew and remained viable after 9 day post-infection. Although damaged forms were noticed in both cell lines, co-localization with lysosomes was low in both cell lines, especially in BJ cells. The invasion of non-phagocytic cells and the lack of cytopathic effects onto the host cells by B. burgdorferi indicated one mechanism of immune evasion for the bacteria. The differences in attachment, pleomorphic form expressions, and the lack of lysosomal involvement between the infected host cells likely explain the ability of a bacterium to adapt to different environments, as well as, a strategy for persistence inside a host.
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Human polymicrobial infections in tick-borne disease (TBD) patients is an emerging public health theme. However, the requirement for holistic TBD tests in routine clinical laboratories is ambiguous. TICKPLEX® PLUS is a holistic TBD test utilized herein to assess the need for multiplex and multifunctional diagnostic tools in a routine clinical laboratory. The study involved 150 specimens categorized into Lyme disease (LD)-positive (n = 48), LD-negative (n = 30), and febrile patients from whom borrelia serology was requested (n = 72, later "febrile patients") based on reference test results from United Medix, Finland. Reference tests from DiaSorin, Immunetics, and Mikrogen Diagnostik followed the two-tier LD testing system. A comparison between the reference tests and TICKPLEX® PLUS produced 86%, 88%, and 87% positive, negative, and overall agreement, respectively. Additionally, up to 15% of LD and 11% of febrile patients responded to TBD related coinfections and opportunistic microbes. The results demonstrated that one (TICKPLEX® PLUS) test can aid in a LD diagnosis instead of four tests. Moreover, TBD is not limited to just LD, as the specimens produced immune responses to several TBD microbes. Lastly, the study indicated that the screening of febrile patients for TBDs could be a missed opportunity at reducing unreported patient cases.
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Although insect herbivores are known to evolve resistance to insecticides through multiple genetic mechanisms, resistance in individual species has been assumed to follow the same mechanism. While both mutations in the target site insensitivity and increased amplification are known to contribute to insecticide resistance, little is known about the degree to which geographic populations of the same species differ at the target site in a response to insecticides. We tested structural (e.g., mutation profiles) and regulatory (e.g., the gene expression of Ldace1 and Ldace2, AChE activity) differences between two populations (Vermont, USA and Belchow, Poland) of the Colorado potato beetle, Leptinotarsa decemlineata in their resistance to two commonly used groups of insecticides, organophosphates, and carbamates. We established that Vermont beetles were more resistant to azinphos-methyl and carbaryl insecticides than Belchow beetles, despite a similar frequency of resistance-associated alleles (i.e., S291G) in the Ldace2 gene. However, the Vermont population had two additional amino acid replacements (G192S and F402Y) in the Ldace1 gene, which were absent in the Belchow population. Moreover, the Vermont population showed higher expression of Ldace1 and was less sensitive to AChE inhibition by azinphos-methyl oxon than the Belchow population. Therefore, the two populations have evolved different genetic mechanisms to adapt to organophosphate and carbamate insecticides.
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A clinical association between idiopathic liver disease and parvovirus B19 infection has been observed. Fulminant liver failure, not associated with other liver-tropic viruses, has been attributed to B19 in numerous reports, suggesting a possible role for B19 components in the extensive hepatocyte cytotoxicity observed in this condition. A recent report by Abe and colleagues (Int J Med Sci. 2007;4:105-9) demonstrated a link between persistent parvovirus B19 genotype I and III infection and fulminant liver failure. The genetic analysis of isolates obtained from these patients demonstrated a conservation of key amino acids in the nonstructural protein 1 (NS1) of the disease-associated genotypes. In this report we examine a conserved residue identified by Abe and colleagues and show that substitution of isoleucine 181 for methionine, as occurs in B19 genotype II, results in the reduction of B19 NS1-induced cytotoxicity of liver cells. Our results support the hypothesis that in the setting of persistent B19 infection, direct B19 NS1-induced cytotoxicity may play a role in idiopathic fulminant liver failure.
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Apoptosis/efectos de los fármacos , Parvovirus B19 Humano/genética , Parvovirus B19 Humano/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/toxicidad , Sustitución de Aminoácidos , Citometría de Flujo , Genotipo , Células Hep G2 , Humanos , Relación Estructura-Actividad , Proteínas no Estructurales Virales/químicaRESUMEN
The present study evaluates the role of graphene oxide's (GO's) peroxidase-like and inherent/carbocatalytic properties in oxidising silver nitrate (AgNO3) to create graphene nanocomposites with silver nanoparticles (GO/Ag nanocomposite). Activation of peroxidase-like catalytic function of GO required hydrogen peroxide (H2O2) and ammonia (NH3) in pH 4.0 disodium hydrogen phosphate (Na2HPO4). Carbocatalytic abilities of GO were triggered in pH 4.0 deionised distilled water (ddH2O). Transmission electron microscope (TEM), scanning electron microscope (SEM), cyclic voltammetry (CV) and UV-Vis spectroscopy aided in qualitatively and quantitatively assessing GO/Ag nanocomposites. TEM and SEM analysis demonstrated the successful use of GO's peroxidase-like and carbocatalytic properties to produce GO/Ag nanocomposite. UV-Vis analysis indicated a higher yield in optical density values for GO/Ag nanocomposites created using GO's carbocatalytic ability rather than its peroxidase-like counterpart. Additionally, CV demonstrated that GO/Ag nanocomposite fabricated here is a product of an irreversible electrochemical reaction. Our study outcomes show new opportunities for GO as a standalone catalyst in biosensing. We demonstrate a sustainable approach to obtain graphene nanocomposites exclusive of harmful chemicals or physical methods.
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There is insufficient evidence to support screening of various tick-borne diseases (TBD) related microbes alongside Borrelia in patients suffering from TBD. To evaluate the involvement of multiple microbial immune responses in patients experiencing TBD we utilized enzyme-linked immunosorbent assay. Four hundred and thirty-two human serum samples organized into seven categories followed Centers for Disease Control and Prevention two-tier Lyme disease (LD) diagnosis guidelines and Infectious Disease Society of America guidelines for post-treatment Lyme disease syndrome. All patient categories were tested for their immunoglobulin M (IgM) and G (IgG) responses against 20 microbes associated with TBD. Our findings recognize that microbial infections in patients suffering from TBDs do not follow the one microbe, one disease Germ Theory as 65% of the TBD patients produce immune responses to various microbes. We have established a causal association between TBD patients and TBD associated co-infections and essential opportunistic microbes following Bradford Hill's criteria. This study indicated an 85% probability that a randomly selected TBD patient will respond to Borrelia and other related TBD microbes rather than to Borrelia alone. A paradigm shift is required in current healthcare policies to diagnose TBD so that patients can get tested and treated even for opportunistic infections.
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Borrelia/inmunología , Coinfección/diagnóstico , Enfermedades por Picaduras de Garrapatas/diagnóstico , Área Bajo la Curva , Borrelia burgdorferi/inmunología , Borrelia burgdorferi/patogenicidad , Antígenos CD57/metabolismo , Coinfección/inmunología , Coinfección/microbiología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Curva ROC , Enfermedades por Picaduras de Garrapatas/inmunología , Enfermedades por Picaduras de Garrapatas/microbiologíaRESUMEN
Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.
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Borrelia burgdorferi is the causative agent of tick-borne Lyme disease. As a response to environmental stress B. burgdorferi can change its morphology to a round body form. The role of B. burgdorferi pleomorphic forms in Lyme disease pathogenesis has long been debated and unclear. Here, we demonstrated that round bodies were processed differently in differentiated macrophages, consequently inducing distinct immune responses compared to spirochetes in vitro. Colocalization analysis indicated that the F-actin participates in internalization of both forms. However, round bodies end up less in macrophage lysosomes than spirochetes suggesting that there are differences in processing of these forms in phagocytic cells. Furthermore, round bodies stimulated distinct cytokine and chemokine production in these cells. We confirmed that spirochetes and round bodies present different protein profiles and antigenicity. In a Western blot analysis Lyme disease patients had more intense responses to round bodies when compared to spirochetes. These results suggest that round bodies have a role in Lyme disease pathogenesis.
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Borrelia burgdorferi/citología , Borrelia burgdorferi/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Actinas/metabolismo , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Western Blotting , Borrelia burgdorferi/química , Citocinas/biosíntesis , Endocitosis , Humanos , Lisosomas/microbiología , Proteoma/análisisRESUMEN
A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.