Comparative proteomics profiling revealed the involvement of GRB2-ROCK2 axis in Lyme neuroborreliosis caused by Borrelia Burgdorferi.

The zoonotic Lyme neuroborreliosis (LNB) disease is caused by Borrelia burgdorferi, with wide distribution, rapid dissemination and high disability rate. However, the molecular mechanism underlying B. burgdorferi mediated neuroborreliosis remains largely unknown. Here, the frontal cortex from rhesus brains was incubated with B. burgdorferi, and proteomics profiling was evaluated by isobaric tag for relative and absolute quantitation. Proteins were identified and quantified, and differentially expressed proteins (DEPs) were isolated by comparing co-cultured samples and control samples. A total of 43, 164 and 368 DEPs were significantly altered after 6, 12 and 24 h treatment with B. burgdorferi respectively. Gene ontology and KEGG pathway analyses revealed that chemokine biological process was significantly enriched. Two genes in chemokine pathway including GRB2 and ROCK2 were significantly up-regulated after B. burgdorferi co-culturing. By in vitro assay, we confirmed that the expression of GRB2 and ROCK2 was increased after B. burgdorferi infection. In conclusion, our study revealed the involvement of chemokine pathway in the pathogenesis of LNB. GRB2 and ROCK2 may be novel biomarkers and therapeutic targets for LNB.

Borrelia burgdorferi basic membrane protein A stimulates murine macrophage to secrete specific chemokines.

In this study, we investigated the mechanisms that lead to the production of proinflammatory mediators by the murine macrophage cell line, RAW264.7, when these cells are exposed in vitro to recombinant Borrelia burgdorferi basic membrane protein A (rBmpA). Using antibody protein microarray technology with high-throughput detection ability for detecting 25 chemokines in culture supernatant the RAW264.7 cell culture supernatants at 12 and 24 h post-stimulation with rBmpA, we identified two chemokines, a monocyte chemoattractant protein-5 (MCP-5/CCL12) and a macrophage inflammatory protein-2 (MIP-2/CXCL2), both of which increased significantly after stimulation. We then chose these two chemokines for further study. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction revealed that with the increase of rBmpA concentration, MCP-5/CCL12 and MIP-2/CXCL2 showed concentration-dependent increases (p <0.01).Our results indicate that the rBmpA could stimulate the secretion of several specific chemokines and induce Lyme arthritis.

Molecular Interactions During Borrelia burgdorferi Migration from the Vector to the Mammalian Nervous System.

Lyme disease (LD) is an infectious disease caused by the spirochetes of genus borrelia, which are transmitted by the ticks of the genus ixodes. LD is transmitted by the spirochete B. burgdorferi sensu lato. Once in contact with the host through a tick bite, the pathogen comes into contact with the host defense, and must escape this machinery to establish LD, thus using a large number of mechanisms involving the vector of the pathogen, the pathogen itself and also the host. The initial diagnosis of the disease can be made based on the clinical symptoms of LD and the disease can be treated and cured with antibiotics if the diagnosis is made early in the beginning of the disease. Contrariwise, if LD is left untreated, the pathogen disseminates throughout the tissues and organs of the body, where it establishes different types of disease manifestations. In the nervous system, the inflammation caused by B. burgdorferi is known as Lyme neuroborreliosis (LNB). LNB is one of the principal manifestations of LD. In this review, we systematically describe the different molecular interactions among B. burgdorferi, the vector (tick) and the mammalian host.

Isoforskolin and Cucurbitacin IIa promote the expression of anti-inflammatory regulatory factor SIGIRR in human macrophages stimulated with Borrelia burgdorferi basic membrane protein A.

Certain natural products, derived from medicinal plants, exhibit anti-inflammatory properties, but the mechanism of action of many remains unclear. Borrelia burgdorferi spirochetes are responsible for causing Lyme arthritis through activation of the Toll-like receptor (TLR) signaling pathway. In this study, we investigated the mechanisms by which Isoforskolin (ISOF) and Cucurbitacin IIa (CuIIa), compounds derived from Chinese herbs, can exert anti-inflammatory effects by modulating single immunoglobulin interleukin-1 receptor-related receptor (SIGIRR; also known as Toll/interleukin-1 receptor 8, TIR8) and thereby inhibiting B. burgdorferi basic membrane protein A (BmpA)-induced TLR signaling in human macrophages, specifically the THP-1 human monocytic cell line. After THP-1 cells were exposed in vitro to: i) recombinant (r)BmpA, ii) rBmpA and ISOF or iii) rBmpA and CuIIa, Cytotoxicity assay (Cell Counting Kit-8, CCK-8) are used to measure the effects of ISOF and CuIIa on cell viability. Meanwhile, real-time polymerase chain reaction and Western blotting were used to quantify SIGIRR mRNA and protein levels, respectively, at 6, 12, 24 and 48 h time points post-stimulation. In addition, proinflammatory cytokine tumor necrosis factor-α (TNF-α) was determined by ELISA analysis. Our study showed that rBmpA stimulation of THP-1 cells resulted in a drop in SIGIRR levels in THP-1 cells. More importantly, SIGIRR levels increased significantly in rBmpA-stimulated THP-1 cells following ISOF or CuIIa administration, and the results of ELISA analysis suggested that ISOF or CuIIa reduced the secretion of the proinflammatory cytokine TNF-α. In conclusion, These results reveal new possibilities for the treatment of Lyme arthritis.

Seroprevalence of Tick-Borne Anaplasma phagocytophilum Infection in Healthy Adult Population and Patients with Acute Undifferentiated Fever from the Yunnan Province of China.

Anaplasma phagocytophilum-the causative agent of human granulocytic anaplasmosis (HGA)-is a tick-borne pathogen transmitted by Ixodid ticks infecting wild and domestic mammals as well as humans. Despite the availability of evidence regarding this emerging infection among vectors, host animals, and individuals in China, there is limited knowledge on the prevalence and distribution of A. phagocytophilum in the Yunnan Province. The aim of this study was to assess the seroprevalence of A. phagocytophilum in healthy adults and patients with acute undifferentiated fever from four regions in the Yunnan Province. The enzyme-linked immunosorbent assay and indirect immunofluorescence assay were used to detect immunoglobulin (Ig) G and IgM antibodies against A. phagocytophilum in sera obtained from 1185 healthy blood donors and 245 patients with acute undifferentiated fever, respectively. Demographic variables were assessed as potential risk factors using the chi-squared test. The rates of seropositivity rates were 7.59% and 4.49% in healthy donors and fever patients, respectively. Analysis of risk factors such as gender, age groups, and place of residence showed statistically significant differences. Infections with A. phagocytophilum occur widely among individuals residing in southwestern China. Our results indicate that there is serological evidence of HGA in this population and presence of acute A. phagocytophilum infections in patients with undifferentiated fever in the Yunnan Province.

Genetic diversity and drug susceptibility patterns of the Mycobacterium tuberculosis complex in Yunnan, China.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) which has been threatening global public health for many years. High genetic diversity is dominant feature of Mtb. Increasing cases of multidrug-resistant (MDR) tuberculosis (MDR-TB) is a serious public health problem to TB control in China. Spontaneous mutations in the Mtb genome can alter proteins which are the target of drugs, making the bacteria drug resistant. The purpose of the present study was to analyze the genotype of Mtb isolates from some areas in Yunnan, China and explore the association between genotypes and MDR-TB. Using spoligotyping, we identified Beijing genotypes, six non-Beijing genotypes and a number of orphan genotypes from 270 Mtb isolates from patients in Yunnan Province during 2014-2016. Of 270 Mtb isolates, 102 clinical Mtb strains were identified as drug-resistant (DR) by drug susceptibility testing (DST), among them, 52 MDR strains. Beijing genotypes occupied the highest MDR proportion (78.85%) followed by the orphan genotypes (15.38%). The characteristics of MDR strains showed high genetic diversity. The results will help to efficiently improve diagnosis and treatment and provide valuable information for Mtb molecular epidemiology.

Borrelia burgdorferi basic membrane protein A initiates proinflammatory chemokine storm in THP 1-derived macrophages via the receptors TLR1 and TLR2.

Lyme disease, reffered to as Lyme borreliosis, is a tick-borne zoonotic disease caused by Borrelia burgdorferi spirochetes. Lyme arthritis, the most common, serious and harmful manifestation during the late stages of Lyme disease, is closely associated with the Borrelia burgdorferi basic membrane protein A (BmpA). Chemokines are also reported to have an important role in Lyme arthritis. Toll-like receptors (TLRs) recognize and bind to pathogen-associated molecules which are structurally conserved among microbes, to activate transcriptional events, including cytokine production, inflammation, and tissue damage. We speculated that BmpA could induce a storm of proinflammatory chemokines via TLRs and downstream moleculars, and that TLR1, TLR2, TLR5, TLR6 and the adaptor protein, MyD88, may be involved in this process. We explored this hypothesis using the human monocytic leukemia cell line, THP-1, and recombinant BmpA (rBmpA). Cell surface TLR1 and TLR2 were neutralized using specific antibodies before stimulation with rBmpA and analysis of chemokine secretion using a chemokine chip. Further, the expressions level of the four TLRs and MyD88 were analyzed following stimulation with rBmpA. Stimulation with rBmpA resulted in elevated levels of seven cytokines. Further, TLR1 and TLR2 antibody treated cells exhibited an overall reduction in rBmpA-induced chemokine expression. TLR1, TLR2, and MyD88 expression levels (both mRNA and protein) increased after stimulation with rBmpA. Our data confirm that TLR1, TLR2, and MyD88 are involved in BmpA-induced proinflammatory chemokines, which may be closely involved in Lyme arthritis pathogenesis.

Immunogenicity of different dosing schedules of the human live attenuate rotavirus vaccine (RV1) in infants and children: a meta-analysis.

Rotavirus immunization strategies have become part of a comprehensive global public health program to control rotavirus-associated gastroenteritis, particularly in infants and children in developing countries. Several studies have reported the efficacy of different rotavirus vaccine dosing schedules, but with mixed findings. Therefore a systematic review of the published literature on rotavirus vaccination dosing schedules using the live attenuated RV1 rotavirus vaccine in infants and children, including randomized controlled clinical trials (RCTs), published between January 1998 to January 2018 was conducted, with meta-analysis of the published data. The literature search was performed using six databases. The initial review identified 495 publications, of which three satisfied the selection eligibility criteria. The three studies that assessed RV1 rotavirus vaccine immunogenicity compared a two-dose vaccination schedule with a three-dose vaccination schedule. The use of a three-dose vaccination schedule did not show a statistically significant seroconversion rate when compared with a two-dose vaccination schedule (OR = 0.87; 95% CI,: 0.65--1.17;, p- = 0.298). Analysis of included studies with one-month follow-up time showed that the three-dose vaccination schedule did not result in have significantly increased geometric mean concentrations (GMCs) compared with the two-dose vaccination schedule (p = 0.311).Rotavirus immunogenicity did not increase significantly with the three-dose schedule at 6, 10 and 14 weeks with the two-dose schedule at 10 and 14 weeks. These findings indicate that further controlled studies should be undertaken to support the optimum immunization schedules for rotavirus in terms of clinical effectiveness and cost-effectiveness, particularly for infants and children in developing countries.

Immunogenicity and Safety of the M72/AS01E Candidate Vaccine Against Tuberculosis: A Meta-Analysis.

Background: Currently, there is no tuberculosis (TB) vaccine recommended for use in latent TB infections and healthy adults. M72/AS01E is a new peptide vaccine currently under development, which may improve protection against TB disease. This vaccine has been investigated in several phase I/II clinical trials. We conducted a meta-analysis to clarify the immunogenicity and safety of the M72/AS01E peptide vaccine. Methods: We searched the PubMed, Embase, and Cochrane Library databases for published studies (until December 2018) investigating this candidate vaccine. A meta-analysis was performed using the standard methods and procedures established by the Cochrane Collaboration. Results: Seven eligible studies-involving 4,590 participants-were selected. The analysis revealed a vaccine efficacy was 57.0%, significantly higher abundance of polyfunctional M72-specific CD4+ T cells [standardized mean difference (SMD) = 2.58] in the vaccine group vs. the control group, the highest seropositivity rate [relative risk (RR) = 74.87] at 1 month after the second dose of vaccination (Day 60), and sustained elevated anti-M72 IgG geometric mean concentration at study end (Day 210) (SWD = 4.94). Compared with the control, participants who received vaccination were at increased risk of local injection site redness [relative risk (RR) = 5.99], local swelling (RR = 7.57), malaise (RR = 3.01), and fatigue (RR = 3.17). However, they were not at increased risk of headache (RR = 1.57), myalgia (RR = 0.97), and pain (RR = 3.02). Conclusion: The M72/AS01E vaccine against TB is safe and effective. Although the vaccine is associated with a mild adverse reaction, it is promising for the prevention of TB in healthy adults.

Using cytometric bead arrays to detect cytokines in the serum of patients with different types of pulmonary tuberculosis.

Cytokines play a crucial role in mediating immune responses to tuberculosis (TB). The aim of this study was to evaluate the levels of cytokines in patients with different forms of pulmonary tuberculosis (PTB) and identify valuable cytokine biomarkers for the diagnosis of PTB. We measured the levels of six cytokines (interleukin (IL-2, IL-4, IL-6, and IL-10), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ)) in the serum of healthy donors (n = 30). Patients with active PTB (n = 46) and those with latent tuberculosis infection (LTBI, n = 38) were examined using cytometric bead arrays. The levels of the six cytokines in the serum samples were measured promptly, sensitively, and simultaneously. The levels of IL-2, IL-6, IL-10, and IFN-γ were significantly higher in the PTB group compared with those reported in the healthy donors ( P < 0.01 or P < 0.05). In addition, significantly higher levels of IL-2, IL-6, IL-10, and IFN-γ were found in the active PTB group compared with those observed in the LTBI group ( P < 0.01 or P < 0.05). However, the levels of IL-4 and TNF-α in the sera of patients from the PTB group did not show a significant correlation with those measured in the healthy donor group. Our data demonstrated that IL-2, IL-6, IL-10, and IFN-γ may be useful in the auxiliary diagnosis of tuberculosis and as biomarkers for distinguishing LTBI from TB.

Rhesus Brain Transcriptomic Landscape in an ex vivo Model of the Interaction of Live Borrelia Burgdorferi With Frontal Cortex Tissue Explants.

Lyme neuroborreliosis (LNB) is the most dangerous manifestation of Lyme disease caused by the spirochete Borrelia burgdorferi which can reach the central nervous system most commonly presenting with lymphocytic meningitis; however, the molecular basis for neuroborreliosis is still poorly understood. We incubated explants from the frontal cortex of three rhesus brains with medium alone or medium with added live Borrelia burgdorferi for 6, 12, and 24 h and isolated RNA from each group was used for RNA sequencing with further bioinformatic analysis. Transcriptomic differences between the ex vivo model of live Borrelia burgdorferi with rhesus frontal cortex tissue explants and the controls during the progression of the infection were identified. A total of 2249, 1064, and 420 genes were significantly altered, of which 80.7, 52.9, and 19.8% were upregulated and 19.3, 47.1, 80.2% were downregulated at 6, 12, and 24 h, respectively. Gene ontology and KEGG pathway analyses revealed various pathways related to immune and inflammatory responses during the spirochete infection were enriched which is suggested to have a causal role in the pathogenesis of neurological Lyme disease. Moreover, we propose that the overexpressed FOLR2 which was demonstrated by the real-time PCR and western blotting could play a key role in neuroinflammation of the neuroborreliosis based on PPI analysis for the first time. To our knowledge, this is the first study to provide comprehensive information regarding the transcriptomic signatures that occur in the frontal cortex of the brain upon exposure to Borrelia burgdorferi, and suggest that FOLR2 is a promising target that is associated with neuroinflammation and may represent a new diagnostic or therapeutic marker in LNB.

Macrophage migration inhibitory factor-794 CATT microsatellite polymorphism and risk of tuberculosis: a meta-analysis.

Tuberculosis (TB) is a chronic infectious disease that has been threatening public health for many years. Several studies have shown the relationship between the macrophage migration inhibitory factor (MIF)-794 CATT (MIF-794 CATT) microsatellite polymorphism and susceptibility to TB. However, the results remain inconclusive. Therefore, we aim to find out the impact of MIF-794 CATT microsatellite polymorphism on risk of TB by a comprehensive meta-analysis. We conducted a systematic study search in PubMed, Embase, the Cochrane Library, and the China National Knowledge Infrastructure (CNKI) up to October 2017. Five studies involving 836 cases and 678 controls were included in the current meta-analysis. We calculated the pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) to estimate the association between the MIF-794 CATT microsatellite polymorphism and risk of TB. The reliability of the results were evaluated with trial sequential analysis (TSA). The results suggested that the MIF-794 CATT microsatellite polymorphism was significantly associated with the susceptibility of TB in all comparisons for allele (7 + 8 compared with 5 + 6, OR = 1.56, 95% CI = 1.31-1.87, P<0.00001) and genotype (7/X + 8/X compared with 5/X + 6/X, OR = 1.81, 95% CI = 1.39-2.36, P<0.0001). Therefore, the meta-analysis indicated the MIF-794 allele CATT7 and CATT8 may be a risk factor to increase the susceptibility of TB, which was confirmed by TSA.

Application of droplet digital PCR to detect the pathogens of infectious diseases.

Polymerase chain reaction (PCR) is a molecular biology technique used to multiply certain deoxyribonucleic acid (DNA) fragments. It is a common and indispensable technique that has been applied in many areas, especially in clinical laboratories. The third generation of polymerase chain reaction, droplet digital polymerase chain reaction (ddPCR), is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify DNA. Droplet digital polymerase chain reaction is now widely used in low-abundance nucleic acid detection and is useful in diagnosis of infectious diseases. Here, we summarized the potential advantages of droplet digital polymerase chain reaction in clinical diagnosis of infectious diseases, including viral diseases, bacterial diseases and parasite infections, concluded that ddPCR provides a more sensitive, accurate, and reproducible detection of low-abundance pathogens and may be a better choice than quantitative polymerase chain reaction for clinical applications in the future.

Chemokine CXC Ligand 13 in Cerebrospinal Fluid Can Be Used as an Early Diagnostic Biomarker for Lyme Neuroborreliosis: A Meta-Analysis.

Lyme neuroborreliosis (LNB), which is the most common neurological manifestation of Lyme disease (LD), seriously impairs both the central and peripheral nervous systems. Current LNB diagnostic methods and criteria are not very effective. Recently, several studies have indicated that a high concentration of the chemokine CXC ligand 13 (CXCL13) in cerebrospinal fluid (CSF) could be used as a new biomarker for the diagnosis of LNB. Thus, we carried out a meta-analysis to systematically analyze the data from these studies to evaluate the value of CXCL13 as an LNB biomarker. After searching for articles in several databases, including PubMed, Embase, the Cochrane Library, and the China National Knowledge Infrastructure (CNKI), we included 7 articles in the meta-analysis with a total of 1299 patients with LNB or other neuroinflammatory diseases. From these 1299 patients, 343 patients with LNB served as the experimental group and 956 patients with other neuroinflammatory diseases or healthy individuals served as the control group. The analyses were performed using Meta-Disc1.4 statistical software. Based on the pooled specificity, sensitivity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and summary receiver operating characteristic curve, we found that CXCL13 indeed has a high sensitivity and specificity for diagnosing LNB, which means that it can be used as a new diagnostic biomarker for the diagnosis of LNB.