Correlation of cytokine level with the severity of severe fever with thrombocytopenia syndrome.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) was an emerging hemorrhagic fever that was caused by a tick-borne bunyavirus, SFTSV. Although SFTSV nonstructural protein can inhibit type I interferon (IFN-I) production Ex Vivo and IFN-I played key role in resistance SFTSV infection in animal model, the role of IFN-I in patients is not investigated. METHODS: We have assayed the concentration of IFN-α, a subtype of IFN-I as well as other cytokines in the sera of SFTS patients and the healthy population with CBA (Cytometric bead array) assay. RESULTS: The results showed that IFN-α, tumor necrosis factor (TNF-α), granulocyte colony-stimulating factor (G-CSF), interferon-γ (IFN-γ), macrophage inflammatory protein (MIP-1α), interleukin-6 (IL-6), IL-10, interferon-inducible protein (IP-10), monocyte chemoattractant protein (MCP-1) were significantly higher in SFTS patients than in healthy persons (p < 0.05); the concentrations of IFN-α, IFN-γ, G-CSF, MIP-1α, IL-6, and IP-10 were significant higher in severe SFTS patients than in mild SFTS patients (p < 0.05). CONCLUSION: The concentration of IFN-α as well as other cytokines (IFN-γ, G-CSF, MIP-1α, IL-6, and IP-10) is correlated with the severity of SFTS, suggesting that type I interferon may not be significant in resistance SFTSV infection in humans and it may play an import role in cytokine storm.

[Correlation Study on Chinese Medical Syndrome Types of Chronic Atrophic Gastritis Patients, Hp, and IL-1ß Polymorphism].

OBJECTIVE: To explore the correlation between Chinese medical (CM) syndrome types of chronic atrophic gastritis (CAG) patients and Helicobacter pylori (Hp) infection, polymorphisms of IL-1B, and IL-1ß. METHODS: Totally 192 CAG patients and 202 healthy subjects (as the healthy control group) were recruited in this case-control study. The Hp infection was tested by 13C-urea breath test and colloidal gold-labeled assay (GICA). The concentration of peripheral blood IL-1ß was measured by ELISA. The polymorphisms of IL-1B gene in the promoter region were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Pi-Wei weakness syndrome (PWWS) was dominant in CAG patients (31.77%, 61/192 cases). The Hp infection ratio in CAG patients was 53.65% (103/192 cases), of which, Pi-Wei damp-heat syndrome(PWDHS, 64.86%, 24/37 cases) and Gan-Wei disharmony syndrome (GWDS, 66.67%, 24/36 cases) were dominant. Compared with the health control group, the plasma concentration of IL-1ß was obviously elevated in CAG patients with PWDHS, GWDS, and static blood obstructing collaterals syndrome (SBOCS) (all P < 0.05). Additionally, there was no difference in the distribution of polymorphisms in the promoter region of IL-1 B gene between the CAG patients and the healthy control group (P > 0.05). CONCLUSIONS: The incidence risk of CAG was not associated with IL-1B polymorphism. But CM syndrome types of CAG patients was associated with Hp infection and peripheral blood IL-1ß levels.

[Effects of pigment epithelial derived factor gene on growth of lung cancer cell and neovascularization: experiments with lung cancer cells and chick embryos].

OBJECTIVE: To investigate the effect of pigment epithelium derived factor (PEDF) on the growth of lung cancer cells and the cancer-related neovascularization. METHODS: The full length of human PEDF gene was amplified by polymerase chain reaction (PCR). Human lung cancer cells of the line SKEMS1 were cultured and transfected with PEDF(exp), An eukaryotic expression vector constructed by recombinant DNA technology, so as to construct the SKMES1(PEDFexp) cells over-expressing PEDF protein. RT-PCR and Western blotting were used to confirm the mRNA and protein expression of PEDF in these cells. Another SKMES1 lung cancer cells were transfected with blank plasmids (SKMES1(pEF/His) cells). The SKMES1 cells not transfected were called SKMES1(WT) cells. The 3 kinds of SKMES1 cells were inoculated in the chorio-allantoic membrane (CAM) of chick embryo hatched for 7 days respectively. The size and weigh of the tumor were measured. The vessels density was examined. RESULTS: The tumor volume of the SKMES1(PEDFexp) group was (0.10 +/- 0.05) cm(3), significantly smaller than that of the control group [(0.17 +/- 0.07) cm(3), P = 0.016], and the mass of the SKME(SPEDFexp) group was (0.008 +/- 0.004) mg, significantly smaller than that of the control group too [(0.024 +/- 0.009) mg, P = 0.006]. The amount of first class neo-vessels of the SKMES1(PEDFexp) group was (15 +/- 3), significantly fewer than that of the control group [(41 +/- 9), P < 0.001]. The amount of second class neo-vessels of the SKMES1(PEDFexp) group was (75 +/- 22), also significantly fewer than that of the control group [(175 +/- 39), P = 0.001]. CONCLUSION: Inhibiting the growth of lung cancer cells and neovascularization, PEDF protein may be used as a potential biological drug to treat lung cancer.

Association of genetic variations in IL-6/IL-6R pathway genes with gastric cancer risk in a Chinese population.

Interleukin-6 (IL-6) and its receptor (IL-6R) were regarded to be responsible for the occurrence of gastric cancer for their regulation roles in the inflammation. The genetic variations in these two genes (IL-6: rs6949149, rs1800796, rs10499563 and IL-6R: rs2228145) have been suggested to be associated with gastric cancer risk. However, the published results were inconsistent among subjects of different ethnicity. To evaluate such an association in Chinese population, we carried out this case-control study based on 473 patients with gastric cancer and 474 healthy controls, whose genotypes were detected by the Sequenom MassARRAY platform, and Helicobacter pylori infection was assessed by immunogold testing kit. This study showed that rs1800796 CG genotype was associated with decreased risk of gastric cancer (adjusted OR=0.75, 95% CI: 0.57-0.99, p=0.043). The stratified analysis revealed that, in the Helicobacter pylori negative infection subgroup, rs2228145 AC (adjusted OR=0.64, 95% CI: 0.42-0.97) and AC/CC (adjusted OR=0.66, 95% CI: 0.45-0.99) genotypes were associated with decreased risk of gastric cancer, respectively. In contrast, in the Helicobacter pylori positive infection subgroup, rs10499563 TC (adjusted OR=0.64, 95% CI: 0.43-0.95), CC (adjusted OR=0.35, 95% CI: 0.14-0.90), TC/CC (adjusted OR=0.59, 95% CI: 0.40-0.87) genotype were associated with decreased gastric cancer risk, respectively. Moreover, in the male subgroup, rs1800796 CG (adjusted OR=0.61, 95% CI: 0.44-0.84) and CG/GG (adjusted OR=0.67, 95% CI: 0.49-0.91) genotype were associated with decreased risk of gastric cancer, respectively. In short, this study suggested that IL-6R rs2228145 and IL-6 rs10499563 genotype were associated with decreased risk of gastric cancer for the individuals with negative and positive Helicobacter pylori infection.

Clinical analysis of 23 cases of steroid-associated osteonecrosis of the femoral head with normal initial magnetic resonance imaging presentation.

To explore the clinical characteristics of steroid-associated osteonecrosis of the femoral head (ONFH) presenting initially normal magnetic resonance imaging (MRI) results.This retrospective study examined data from 23 cases that suffered from ONFH but presented a normal image at the first MRI examination after corticosteroid therapy from June 2005 to December 2013. Data on protopathy, age, sex, time of pain onset, MRI examination, and initial diagnosis were collected and analyzed.Average time from steroid therapy to first MRI examination was 45.7 ±â€Š25.5 days (range, 10-94 days). Average time to final diagnosis was 199.9 ±â€Š165.8 days (range, 32-762 days). Of the 23 cases, 21 cases complained of discomfort and were misdiagnosed because of a normal initial MRI scan. Twelve hips progressed to collapse and 1 hip received lumbar discectomy when got the final diagnosis. Cases with continuous pain (9/21) presented with pain at a later time than those with intermittent pain (12/21), although the continuous pain cases were diagnosed earlier.MRI performed 2 to 3 months after steroid therapy may present normal images. Another MRI examination is necessary to make a definite diagnosis.

Screening and identification of proteins interacting with nucleostemin.

AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS. METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and beta-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially. RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A. CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

[Establishment of a stable and regulable 293 cell line expressing mycoplasma hyorhinis protein P37].

OBJECTIVE: To establish a stable cell line, which can express P37 protein of mycoplasma hyorhinis and be regulated by tetracycline, for investigating the effect of p37 on phenotype of cells and its mechanism. METHODS: Recombinant plasmid PcDNA5/FRT/TO-p37 was constructed and cotransfected with pOG44 into Flp-In-T-REx-293 cells by lipofectamine. Positive clones were screened with Hygromycin and Blasticidin. RT-PCR and Western blot were used to exam the mRNA and protein expression in selected clones. The expression level at different inducing times and concentrations of tetracycline were examined. MTT assay was used to observe the effect of P37 on proliferation of 293 cells. RESULTS: P37 protein, which is 43.5x10(3), was expressed in the selected clone as well as secreted from cells. Tetracycline showed a good regulation on the expression of P37 protein, which was not detectable without tetracycline induction. When induced with 2 mg/L tetracycline for 60 hours, the P37 protein expression reached maximum level. Cell growth was promoted after being transfected with p37. CONCLUSION: A stable cell line expressing P37 regularly was established, which provides a good cell model for studying p37 function and its molecular mechanism.

[Characterization of the interaction between low density lipoprotein receptor-related protein-associated protein 1 and the cancer and embryo expression protein 65].

OBJECTIVE: To understand the mechanism of cancer and embryo expression protein 65 (CEP65) in cancer development. METHODS: CEP65 was used as a bait protein to isolate the partners of CEP65 from a human placenta cDNA library with yeast two hybrid system. The DNA fragments from positive clone were expressed prokaryotic and mammalian cells respectively, the GST pull-down experiment was performed to ascertain the binding activity. RESULTS: After screening a human placenta cDNA library, the low density lipoprotein receptor-related protein-associated protein 1 (RAP) was isolated and shown to interact with CEP65. GST pull-down assay results indicated that His-CEP65 and GST-RAP expressed by prokaryotic system were immunoprecipitated, and so were Myc-RAP and GST-P65 expressed by mammalian cells. CONCLUSION: RAP can interact with CEP65 and RAP may be a candidate to mediate the function of CEP65.

[Effect of the pre-hospital systematic treatment on prognosis patients of with severe acute organophosphorus pesticide poisoning].

OBJECTIVE: To investigate if the duration from poisoning to treatment (no treatment period) is related to the prognosis of patients with severe acute organophosphorus pesticide poisoning (SAOPP). METHODS: One hundred and seventy-four patients with the pre-hospital systematic treatment served as the treatment group while 160 patients going to the hospital by themselves without treatment or rejecting gastrolavage served as the control group. Patients in both groups were treated by gastrolavage, pralidoxime chloride, atropine and other expectant treatment. The duration of no treatment period, death, and severe complication were observed. The time of disappearance of symptoms, the recovery time of acetyl cholinesterase (AChE), atropinization time, atropine dosage, pralidoxime chloride dosage, naloxone dosage, hospitalization days and other targets were also observed. RESULTS: The duration of no treatment period in treatment group [(1.2 +/- 0.3) h] was significantly shorter than that in control group [(2.8 +/- 0.5) h, (P < 0.01)]. The mortality rate in treatment group was 6.32% while that in control group 22.5% (P < 0.01). The incidence of respiratory failure, heart injury, brain injury, atropine poisoning, intermediate syndrome, liver injury in treatment group (12.64%, 5.75%, 8.62%, 1.72%, 4.60%, 5.17% respectively) were lower than those in control group (25.63%, 13.75%, 17.50%, 6.25%, 7.50%, 9.38% respectively, P < 0.05 or P < 0.01). The time of symptoms disappearance, the recovery time of AChE, atropinization time, atropine dosage, pralidoxime chloride dosage, naloxone dosage, hospitalization days in treatment group were significantly superior to those in control group (P < 0.05 or P < 0.01). CONCLUSION: The pre-hospital systematic treatment can improve the prognosis of the patients with SAOPP, which is worth popularizing and using.

L-selectin and E-selectin expressed on monocytes mediating Ehrlichia chaffeensis attachment onto host cells.

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is an obligatory intracellular bacterium that exhibits monocytic host cell tropism. Ehrlichiae must enter the host cell, and then establish infection. The tropism of E. chaffeensis for monocytes suggests that the cell contains some specific surface components that mediate E. chaffeensis attachment and entry into host cells. In this study, host cell surface components that play a role in ehrlichial attachment were identified using a human monocyte/macrophage cell line, THP-1. E. chaffeensis attachment to THP-1 cells was partially blocked in the presence of antibodies to E-selectin and L-selectin, but not by antibodies to P-selectin, integrin alpham, integrin alphax, or normal mouse IgG as determined by real time polymerase chain reaction. Conversely, in HeLa cells that do not exhibit surface expression of E-selectin and L-selectin, antibodies to these cell surface proteins did not inhibit E. chaffeensis attachment. These findings indicate that E-selectin and L-selectin are cell surface proteins that might act as co-receptors and contribute to E. chaffeensis attachment and entry into THP-1.

Experimental Ehrlichia chaffeensis infection in beagles.

A canine model for human monocytic ehrlichiosis was used to assess persistent infection and antigenic variation of Ehrlichia chaffeensis. Two beagle dogs were infected subcutaneously with E. chaffeensis Arkansas strain. The dogs were observed for 6 months after inoculation for clinical signs, blood chemistry changes, antibodies to E. chaffeensis and presence of E. chaffeensis in the blood. Both dogs developed thrombocytopenia, but exhibited normal body temperatures during the entire course of infection. In one dog, E. chaffeensis was cultivated for up to 74 days post-inoculation and E. chaffeensis DNA was detected in the dog's blood for up to 81 days. In the other dog, E. chaffeensis was cultured for up to 102 days and E. chaffeensis DNA was detected in the blood for up to 117 days. PCR amplification and DNA sequence analysis indicated that there was no genetic variation in the 120 kDa outer-membrane glycoprotein gene of E. chaffeensis during infection of the dogs. The dogs developed antibodies to the immunodominant proteins of E. chaffeensis, including the 175, 140, 120, 80, 50 and 28 kDa proteins, starting in the fifth week post-inoculation. The dogs maintained high antibody titres throughout the 6-month study period. These results indicate that dogs become carriers of E. chaffeensis for 2-4 months after infection without exhibiting signs of clinical disease, suggesting that dogs may serve as a natural host for E. chaffeensis.

Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells.

Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.

The developmental cycle of Ehrlichia chaffeensis in vertebrate cells.

Ehrlichia chaffeensis, an obligatory intracellular bacterium, has two forms in mammalian cells: small dense-cored cells (DC) with dense nucleoid and larger reticulate cells (RC) with uniformly dispersed nucleoid. We have determined by electron microscopy that DC but not RC attaches to and enters into the host cells and RC but not DC multiples inside the host cells. Analysis of outer membrane protein expression by confocal microscopy showed that RC expressed the 28 kDa outer membrane protein (p28), the intermediate form, which were transforming from RC to DC, expressed both gp120 and p28, and the mature DC expressed gp120 only. The TCID50 of DC is 6 log10 higher than RC. We conclude that E. chaffeensis has a developmental cycle, in which the DC attaches to and enters into the host cells, and transforms into RC and the RC multiplies by binary fission for 48 h and then matures into DC at 72 h.

A mutation inactivating the methyltransferase gene in avirulent Madrid E strain of Rickettsia prowazekii reverted to wild type in the virulent revertant strain Evir.

Rickettsia prowazekii Madrid E (E) strain is an effective vaccine, but can revert to virulent status when passaged in animals. The aim of this study is to identify the reverse mutation that may determine the virulence of R. prowazekii by comparing the genetic structures of E strain and its virulent revertant Evir strain. We determined that the gene (Rp028/Rp027) encoding the methyltransferase was mutated by frameshift in avirulent E strain but not in virulent revertant Evir strain and wild type virulent Breinl strain. We conclude that the mutation in the E strain gene reverts to wild type in the virulent revertant Evir strain. Whether the mutation plays an essential role in the attenuation of E strain needs to be further investigated.

Lipoprotein p37 from Mycoplasma hyorhinis inhibiting mammalian cell adhesion.

p37 protein is a membrane lipoprotein of Mycoplasma hyorhinis, and our previous work showed that there was high ratio of M. hyorhinis infection in human gastric carcinoma. To investigate the possible functions of p37 in cancer development, the nucleotide sequence of p37 gene was modified and expressed well in transfected cells. We found that p37 localized at the Golgi apparatus and could be secreted out of the cell. Human gastric cancer cells AGS, after being transfected with the p37 gene, were smaller, more spherical and easy to detach from each other. Their adhesion to matrix was also diminished and cytoskeleton in these stable p37 AGS cell was rearranged and transcription co-factor beta-actin was transferred to nucleolus with down-regulation of ICAM-1 and integrin beta1. These findings will be helpful for us to elucidate the effects of p37 on eukaryotic cells as well as to better understand the potential relationship between cancer and mycoplasma infection.

Expression of members of the 28-kilodalton major outer membrane protein family of Ehrlichia chaffeensis during persistent infection.

The 28-kDa immunodominant outer membrane proteins (P28 OMPs) of Ehrlichia chaffeensis are encoded by a multigene family. As an indirect measure of the in vivo expression of the members of the p28 multigene family of E. chaffeensis, sera from two beagle dogs experimentally infected with E. chaffeensis were evaluated for the presence of specific antibodies to P28 OMPs by enzyme-linked immunosorbent assay. Antigenic peptides unique to each of the P28s were identified within the first hypervariable region of each P28 OMP. Serological responses to peptides derived from all P28 OMPs were detected from day 30 postinoculation to day 468 and from day 46 until day 159 in the two beagles. Although antibody titers to the peptides fluctuated, the peak response to all of the peptides appeared simultaneously in each dog. The antibody responses to another outer membrane protein of E. chaffeensis (GP120) showed similar temporal and quantitative changes. These data suggest that the P28 OMPs are expressed concurrently during persistent Ehrlichia infection.

Survival strategy of obligately intracellular Ehrlichia chaffeensis: novel modulation of immune response and host cell cycles.

Ehrlichia chaffeensis is an obligatory intracellular bacterium which resides in an early endosome in monocytes. E. chaffeensis infection in a human monocyte cell line (THP1) significantly altered the transcriptional levels of 4.5% of host genes, including those coding for apoptosis inhibitors, proteins regulating cell differentiation, signal transduction, proinflammatory cytokines, biosynthetic and metabolic proteins, and membrane trafficking proteins. The transcriptional profile of the host cell revealed key themes in the pathogenesis of Ehrlichia. First, E. chaffeensis avoided stimulation of or repressed the transcription of cytokines involved in the early innate immune response and cell-mediated immune response to intracellular microbes, such as the interleukin-12 (IL-12), IL-15, and IL-18 genes, which might make Ehrlichia a stealth organism for the macrophage. Second, E. chaffeensis up-regulated NF-kappaB and apoptosis inhibitors and differentially regulated cell cyclins and CDK expression, which may enhance host cell survival. Third, E. chaffeensis also inhibited the gene transcription of RAB5A, SNAP23, and STX16, which are involved in membrane trafficking. By comparing the transcriptional response of macrophages infected with other bacteria and that of macrophages infected with E. chaffeensis, we have identified few genes that are commonly induced and no commonly repressed genes. These results illustrate the stereotyped macrophage response to other pathogens, in contrast with the novel host response to obligate intracellular Ehrlichia, whose survival depends entirely on a long evolutionary process of outmaneuvering macrophages.