| BackgroundSyphilis is a long-term chronic sexually transmitted disease caused by treponema pallidum(T.pallidum), which might even cause irreversible damage in some parenchymatous organs including nervous system and result in severe complications. Among them, neurosyphilis is one of the most complications. In recent years, the incidence of syphilis has rapidly increased and greatly threatened the public health with an estimated 11 million people infected each year, which is widely noticed in the world. Currently, what is especially worth noticing is the increasing rate of HIV and syphilis co-infection. Therefore, we will take effective measures to control the up trend in 5 years and reduce incidence of syphilis in 10 years, basically eliminate congenital syphilis and establish the preventive system for control of HⅣ and syphilis infection according to the programme for Moniloring and Control of Syphilis Infection from 2010 to 2020 years made by National Ministry of Health. The prevalence of neurosyphilis increases significantly on immune activation by HⅣ and syphilis infection which damage the mucosa and epithelial barrier. It is significant to deeply understand the epidemic characteristics of neurosyphilis among HⅣ-infected patients with syphilis in China because we can use it to guide the early diagnosis, the prevention and treatment of neurosyphilis.Currently, there is no gold standard for the diagnosis of neurosyphilis which the sensitivity and specificity were both high. It is generally accepted that a reactive Venereal Disease Research Laboratory (VDRL) test in cerebrospinal fluid uses as a diagnostic criteria of neurosyphilis. When the CSF-VDRL is negative, the diagnosis of neurosyphilis may be based on pleocytosis and/or elevated protein concentration in cerebrospinal fluid (CSF) collected by lumbar puncture. The diagnosis of neurosyphilis in HIV-infected patients with syphilis has more difficulties, because CSF pleocytosis is probably induced by HⅣ infection. Generally, HⅣ-infected patients with syphilis were advised to examine CSF to rule out neurosyphilis. Guidelines from the Centers for Disease Control and Prevention (CDC) recommended CSF examination in HⅣ-infected patients with syphilis associated with neurological or ocular symptoms or signs, or late latent syphilis, or syphilis of unknown duration, or serologic treatment failure. However, whether all HⅣ-infected patients with syphilis should be performed lumbar puncture is still controversial. It is important and great significant to explore the value clinical and laboratorial factors for the diagnosis of neurosyphilis and decide to do CSF examination. To our knowledge, there was no report on correlation between neurosyphilis and clinical or laboratorial features among HⅣ-infected males patients with syphilis in China.Although it is known that the immune status plays an important role in the clearance of pathogenic microorganism, an inappropriate immune response causes to inflammation and damage the human tissue including the central nervous system. Whether central nervous system infection caused by Tp has the similar mechanism is unclear. Microglia is the immune cells and low in the central nervous system. When the central nervous system suffered external injuries, infection and dysfunction, microglia was activated under the stimulation of inflammatory factors and participated in the pathogenesis of a variety of diseases of central nervous system (CNS) such as Alzheimer’s disease (AD). GPI in cognitive impairment caused by Treponema pallidum infection was similar with AD. When Treponema pallidum invades the central nervous system, resting microglia is activated and releases inflammatory mediators. Whether it causes neurotoxicity and leads to "secondary damage" is worthy of our attention.We use the technology of primary neural microglia culture to conduct above studies. Study on microglia involved in the pathogenesis of a variety of diseases of CNS depends on primary culture of microglia. Primary microglia culture in vitro was established by Gulian and McCarthy. But this method of the primary microglia culture has some limitations including multiple complicated steps, less amount of microglia and lower purity. Later, many methods of primary microglia culture have shown some improvement based on this method, but it still do not establish a stable and efficient standard method of primary microglia culture.ObjectivePart11. One objective of our present study is to investigate the prevalence and characteristics of neurosyphilis among HⅣ-infected male patients with syphilis in China.2. Another objective of our present study is to analyze the correlation between neurosyphilis and clinical or laboratorial features.Part21. We aim to establish a reliable and efficient method of primary microglia culture, which lays a foundation for the subsequent experiments.2. Preliminary exploration on the activation of microglia stimulated by Treponema pallidum.MethodsPart1Between January 2009 and August 2014, a total of 178 HⅣ-infected male patients with syphilis who had no history of neurosyphilis and underwent lumbar puncture to rule out neurosyphilis were enrolled in our study from the Shanghai Skin Disease Hospital in Shanghai, in China. Interviewer-administered interviews were conducted to collect information of social-demographic and behavioral characteristics. Venous blood and cerebrospinal fluid specimens were collected to test for syphilis and/or neurosyphilis. Correlation analysis of neurosyphilis and clinical and laboratory features of all participants was conducted.Part21. We used neonatal C57BL/6J mice for this study and approximately 8-16 pups can be processed at a time. ALL cortex tissues of neonatal mice were isolated and blew into single cells. Mixed glial cells were cultured in the complete culture medium. For subsequent medium changes once every 3 days,1/2 medium was removed from the culture dish and with the complete medium. At 10 days after plating, mixed glia cultures will be. The culture is ready to be shaken to obtain microglia. So we placed 25cm2 culture flask with mixed glia cells on the shaker for 2h at 260r.p.m at 37 ℃ to harvest microglia cells. Isolated microglia cells were calculated with flow cytometry after being stained with anti-mouse CD11b+ antibody (PE).2. The obtained microglia at 1×105 were inoculated in 6 wells of 12-well culture plate and cultured for 24 hours which were divided three groups:positive group, experimental group with 2 wells in each groups. After culture for 24 hours, we then added 1μg/ml LPS in positive group and 1×107 cells/ml Tp in experimental group for 24 hours. After digesting, the reactive microglia cells were collected and calculated with flow cytometry after being stained with anti-mouse CD11b+ and anti-mouse CD80 fluorescent antibody.ResultsPart11.178 eligible participants were males and the median age of participants was 31 years (range,18-75 years). More than half of participants (56.7%) were MSM. The number of patients with secondary syphilis was the highest among all cases composed 96(54%) cases of secondary syphilis,4(2.2%) case of primary syphilis,7(3.9%) of early latent syphilis and 71(39.8%) cases which includes patients with syphilis of unknown duration, late latent syphilis and tertiary syphilis. The prevalence of neurosyphilis was 25.0% in patients with secondary syphilis,14.3% in patients with early latent syphilis and 22.5% in patients with late syphilis including syphilis of unknown duration.2. Of all cases,41 (23.0%) cases met diagnostic criteria for neurosyphilis. Neurosyphlis was kept in significantly association with a peripheral blood CD4+ T cell count ≤350cells/μL(OR,8.08; 95% CI,2.12-30.78;P<0.05) and previous syphilis treatment over one year before entry (OR,5.97;95%CI,1.59-22.44; P＜0.05) (Table 3). There was no significant correlation between serum RPR titer ≥1:32, stage syphilis, neurologic manifestation, visual manifestation and neurosyphilis.Part21. We achieved a yield of 1 × 106 cells per 25cm2 culture flask and reached a purity of more than 88% after purification by the present improved method.2. The expression of CD80 in the negative control group, positive control group and the experimental group were 0.44%,0.79%,1.01%, respectively. The amount of CD80 in positive group was significantly higher than that in negative group (P<0.05). However, the activation of microglia stimulated by Treponema pallidum was not obvious.ConclusionsPart1We concluded that the peripheral blood CD4 T cell counts ≤350 cells /μl was a risk factor for neurosyphilis among HⅣ-infected male patients with syphilis in China. The single serum RPR titer of 1/32 might not be used as a cutoff point for lumbar puncture. Furthermore, identification and screening of sexual partners is important for controlling the incidence and prevalence of HⅣ-infected male patients with syphilis in China.Part21. We have established a reliable and efficient method of primary microglia culture and lay the foundation for the subsequent experiments.2. We concluded that resting microglia stimulated by LPS transformed into mature by expressed high levels of CD80. However, Treponema pallidum might not induce upregulation of the expression of co-stimulatory molecule CD80 on microglia. |
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