Hemofagocitose em cultura de sangue periférico precedendo hemofagocitose em biópsia de medula óssea na síndrome hemofagocítica secundária / Hemophagocytosis in peripheral blood culture preceding hemophagocytosis in bone marrow biopsy in secondary hemophagocytic syndrome

A síndrome hemofagocítica é determinada por desregulação do sistema imunológico, caracterizada por ativação excessiva de macrófagos, resultando em fagocitose de células sanguíneas normais no fígado, baço e medula óssea. Pode ser primária (genética) ou secundária (adquirida). Em adultos quase sempre é secundária, tendo infecções, neoplasias e doenças autoimunes como frequentes desencadeadores. Entre as principais manifestações da síndrome estão febre prolongada e hepatoesplenomegalia. O diagnóstico até o momento é confirmado pelo achado de hemofagocitose em biópsia de medula óssea. Entretanto, é descrito que a biópsia de medula óssea é normal nos primeiros dias de manifestações da síndrome. O presente relato tem como objetivo mostrar a observação de hemofagocitose em cultura de células de sangue periférico de paciente de 29 anos precedendo a hemofagocitose em biópsia de medula óssea. A paciente apresentava diferentes infecções, com grave comprometimento do estado geral e sem melhora com o tratamento das infecções. O achado laboratorial permitiu o tratamento precoce da síndrome hemofagocítica e a melhora da paciente. No presente relato a técnica utilizada está descrita detalhadamente para que possa ser reproduzida, além de ser apresentada uma revisão não sistemática da literatura sobre a síndrome.

Hemophagocytic syndrome, which is caused by dysregulation of the immune system, is characterized by excessive macrophage activation, resulting in phagocytosis of normal blood cells in the liver, spleen, and bone marrow. It can be primary (genetic) or secondary (acquired). In adults, it is almost always secondary, with infections, neoplasms, and autoimmune diseases as frequent triggers. The main manifestations of this syndrome are prolonged fever and hepatosplenomegaly. Currently, diagnosis is confirmed through finding hemophagocytosis in a bone marrow biopsy. However, it has been reported that bone marrow biopsy results are still normal on the first day the syndrome manifests. Here we report observing hemophagocytosis in cultured peripheral blood cells from a 29-year-old patient prior to finding hemophagocytosis in bone marrow biopsy. The patient had various infections and a poor general condition, which did not improve after treating the infections. The laboratory findings allowed early treatment of hemophagocytic syndrome and the patient improved. We describe our technique in detail so it can be reproduced, and we provide a non-systematic review of the literature on the syndrome.

Correlation between persistent high-risk human papillomavirus infection and vaginal microecology and cervical lesions / 中国热带医学

@#Abstract: Objective To investigate the correlation between persistent and non-persistent HPV infection and vaginal microecology and cervical lesions, and to provide the basis for HPV prevention and treatment. Methods In this prospective study, 229 female patients with high-risk type (HR-HPV) were selected for cervical cytology and vaginal microecological examination in the gynecological outpatient department of Baise Maternal and Child Health Hospital from January 2018 to June 2021. The patients were followed up for 1 year to detect persistent HR-HPV infection. The relationship between HR-HPV persistent infection and vaginal microecology and cervical lesions was analyzed using the HPV-negative group as a control. Results Among 229 patients with HR-HPV, there were 109 patients with persistent HR-HPV infection and 120 patients with non-persistent HR-HPV infection in 1-year follow-up, and the incidence of persistent HR-HPV infection was 47.6%. In the HR-HPV persistent and non-persistent infection and HPV-negative groups, the bacterial vaginal incidence was 20.2%, 15.0% and 8.6%, respectively; vulvovaginal candidiasis was 19.3%, 13.3% and 7.9%, respectively; trichomoniasis vaginitis was 12.8%, 9.2% and 4.5%, respectively; mixed infection was 10.1%, 6.7% and 2.7%; H2O2 detection rate was 24.8%, 18.3% and 12.0%,the positive rate of pH value was 52.3%, 40.8% and 36.4%, and microecological normal detection rate was 22.9%, 32.7% and 40.2%, respectively. There were significant differences among the three groups (χ2=10.634, 10.522, 9.010, 9.374, 10.054, 8.268, P<0.01). In the HR-HPV persistent and non-persistent infection groups, the rates of atypical squamous cell detection were 12.8% and 10.0%, and 8.3% and 4.2% for low-grade squamous cell lesions, and 4.6% and 1.7% for high-grade squamous cell carcinoma, 2.8% and 0 for squamous cell carcinoma, respectively. There was no significant difference in the composition of atypical squamous cells between the two groups (χ2=4.358, P>0.05), there were significant differences in the composition of low-grade, high-grade and squamous cell carcinoma (χ2=11.472, 12.685, 11.378, P<0.01). Spearman rank correlation analysis showed that the presence or extent of HPV infection was positively correlated with bacterial vaginosis, vulvovaginal candidiasis, trichomonal vaginitis and mixed infection (P<0.05), positively correlated with H2O2, sialdase, leucocyte esterase,pH positive and positive for all four items (P<0.05), negatively correlated with microecology (P<0.01), positively correlated with low grade, high grade and squamous cell carcinoma (P<0.01), and not significantly correlated with atypical squamous cell carcinoma (P>0.05). Conclusion Persistent cervical HPV infection is an important factor of dysregulation in vaginal microecology and aggravates the degree of dysregulation in vaginal microecology, which is related to the development of cervical lesions.

CD8 + T cell exhaustion statuses in patients with human immunodeficiency virus infection, Mycobacterium tuberculosis infection and co-infection / 中华传染病杂志

Objective:To analyze the statuses of CD8 + T cell exhaustion in patients with human immunodeficiency virus (HIV) infection, Mycobacterium tuberculosis (MTB) infection and co-infection. Methods:A total of 87 patients infected with HIV and/or MTB in Wuxi Fifth People′s Hospital and Taicang First People′s Hospital from August 2019 to January 2020 were enrolled, including 18 cases of HIV infection, 34 cases of active tuberculosis (ATB), 19 cases of latent tuberculosis infection (LTB), seven cases of HIV coinfected with ATB, and nine cases of HIV coinfected with LTB. Another 11 healthy controls were also included. The peripheral blood of all subjects was collected for cell surface staining and intracellular cytokine staining, and flow cytometry was used to detect the expressions of activation molecules including CD62 ligand, CD44 and CD127, the transcription factor like eomesodermin (EOMES), T cell factor 1 (TCF-1), T-box expressed in T cells (T-bet), B lymphocyte-induced maturation protein 1 (Blimp-1), inhibitory receptors including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin domain 3 (Tim-3) on CD8 + T cells. Mann-Whitney U test was used for statistical analysis. Results:The mean fluorescence intensities (MFIs) of the activation molecules CD62 ligand and CD44 in the HIV group were lower than those in the healthy control group, while the inhibitory receptor Tim-3 was higher than that in the healthy control group. The differences were all statistically significant ( U=31.00, 1.00 and 0.00, respectively, all P<0.010). The MFIs of CD62 ligand and CD44 in HIV coinfected with LTB group were lower than those in LTB group, while PD-1 and Tim-3 were higher than those in LTB group. The differences were all statistically significant ( U=4.00, 26.00, 6.00 and 3.00, respectively, all P<0.010). The MFIs of CD62 ligand, CD44 and CD127 in HIV coinfected with ATB group were lower than those in ATB group, while PD-1 and Tim-3 were higher than those in ATB group. The differences were all statistically significant ( U=9.00, 40.00, 45.50, 28.00 and 7.00, respectively, all P<0.010). The proportion of terminal effector CD8 + T cells in the HIV group was higher than that in the healthy control group, while the proportion of central memory CD8 + T cells was lower than that in the healthy control group. The differences were both statistically significant ( U=15.00 and 33.00, respectively, both P<0.010). The proportion of terminal effector CD8 + T cells in the HIV coinfected with LTB group was higher than the LTB group, while the proportion of central memory CD8 + T cells was lower than that in the LTB group. The differences were both statistically significant ( U=7.00 and 20.00, respectively, both P<0.010). The proportion of terminal effector CD8 + T cells in the HIV coinfected with ATB group was higher than that in ATB group, while the proportion of central memory CD8 + T cells was lower than that in ATB group. The differences were statistically significant (both U=7.00, P<0.001). The expression level of PD-1 + Tim-3 + T cells in HIV group was higher than that in healthy control group, that in HIV coinfected with LTB group was higher than that in LTB group, and that in HIV coinfected with ATB group was higher than that in ATB group. The differences were all statistically significant ( U=21.00, 6.00 and 5.50, respectively, all P<0.001). The MFI of transcription factors EOMES and TCF-1 in HIV coinfected with LTB group were lower than those in HIV group, while the MFI of T-bet was higher than that in HIV group. The differences were all statistically significant ( U=3.00, 4.00 and 9.00, respectively, all P<0.001). The MFI of EOMES and TCF-1 in HIV coinfected with ATB group were lower than those in HIV group, while the MFI of T-bet and Blimp-1 were higher than those in the HIV group. The differences were all statistically significant ( U=11.00, 14.00, 7.00 and 22.00, respectively, all P<0.050). Conclusions:MTB co-infected with HIV patients present lower immune function and a higher degree of CD8 + T cell exhaustion. In addition, HIV patients co-infected with LTB and ATB have a higher degree of CD8 + T cell exhaustion than HIV infected patients.

Various features of Chlamydia trachomatis aberrant reticulate body under different induction conditions in vitro and the possible mechanisms / 中华微生物学和免疫学杂志

Persistent Chlamydia trachomatis urogenital tract infection may lead to pelvic inflammatory disease, ectopic pregnancy and tubal infertility in women, and urethritis, epididymitis and other complications in men, which is a hotspot and chanllenge in disease control at home and abroad. In recent years, many researches have shown that Chlamydia trachomatis aberrant reticulate body may be one of the major causes of persistent infection. This review summarized the genomic and proteomic characteristics of Chlamydia trachomatis aberrant reticulate body induced under various conditions in vitro, aiming to elucidating its role in antimicrobial resistance. The identification of persistent infection-related factors, providing new diagnostic targets for the detection of subclinically refractory long-term infections, is a prerequisite for finding appropriate methods to diagnose and treat the complications of Chlamydia trachomatis infection and is crucial for identifying new targets in the post-genomic era.

Prevalence of bovines persistently infected with bovine viral diarrhea virus (BVDV) in dairy cattle herds in Paraná State, Brazil / Prevalência de bovinos persistentemente infectados com o vírus da diarreia viral bovina (BVDV) em rebanhos leiteiros no estado do Paraná, Brasil

This study aimed to establish the prevalence of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) in dairy farms at Parana State, Brazil. Samples were collected from 6,465 female Holstein Friesian Dairy Cattle, including animals less than two years old, females over two years old who had not given birth at the farm, and mothers of calves diagnosed as persistently infected. The cattle came from 40 dairy herds distributed in 10 municipalities in the State of Paraná. The samples were obtained from May 2015 to August 2018. The diagnosis of PI animals was made with an antigen-capture ELISA test. We detected PI animals in fifteen herds sampled (37.5%), ranging from one to sixteen animals per herd. The prevalence in Parana State's municipalities was 1.78%, ranging from 0.3 to 8.9% at positive herds. The analysis of the individual herds shows significant dissemination of the BVDV in Parana's municipalities, including endemic areas. With this, we highlight the need for measures to raise awareness among producers about the existence and importance of bovine viral diarrhea (BVD) in dairy herds, reinforcing the PI animals' role in disease epidemiology and the economic impact caused by the maintenance of these farm animals.(AU)

Com o intuito de se estabelecer a prevalência de animais persistentemente infectados (PI) com o BVDV em propriedades leiteiras no estado do Paraná. Foram coletadas amostras de 6.465 bovinos, fêmeas, da raça Holandês Preto e Branco (HPB). Amostraram-se animais com idade inferior a dois anos, fêmeas com mais de dois anos que não haviam tido partos na propriedade, e mães de bezerros que foram diagnosticados como persistentemente infectados. Os bovinos foram provenientes de 40 rebanhos leiteiros, distribuídos em 10 municípios no Estado do Paraná. A coleta deu-se no período de maio de 2015 a agosto de 2018. O diagnóstico dos animais PI foi feito por meio do teste de ELISA de captura de antígeno. Animais PI foram detectados em quinze rebanhos amostrais (37,5%), oscilando entre um e dezesseis animais por rebanho. A prevalência nos municípios do estado Paraná foi de 1,78%, oscilando entre 0,3 a 8,9% nos rebanhos positivos. Com a alta prevalência de animais PI observada, quando analisados os rebanhos amostrais individualmente, é possível afirmar que há uma disseminação importante do BVDV em municípios paranaenses, destacando inclusive áreas endêmicas. Com isso, vê-se a necessidade de medidas de conscientização dos produtores sobre a existência e importância da BVD nos rebanhos, destacando o papel dos animais PI na epidemiologia da doença, bem como o impacto econômico causado pela manutenção desses animais nos rebanhos.(AU)

Prevalence of bovines persistently infected with bovine viral diarrhea virus (BVDV) in dairy cattle herds in Paraná State, Brazil

ABSTRACT: This study aimed to establish the prevalence of animals persistently infected (PI) with bovine viral diarrhea virus (BVDV) in dairy farms at Parana State, Brazil. Samples were collected from 6,465 female Holstein Friesian Dairy Cattle, including animals less than two years old, females over two years old who had not given birth at the farm, and mothers of calves diagnosed as persistently infected. The cattle came from 40 dairy herds distributed in 10 municipalities in the State of Paraná. The samples were obtained from May 2015 to August 2018. The diagnosis of PI animals was made with an antigen-capture ELISA test. We detected PI animals in fifteen herds sampled (37.5%), ranging from one to sixteen animals per herd. The prevalence in Parana States municipalities was 1.78%, ranging from 0.3 to 8.9% at positive herds. The analysis of the individual herds shows significant dissemination of the BVDV in Paranas municipalities, including endemic areas. With this, we highlight the need for measures to raise awareness among producers about the existence and importance of bovine viral diarrhea (BVD) in dairy herds, reinforcing the PI animals role in disease epidemiology and the economic impact caused by the maintenance of these farm animals.

RESUMO: Com o intuito de se estabelecer a prevalência de animais persistentemente infectados (PI) com o BVDV em propriedades leiteiras no estado do Paraná. Foram coletadas amostras de 6.465 bovinos, fêmeas, da raça Holandês Preto e Branco (HPB). Amostraram-se animais com idade inferior a dois anos, fêmeas com mais de dois anos que não haviam tido partos na propriedade, e mães de bezerros que foram diagnosticados como persistentemente infectados. Os bovinos foram provenientes de 40 rebanhos leiteiros, distribuídos em 10 municípios no Estado do Paraná. A coleta deu-se no período de maio de 2015 a agosto de 2018. O diagnóstico dos animais PI foi feito por meio do teste de ELISA de captura de antígeno. Animais PI foram detectados em quinze rebanhos amostrais (37,5%), oscilando entre um e dezesseis animais por rebanho. A prevalência nos municípios do estado Paraná foi de 1,78%, oscilando entre 0,3 a 8,9% nos rebanhos positivos. Com a alta prevalência de animais PI observada, quando analisados os rebanhos amostrais individualmente, é possível afirmar que há uma disseminação importante do BVDV em municípios paranaenses, destacando inclusive áreas endêmicas. Com isso, vê-se a necessidade de medidas de conscientização dos produtores sobre a existência e importância da BVD nos rebanhos, destacando o papel dos animais PI na epidemiologia da doença, bem como o impacto econômico causado pela manutenção desses animais nos rebanhos.

Pegivirus humano: potencial patogénico y riesgo de desarrollo de linfoma no Hodgkin / Human Pegivirus: pathogenic potential and non-Hodgkin lymphoma development risk

Resumen El pegivirus humano (HPgV) es un virus ARN que fue identificado en el año 1995. Actualmente se encuentra clasificado dentro de la familia Flaviviridae, género Pegivirus, relacionado filogenéticamente con el virus de la hepatitis C (VHC). El HPgV es un virus linfotrópico, con replicación en médula ósea, tejidos linfoides, y en células mononucleares de sangre periférica. Este virus se transmite por vía parenteral y sexual. Según estimaciones realizadas, en el mundo existen alrededor de 750 millones de personas infectadas por este agente. Se ha evidenciado que hasta en 25% de los casos se presenta una infección persistente, y aunque se considera que el HPgV es un virus no patogénico, existen evidencias epidemiológicas que sugieren una relación con el desarrollo de desórdenes linfoproliferativos, particularmente linfoma no Hodgkin (LNH). Algunos estudios han reportado una alta prevalencia de HPgV en pacientes con LNH comparado con donantes de sangre y/o pacientes con enfermedades hematológicas no malignas, lo que se asocia a un incremento en el riesgo relativo para el desarrollo de LNH en personas infectadas. De otra parte, existen estudios epidemiológicos que contradicen esta asociación, por lo que el rol de HPgV en la aparición de desórdenes lifoproliferativos es un tema actual de debate. En el presente manuscrito se discute el potencial patogénico derivado de los mecanismos de infección persistente del HPgV, así como las principales evidencias sobre la relación entre el HPgV y el riesgo de desarrollo de LNH.

The human pegivirus (HPgV), classified in the Flaviviridae family - Pegivirus genus, is an RNA virus identified in 1995. HPgV is a lymphotrophic virus, with replication sites in bone marrow and lymphoid tissue, as well as in peripheral blood mononuclear cells (PBMCs). Transmission is through sexual and parenteral routes, and recent estimations suggest nearly 750 million people are infected with HPgV worldwide. Almost 25% of infected individuals can develop persistent infection. Until now, HPgV has been considered a non-pathogenic virus; however, epidemiological studies suggest a potential role in lymphoproliferative diseases, particularly in the development of non-Hodgkin lymphoma (NHL). The evidence of this is controversial and the role of HPgV in lymphomagenesis has not yet been demonstrated. Several studies report a high prevalence of HPgV infection in patients with NHL compared to controls and patients with other hematological diseases. Therefore, analytic studies show that HPgV could be related to an increased risk of NHL development. Conversely, other studies indicate no association between HPgV and NHL, so the role of HPgV in lymphomagenesis is not clear. This review summarizes the main findings related to HPgV's pathogenic potential and association with NHL.

Expression of IL-17 and estrogen and its receptor in HPV16 continuously infected patients / 局解手术学杂志

Objective To understand the role of estrogen,estrogen receptor and IL-17 in human papillomavirus (HPV) infection and clearance.Methods We selected the clinic or the hospitalized patients in People' s Hospital of Pudong New District hospital with HPV negative or single-HPV16 positive during the period of August 2014 to January 2017.Cervical exfoliated cells were harvested by Thinprep cytologic test (TCT).The reverse spot hybridization technique was carried out for HPV subtype analysis.RT-PCR was employed for HPV16 DNA viral load.The levels of E2 and IL-17 were detected by ELISA.Immunohistochemical was used to detect ER expression.The experiments were repeated every three months.According to the difference in the outcome of human papilloma virus 16 infection,the experiment was divided into three groups.The first group was HPV negative,which was negative for one year.The second group was HPV16 positive,but within one year the difference was gradually cleared.The third group was HPV16 positive,but the HPV16 persistently existed after one year.Results The results of the last test were compared with the first test results.We found that There was no significant difference among the three groups in the expression of E2 and ER.Also,there was no significant difference between the HPV negative group and the HPV16 persistently infected group in variation of IL-17 concentration.But the difference between the HPV16 cleared group and the anyone of the two groups above was significantly.The variation of IL-17 concentration was significantly higher in the HPV16 cleared group.Conclusion In normal cytology,and only HPV16 infected stage,endogenous E2 and ER of cervix may not play a role in HPV infection,or they may have little effect on HPV infection.Local immune factor IL-17 plays an important role in the process of HPV16 removal,and the increase of IL-17 concentration can help to eliminate HPV16.

Regulation of apoptosis of host cells by in vitro azithromycin-induced persistent Chlamydia trachomatis infection / 中华皮肤科杂志

Objective To evaluate the regulatory role of azithromycin-induced persistent Chlamydia trachomatis (Ct) infection in the apoptosis of Hela229 cells.Methods Hela229 cells were firstly co-cultured with Ct for 22 hours,and then cultured with Dulbecco's modified Eagle's medium (DMEM) containing 0.08 mg/L azithromycin for 26 hours to establish a cell model of persistent Ct infection (persistent infection group).These infected Hela229 cells cultured with azithromycin-free DMEM served as a cell model of acute Ct infection (acute infection group).After 48-hour infection with Ct,azithromycin was removed,and infected Hela229 cells in the above 2 groups were successively cultured with DMEM for the resurgence of Ct.Immunofluorescence assay and electron microscopy were performed to verify the persistent Ct infection model.The Hela229 cells in the persistent infection group and acute infection group as well as uninfected Hela229 cells (control group) were treated with staurosporine (STS) for 4 hours to induce the apoptosis,and then cell apoptosis was detected by Hoechst 33258 staining,annexin V/propidium iodide staining and flow cytometry.Results After the treatment with azithromycin,atypical inclusions with aberrant reticulate bodies appeared in the Ct-infected cells.After removing azithromycin,cells were cultured until 96 hours after infection,and infectious elementary bodies reappeared in the Ct inclusions.After the treatment with STS,Hoechst staining showed that there was loose chromatin in the persistently infected cells,while chromatin condensation was observed in the uninfected cells.After 24-hour infection with Ct and 4-hour induction with STS,the apoptosis rate was significantly higher in the persistent infection group (45.567％ ± 2.631％) than in the acute infection group (38.567％ ± 1.701％,t =2.686,P =0.028),but significantly lower in the persistent infection group than in the uninfected group (69.800％ ± 2.835％,t =8.187,P ＜ 0.001).After 48-hour infection with Ct and 4-hour induction with STS,there was a significant difference in the apoptosis rate between the persistent infection group (46.700％ ± 5.257％) and acute infection group (61.767％ ± 1.815％,t =5.781,P ＜ 0.001),as well as between the persistent infection group and the uninfected group (68.667％ ± 3.156％,t =7.421,P ＜ 0.001).Conclusion This study showed that azithromycin-induced persistent Ct infection regulated the apoptosis of host cells,and this effect lasted 48 hours.

Expression of T helper cell 17-related immune factors in women of child bearing age with human papillomavirus subclinical cervical infection / 上海交通大学学报（医学版）

Objective • To investigate the change of immune microenviroment in the lower genital tract of women who had early papillomavirus (HPV) infections. Methods • One hundred and twenty women infected with high-risk HPV were enrolled in the study, who were at the age of 25-46, with no cervical intraepithelial neoplasia (CIN - III ) or cervical carcinoma. Twenty HPV-negative women were used as control, who did not have abnormal cervical squamous epithelial lesions screened by ThinPrep cytologic test. Vagina douche was collected, and the expression of T helper cell 17 (Th17)-related cytokines and interleukin 10 (IL-10)/IL-4 was tested by suspension micro phase protein chip technology. Results • The expression of IL-17, IL-6, TGF-β and IL-4 in HPV-positive patients was significantly higher than that in HPV-negative patients. The expression of IL-4 and IL-10 in HPV16/18-positive patients was significantly lower than that in patients infected with other HPV types. The expression of IL-17, IL-6 and TGF-β in patients with persistent HPV infection was insignificantly higher than that in patients with temporary HPV infection, while the expression of IL-17, IL-6 and TGF-β in patients with persistent HPV infection was significantly higher than that in HPV-negative patients. Conclusion • HPV infection can stimulate the body's immune response and change the local immune microenvironment in early stage. Th17 related cytokines are closely related to the persistent HPV infection. Maybe it can provide a new direction on the guidance of HPV treatment and prevention of persistent HPV infection.

Efeito antimicrobiano do bacteriófago geneticamente modificado φEf11/φFL1C(Δ36)PnisA sobre cepas de E. faecalis em biofilme estático e em canais radiculares infectados / Antibacterial effect of a Genetically-Engineered Bacteriophage φEf11/φFL1C(Δ36)PnisA on static biofilms and on Dentin Infected with E. faecalis

A presença de micro-organismos no sistema de canais radiculares (SCR) tem sido apontada como uma das principais causas de insucesso da terapia endodôntica. A capacidade de formar biofilme e penetrar nos túbulos dentinários são fatores de sobrevivência que favorecem a perpetuação de micro-organismos no interior do SCR. Terapias que promovam a desorganizazão de biofilmes e eliminação de bactérias dentro dos túbulos dentinários são fundamentais para a desinfecção endodôntica. Uma terapia descoberta há um século, denominada de Bacteriofagoterapia, baseia-se na utilização de vírus capazes de infectar e matar bactérias. Esta abordagem antimicrobiana tem recebido bastante atenção atualmente por representar uma alternativa para o tratamento de doenças causadas por bactérias multirresistentes aos antibióticos. O objetivo deste estudo foi avaliar a eficácia de um bacteriófago modificado geneticamente, φEf11/φFL1C(Δ36)PnisA, para eliminar biofilmes de duas cepas de E. faecalis: JH2-2 (sensível à vancomicina e resistente ao ácido fusídico e à rifampicina) e V583 (resistente à vancomicina). Este estudo foi dividido em dois experimentos distintos. No primeiro experimento, biofilmes estáticos de 48 horas de cepas de E. faecalis JH2-2 (pMSP3535 nisR / K) ou V583 (pMSP3535 nisR / K) formados em lâminulas de vidro (coverslips) foram inoculados por suspensão do bacteriófago φEf11/φFL1C(Δ36)PnisA. Após 48 horas de incubação, a biomassa bacteriana foi fotografada por microscopia confocal e as células viáveis foram quantificadas por medição de unidades formadoras de colônias (UFC). No segundo experimento, segmentos radiculares de dentes humanos extraídos foram cimentados em dispositivos vedáveis de duas câmaras para formar modelos ex vivo com dentina infectada, contendo solução tampão na câmara inferior. Os modelos foram inoculados com uma suspensão de E. faecalis V583 ou E. faecalis JH2-2. Após sete dias de incubação a 37°C, adicionou-se ao canal de cada segmento de dentina infectada dos grupos 2 e 5 uma suspensão do fago geneticamente modificado, φEf11/φFL1C(Δ36)PnisA e manteve-se a incubação por mais 72 horas. Os segmentos de dentina foram instrumentados com Gates Glidden e a solução tampão foi aliquotada para semeadura e contagem de UFC e aferição do título residual de células de E. faecalis. Os resultados do primeiro experimento mostraram uma diminuição de 10-100 vezes (p≤ 0,05) das células viáveis (UFC / biofilme) após tratamento com bacteriófago, o que foi consistente com a comparação das imagens de biofilme tratado e não tratado visualizadas com projeções máximas da série Z. No segundo experimento a titulação de E. faecalis verificada após tratamento com o bacteriófago foi reduzida em 18% para os modelos infectados com JH2-2 e em 99% (p≤ 0,05) nos modelos infectados com V583. Com base nesses resultados, pode-se concluir que a biomassa dos biofilmes de E. faecalis, tanto sensíveis quanto resistentes à vancomicina, foi significantemente reduzida após a infecção pelo bacteriófago φEf11/φFL1C(Δ36)PnisA. Além disso, o tratamento da dentina infectada por E. faecalis com bacteriófago φEf11/φFL1C(Δ36)PnisA resultou em diminuição da população bacteriana residual de cepas sensíveis e resistentes à vancomicina, alcançando significância estatística no grupo que utilizou a cepa V583.

Residual microorganisms in the root canal system (RCS) have been identified as the main cause of endodontic therapy failure. The ability to form a biofilm and penetrate the dentin tubules are survival factors that favor the perpetuation of microorganisms within the RCS. Therapies that promote the disorganization of biofilms and elimination of bacteria within the dentinal tubules are essential for endodontic disinfection. A therapy discovered a century ago called Bacteriophage Therapy is based on the use of viruses capable of infecting and killing bacteria. Recently, this antimicrobial approach has been receiving considerable attention since it represents an alternative for the treatment of diseases caused by multiresistant antibiotic bacteria. The aim of this study was to evaluate the efficacy of a genetically engineered bacteriophage, φEf11/φFL1C(Δ36)PnisA, to disrupt biofilms of two Enterococcus faecalis strains: JH2-2 (vancomycin sensitive) and V583 (vancomycin resistant). This study was divided into two separate experiments. In the first experiment, 24 hour static biofilms of E. faecalis strains JH2-2(pMSP3535 nisR/K) and V583(pMSP3535 nisR/K) formed on cover slips were inoculated with bacteriophage φEf11/φFL1C(Δ36)PnisA. After 24 and 48 hours incubation, the bacterial biomass was imaged by confocal microscopy and viable cells were quantified by colony forming unit (CFU) measurement. In the second experiment, extracted human dentin root segments were cemented into sealable two-chamber devices, fabricated from syringe needle caps to form in vitro infected-dentin models. The models were inoculated with an overnight suspension of either E. faecalis V583 (vancomycin resistant strain) or E. faecalis JH2-2 (fusidic acid and rifampin resistant, vancomycin sensitive strain). After 7 days of incubation at 37°C, a suspension of a genetically engineered phage, φEf11/φFL1C(Δ36)PnisA, was added to the root canal of each infected dentin segment, and the incubation was continued for an additional 72-hours. Dentin was harvested from the walls of each root canal and assayed for the residual titer of E. faecalis cells. The results from the first experiment showed a 10-100-fold fewer decrease in viable cells (CFU/biofilm) after bacteriophage treatment, which was consistent with comparisons of treated and untreated biofilm images visualized as max projections of the Z-series. On the second experiment, the recovered E. faecalis titer was reduced by 18% for the JH2-2 infected models, and by 99% for the V583 infected models. These results suggest that the biomass of E. faecalis biofilms, both sensitive and resistant to vancomycin, was significantly reduced after infection by bacteriophage φEf11/φFL1C(Δ36)PnisA. In addition, treatment of E. faecalis-infected dentin with the phage resulted in the decrease of the residual bacterial population for both susceptible and vancomycin resistant strains, reaching statistical significance in strain V583 group.

Differences in Rab protein expressions in McCoy cells with acute versus persistent Chlamydia trachomatis infection / 中华皮肤科杂志

Objective To investigate differences in Rab protein expressions in McCoy cells with acute versus persistent Chlamydia trachomatis (Ct) infection.Methods Cultured McCoy cells were infected with different amounts (400,500,550 μl/well) of Ct strain D suspensions,then cultured with the medium containing 100 U/ml penicillin G (persistent Ct infection groups) or that without penicillin G (acute Ct infection groups).Ct-uninfected McCoy cells receiving no penicillin G treatment served as the blank control group,and those receiving penicillin G treatment as the penicillin group.Mter 48-hour culture,McCoy cells were lysed,proteins were collected,and total RNA was extracted from the cells.Enzyme-linked immunosorbent assay (ELISA) was conducted to measure protein levels of Rab4A,Rab6A,Rab10,Rab11A and Rab14,and fluorescence-based quantitative PCR to quantify mRNA expressions of Rab4A and Rab14 (expressed as 2-ΔΔα).Results Protein levels of Rab4A,Rab6A,Rab10,Rab11A and Rab14 were all significantly lower in the acute than in the persistent Ct infection groups (all Z =3.621,P ＜ 0.001),and lower in the persistent and acute Ct infection groups than in the blank control group (all P ＜ 0.008 3),but insignificantly different between the blank control group and penicillin group (all P ＞ 0.05).In addition,the expressions of Rab4A and Rab14 mRNAs were consistent with those of their proteins in these groups.Conclusion The transcriptional and expression levels of Rab proteins are higher in McCoy cells persistently infected with Ct than in those acutely infected with Ct.

The associations between human papilloma viruse persistent infection and Stathmin-1 expression and the clinical significance in cervical intraepithelial neoplasia / 中国医师进修杂志

Objective To explore the relationship between Stathmin-1 and human papilloma viruse (HPV) persistent infection after conization of uterine cervix, and to show the clinical significance to recurrent of cervical intraepithelial neoplasia (CIN). Methods One hundred and six patients who were treated with conization of uterine cervix for CIN 2-3 grades were enrolled. Thirty-six recurrent patients were enrolled in recurrence group, and the others were enrolled in control group. The expression of Stathmin-1 in primary CIN tissues in two groups was detected by immunohistochemistry. The HPV infection was detected by HPV-DNA test. The relationship of HPV persistent infection and recurrence was analyzed. Results The positive expression rate of HPV persistent infection and HPV persistent infection rate in recurrence group were 88.89%(32/36), 83.33%(30/36), in control group were 34.29%(24/70) and 22.86%(16/70), and there were significant difference (P 0.05). Conclusions Stathmin-1 positive expression is related to HPV persistent infection. The two factors can affect the prognosis of high-grade CIN, and can provide new cues and theory basis for the prevention of recurrence.

Detección molecular de coinfección con los virus de la diarrea viral bovina (BVDV) en búfalos de agua (Bubalus bubalis) serológicamente negativos / Molecular analyses detect natural coinfection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) in serologically negative animals

La infección de los búfalos de agua (Bubalus bubalis) con los virus de la diarrea viral bovina (BVDV) ha sido confirmada mediante técnicas serológicas y moleculares en trabajos anteriores. Con el fin de determinar la presencia de animales persistentemente infectados y las especies y subtipos circulantes de BVDV en esta especie animal se realizó un estudio sobre una manada de búfalos de producción mixta con ganado bovino (Bossp.). Nuestros resultados serológicos mostraron un alto nivel de positividad frente a BVDV-1 y BVDV-2 dentro de la manada de búfalos. El análisis molecular sobre muestras de sangre de los animales serológicamente negativos reveló la presencia de ácido nucleico viral, lo que confirma la existencia de búfalos persistentemente infectados. El clonado y la secuenciación de la región 5 'UTR de algunas de las muestras obtenidas de búfalo reveló la presencia de coinfección natural con al menos dos subtipos diferentes de BVDV (1a y 1b) y con las especies virales BVDV-1 y BVDV-2

Infection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) has been confirmed in several studies by serological and molecular techniques. In order to determine the presence of persistently infected animals and circulating species and subtypes of BVDV we conducted this study on a buffalo herd, whose habitat was shared with bovine cattle (Bossp.). Our serological results showed a high level of positivity for BVDV-1 and BVDV-2 within the buffalo herd. The molecular analyses of blood samples in serologically negative animals revealed the presence of viral nucleic acid, confirming the existence of persistent infection in the buffaloes. Cloning and sequencing of the 5' UTR of some of these samples revealed the presence of naturally mix-infected buffaloes with at least two different subtypes (1a and 1b), and also with both BVDV species (BVDV-1 and BVDV-2)

Primary study on change of STAT3-TLR2 signaling axis in Chlamydia trachomatis persistent infection / 中国免疫学杂志

Objective:To explore in state of Chlamydia trachomatis persistent infection,the STAT3-TLR2 axis may be activated and mediating abnormal secretion of inflammatory cytokines.Methods: We established acute infection and IFN-γinduced persistent infection model of Ct in HeLa cell.Gene transcription, cytokine secretion and protein expression were detected by using qRT-PCR, ELISA and Western blot respectively in STAT3-TLR2 signaling axis in each Ct infection model.Results: Persistent Ct infections upregulated the transcription of TLR2,significantly increased both the secretion of inflammatory cytokine IL-1αand the expression of STAT3 and TLR2,moreover,enhanced the activation of STAT3 simultaneously.Conclusion: In the Ct persistent infection induced by IFN-γ,the STAT3-TLR2 signaling axis activated significantly in HeLa cell.

Genetic characterization of bovine viral diarrhea virus strains in Beijing, China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5'-untranslated region (5'-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-alpha (IFN-alpha), IFN-beta, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-alpha, and IFN-beta mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.

The effects of human cytomegalovirus persistent infection on the central nervous system of BALB/c mice / 中华微生物学和免疫学杂志

Objective To investigate the effects of human cytomegalovirus ( HCMV) persistent in-fection on the central nervous system of BALB/c mice.Methods Thirty specific-pathogen-free mice of 6-8 weeks old were randomly divided into three groups including HCMV infected group , inactivated HCMV group and human embryo fibroblast ( HF) control group .Each mouse in the three groups was intraperitoneally inoc-ulated with 1.8 ×107 PFU of HCMV, 1.8 ×107 PFU inactivated HCMV and 1 ×105 HF cells, respectively. All mice were housed in microisolator cages for three months and their behavior and body weight were ob -served.Then three tests including autonomic activities test , Morris Water Maze and step-down passive avoid-ance task were performed on all mice to evaluate the changes of their behavior .Cerebral cortex tissues were collected from all mice to detect HCMV and to conduct polymerase chain reaction (PCR) analysis.Brain tis-sues were stained by HE method to evaluate the pathological damages .Transmission electron microscope was used to observe the ultrastructure of neuron cells and the existence of virus particles .Results (1) The body weight of mice showed no significant differences among the three groups ( P>0 .05 ) .( 2 ) The frequency of autonomic activities were decreases in HCMV infected group in comparison with other two groups , but there was no significant differences among the three groups (P>0.05).(3)The place navigation test demonstra-ted that the escape latency of mice from HCMV infected group as well as HF group showed significant differ -ence after training for different periods of time (P0.05).Compared with the mice in two control groups , the mice in HCMV infected group showed a lower frequency of crossing the quadrant where the platform had been located on pre -vious trials in the probe trial test (P<0.05).Moreover, the time of first crossings was also longer than that of mice from two control groups (P<0.05).(4)In the learning phase the mice from HCMV infected group showed a high frequency of mistakes in comparison with that from two control groups in step -down passive avoidance task (P<0.05).After 24 hours, the frequency of mistakes was decreased in each group , and the differences were significant (P<0.05).The latency was very shorter in mice from HCMV infected group than that observed in two control groups (P<0.05).(5)The HCMV infection was identified in six mice from HCMV infected group .And the positive HCMV UL83 gene could be only detected in HCMV infected group as indicated by PCR analysis .(6)The pathological changes including cell swelling , loosen cytoplasm, decreased cell layers and vacuolization were observed in the cerebral cortex and hippocampus of mice from HCMV infected group .HCMV-specific intracytoplasmic inclusion bodies and herpes virus like particles were detected by using transmission electron microscope .No obvious abnormalities were observed in two control groups.Conclusion HCMV persistent infection could damage the central nervous system of mice .The au-tonomic activities of mice with HCMV persistent infection were not affected , but the learning and memory ca-pability of them were damaged at some extent .

Reproduction of Epstein-Barr Virus Infection and Pathogenesis in Humanized Mice

Epstein-Barr virus (EBV) is etiologically associated with a variety of diseases including lymphoproliferative diseases, lymphomas, carcinomas, and autoimmune diseases. Humans are the only natural host of EBV and limited species of new-world monkeys can be infected with the virus in experimental conditions. Small animal models of EBV infection, required for evaluation of novel therapies and vaccines for EBV-associated diseases, have not been available. Recently the development of severely immunodeficient mouse strains enabled production of humanized mice in which human immune system components are reconstituted and express their normal functions. Humanized mice can serve as infection models for human-specific viruses such as EBV that target cells of the immune system. This review summarizes recent studies by the author's group addressing reproduction of EBV infection and pathogenesis in humanized mice.

Perfil da infecção pelo vírus da diarreia viral bovina (BVDV) em um rebanho bovino leiteiro de alta produção e com programa de vacinação contra o BVDV / Bovine viral diarrhea virus (BVDV) infection profile in a high production dairy herd with vaccination program against BVDV

A infecção pelo vírus da diarreia viral bovina (BVDV) foi avaliada em um rebanho bovino leiteiro de alta produção com histórico de problemas reprodutivos e de vacinação regular contra o BVDV. A identificação do vírus foi realizada por RT-PCR em soro sanguíneo e o perfil sorológico por vírus-neutralização. Inicialmente, 100% (n=692) dos animais do rebanho foram avaliados com relação à presença de infecção ativa pelo BVDV por meio da RT-PCR. Quatro meses após, todos os animais positivos (n=29) na primeira avaliação foram avaliados novamente pela RT-PCR, assim como todos os animais que nasceram (n=72) e os que apresentaram problemas reprodutivos (n=36) no intervalo entre a primeira e a segunda colheita de sangue. Os resultados finais do estudo possibilitaram identificar 27 animais transitoriamente infectados e três animais persistentemente infectados (PI). A sorologia, realizada apenas nos animais positivos na primeira avaliação pela RT-PCR e nas vacas que apresentaram problemas reprodutivos entre a primeira e a segunda RT-PCR, demonstrou grande flutuação nos títulos de anticorpos neutralizantes, além de soroconversão na maioria dos animais. Foram identificados aumentos nos títulos de anticorpos neutralizantes que variaram entre 3 e 8 log2, indicando infecção ativa no rebanho. A circulação viral no rebanho avaliado foi responsável pela expressão de sinais clínicos da esfera reprodutiva em animais com baixo título de anticorpos e consequente falha na proteção fetal. Os resultados demonstram que o controle da infecção pelo BVDV apenas por meio da vacinação regular em rebanhos com animais PI pode não ser eficaz na profilaxia dessa virose.

The profile of bovine viral diarrhea virus (BVDV) infection was studies in a high production dairy herd selected based on a history of reproductive failures and regular vaccination against BVDV. Virus identification was performed by RT-PCR and serological profile was determined by virus-neutralization (VN). Initially, 100% (n=692) of the animals in the herd were monitored for identification of an active infection by RT-PCR in sera. Four months later, all positive animals (n=29) were retested by RT-PCR, along with newly born animals (n=72), or those that had reproductive failures (n=36) in the interval. The RT-PCR assay identified 27 transiently infected animals and three persistently infected (PI). Serology performed only in positive animals in the first RT-PCR and in cows with reproductive failures between the first and second RT-PCR analysis, showed large variation VN antibody titers and seroconversion in most animals. Increases in VN titers were demonstrated, with variation between 3 and 8 log2, indicating virus circulation within the herd. Virus circulation in the vaccinated herd evaluated in this study was likely responsible for reproductive failures observed in cows with low VN titers and for fetal infections. These results demonstrate that control of BVDV infection by regular vaccination in dairy cattle herds with PI animals represents a great challenge for the prophylaxis of this infection.

Análise da diversidade microbiana em infecções endodônticas persistentes / Microbial diversity analysis in persistent root canal infections

O presente trabalho teve por objetivo investigar a microbiota de canais radiculares relacionadas ao insucesso do tratamento endodôntico, buscando a identificação e a quantificação destes micro-organismos. Foram selecionados 36 dentes com infecção endodôntica persistente. O material obturador foi removido do canal radicular e amostras microbiológicas foram coletadas dos canais com o auxílio de limas tipo Hedströen e cones de papel absorvente estéril. A técnica do Checkerboard DNA-DNA hybridization foi utilizada para detecção de até 79 espécies bacterianas em cada amostra, utilizando sondas de DNA específicas. Os dados microbiológicos foram expressos em percentagem média (prevalência), proporção e nível médio de cada espécie em cada amostra. Os testes t independente e de correlação de Pearson foram usados para correlacionar a contagem das bactérias testadas com os dados clínicos (p≤ 0,05). Foi encontrada uma média de 11 espécies por amostra. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) e S. warneri (28%) foram as espécies mais prevalentes, e as espécies encontradas em níveis médios mais altos foram E. faecium, D. pneumosintes, S. epidermidis, H. pylori e C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%), S. ixodetis (3%) apresentaram prevalências mais baixas. E. faecium e S. epidermidis apresentaram os maiores valores de prevalência, níveis médios e proporção. Não houve correlação entre a microbiota detectada nas amostras com os sinais e sintomas clínicos apresentados pelos pacientes, porém nas lesões periapicais de maior área foi detectada contagem significativamente maior de bacilos e espécies Gram-negativas (p<0,05). Baseado nos resultados obtidos é possível concluir que a microbiota presente em dentes com ...

The present study investigated the composition of the root canal microbiota in endodontic failures, aiming to identify and quantify these microorganisms. Thirty six teeth with persistent endodontic infection were selected. The root-filling materials were removed and microbiological samples were taken from the root canals with a Hedströen-type file and sterile paper points. The Checkerboard DNA-DNA hybridization technique was used for the detection of 79 bacterial species in each sample, using specific DNA probes. Microbiological data were express in mean prevalence, proportions and levels of each species in each sample. t independent test and Pearson correlation test were use to correlate bacterial counts and clinical conditions (p≤ 0,05). There were found a mean of 11 different species per sample. E. faecium (36%), S. epidermidis (36%), E. saburreum (28%), P. micra (28%), S. sanguis (28%), C. sputigena (28%), L. buccalis (28%), E. faecalis (28%) and S. warneri (28%) were the most prevalent species, and the species found in highest mean levels were E. faecium, D. pneumosintes, S. epidermidis, H. pylori and C. sputigena. T. socranskii (3%), F. periodonticum (3%), C. gingivalis (3%) and S. ixodetis (3%) were found in low prevalence. E. faecium and S. epidermidis presented the highest values of prevalence, means levels and proportions. No correlation was found between the detected microbiota and clinical findings; however in periapical lesions with highest areas, higher levels of rods and Gram-negative species were detected (p<0.05). Based on these results it may be concluded that the microbiota in teeth with persistent apical periodontitis presents a mixed and complex profile, and periapical lesions with larger area might be high associated with higher counts of rods and Gram-negative species.