685 research outputs found
Molecular characterisation of a Chlamydia suis isolate from a swine in Italy
No full textThe present study described the isolation and the molecular characterization of a Chlamydia suis strains, MS04, obtained from a swine reared in an intensive farm in Italy.Nucleotide sequence analysis of 16S/23S amplicon of the isolate showed 95% similarity with the same region of the S45 C. suis reference strain
Comparative in vitro elicitation of reactive oxygen/nitrogen species and cytokines in Chlamydia pneumoniae and Chlamydia trachomatis infected human monocytes.
No full textComparative in vitro elicitation of reactive oxygen/nitrogen species and cytokines in Chlamydia pneumoniae and Chlamydia trachomatis infected human monocytes.
Marangoni A.1, Bergamini C.2, Fato R.2, Cavallini C.3, Foschi C 1, Russo I.1, Nardini P.1, Cevenini R.1
1Section of Microbiology-DESOS, 2Dipartimento di Biochimica “G. Moruzzi”, and 3Cardiovascular Department, University of Bologna, Bologna, Italy
INTRODUCTION
An increasing number of in vitro studies suggests that chlamydial species can infect immune cells, at least at low level. These infections may alter immune cell function, promote chlamydial persistence in the host and contribute to the progression of several chronic inflammatory diseases.
The limited infection that occurs can affect antigen processing, cytokine production and susceptibility to apoptosis. Regarding that, some authors reported a copious release of reactive oxygen species in Chlamydia pneumoniae-infected macrophages and others the induction of various interleukins and TNF- in blood mononuclear cells after the infection.
The aim of this study was to elucidate in vitro infection characteristics, evaluate reactive oxygen (ROS) and reactive nitrogen species (RNS) production and cytokine release in human blood monocytes infected by human pathogens C. pneumoniae (CP) and C. trachomatis (CT).
METHODS
Human monocytes were isolated from healthy volunteer peripheral blood by centrifugation over Ficoll-Paque and cultured in RPMI 1640. Fresh isolated monocytes were seeded on glass coverlips in 24 well-plates and infected with viable CP and CT elementary bodies (infectivity ratio: 5 EBs/cell). At various time, infectivity rate was evaluated by recultivating disrupted monocytes in LL-CMK2. Infection by CT and CP was carried out as control in the same cellular line. Infected cells were fixed and stained with a fluorescence-labelled MAb to the LPS of Chlamydia (Meridian Diagnostics) at 24, 48, 72 and 96 hours post infection. The number of inclusion was determined by fluorescence microscopy.
In order to evaluate the production of ROS and RNS, monocytes were infected with viable CT and CP EBs in the presence of 10μM DCFDA or 5μM DAF, respectively ROS and RNS fluorescent probes. Phorbol and LPS treatment was used as positive control, for ROS and RNS respectively. Cell fluorescence increase was measured with a spectrofluorometer (Wallac Victor multilabel counter).
Cytokine production was evaluated by measuring mRNA expression of IFNs and TNF-α at different times after infection: CT and CP-infected monocytes were lysed by adding Trizol (Invitrogen) and total RNA was isolated (RNeasy Micro Kit, Qiagen). cDNA was synthesized and a Real-time RT-PCR was performed in a Lightcycler system (Roche Diagnostics), with the SYBR Green fast start kit (Lyghtcycler® FastStart DNA MasterPLUS SYBR Green I). Primers used in the reaction mixture to assess IFN-α, IFN-β, IFN-γ and TNF-α levels were from SuperArray (SABiosciences Corporation). Fluorescence was measured at the end of each extension step and samples were run in duplicate, using for calculations the average threshold cycle (Ct). A positive control was included by stimulating monocytes with 1 μg/ml LPS.
RESULTS
Infectivity rate of CP-infected monocytes was 12% at 24 hours after the infection, 2% at 48 hours and 0% at 72 and 96 hours, in comparison with control infected permissive LL-CMK2 cells. On the contrary, we were not able to recover infectious CT at any time after the infection.
At 3 hours after infection, CT-infected monocytes were able to produce higher level of both ROS and RNS, in comparison with untreated control and CP-infected monocytes (P=0.024). At later time points there were not significant differences between CT and CP-stimulated monocytes.
The expression of IFN-α, IFN-β, IFN-γ was only slightly increased at 4 and 6 hours after infection in CT-infected monocytes, compared to negative control. On t..
Usefulness of Positron Emission Tomography for genital Chlamydia infection assessment in the murine model.
No full textUsefulness of Positron Emission Tomography for genital Chlamydia infection assessment in the murine model.
Marangoni A.1, Nanni C. 2, Quarta C. 2, Foschi C. 1, Russo I. 1, Nardini P. 1, D’Errico A. 3, Rosini F. 3, Aldini R.4, Cevenini R.1
1Section of Microbiology-DESOS, 3Med. Interna, Dell’invecchiamento e Malattie nefrologiche, 4SMETEC Dpt., University of Bologna.
2Nuclear Medicine, Azienda Ospedaliero-Universitaria di Bologna, Policlinico S.Orsola-Malpighi, Bologna, Italy.
OBJECTIVES
Untreated Chlamydia trachomatis infection can wreak havoc on the reproductive organs profoundly affecting fertility in women. Taken together, the high rate of asymptomatic infections and the severity of the infection related pathology indicate that control of chlamydial infections would require the development of new diagnostic non-invasive techniques for genital infection. The aim of this study was to explore the feasibility of 11C-choline Positron Emission Tomography (PET) in the assessment of the degree of inflammation in a C. muridarum genital infection mouse model.
METHODS
Model of infection. All the experiments were approved by the Ethical Committee of the University of Bologna. Animals used were 43 female Balb/c mice, 6-8 weeks old. All animals received 2.5 mg of medroxyprogesterone acetate i. m. 9 and 2 days prior the infection.
Twenty-one mice were infected by placing 15 μl of sucrose-phosphate-glutamic acid (SPG) buffer containing 106 inclusion forming units (IFUs) of C. muridarum into the vaginal vault. Twelve animals were treated with 15 μl of SPG containing heat-inactivated 106 IFUs of C. muridarum. As controls of inflammation, 7 animals were challenged with 15 μl of SPG. The Experimental design. Twenty-one infected animals were randomly allotted into two groups. Group A: 9 animals underwent a 11C-Choline PET at day 5, 10 and 20 post-infection. Group B: 12 animals were sacrificed at 5, 10, 15, 20 days for culture and histological analysis (3 animals for each time point). The 12 mice treated with inactivated chlamydiae were divided in two groups, as well. Group C: 4 animals underwent a 11C-Choline PET at day 5, 10 and 20 post-inoculation. Group D: 8 animals were sacrificed at 5, 10, 15, 20 days for culture and histological analysis (2 animals for each time point). Five control animals underwent 11C-Choline PET at the same days of the infected animals. Similarly, the remaining two control animals were sacrificed for histological analysis, one at 10 days and the other one at 20 days post-inoculation.
PET. 11C-Choline PET was carried out as follows: each animal was anaesthetized with gas anesthesia (Sevofluorane 3–5% and oxygen 1 l/min) and was injected with approximately 20 MBq of 11C-Choline, in a volume of 0.1 ml. The residual dose was measured to verify the effective dose injected. The animal was subsequently placed on the scanner bed in the prone position, after an uptake time of 10 minutes. Images were acquired with a small animal PET tomograph for a total acquisition time of 15 min. 11C-Choline PET images were reconstructed iteratively (OSEM 2D) and read in three planes (axial, sagittal and coronal).
Semi-quantitative analysis was carried out using the parameter SUV (standardized uptake value) representing mean radioactive counts per gram of tissue, divided by injected dose of radioactivity per gram of animal weight. The target region of interest (ROI) was placed on the pelvis at the level of the cervical-vaginal region.
Histology. The specimens obtained from sacrificed animals were preserved in 10% neutral buffered formalin for about 48 h and put in embedding cassettes, then processed in automatic tissue processor. After dehydration they were infiltrated with molten paraffin wax. Four-μm thick sections were cut with microtome, stained with haematoxylin-eosin and mounted on glass microscope slides.
The uterine horns were scored with respect to inflammation using the following criteria: 0: No inflammation. 1..
Chlamydia trachomatis antimicrobial susceptibility in colorectal and endocervical cells
No full textBACKGROUND:
Rectal Chlamydia trachomatis infections represent one of the most common sexually transmitted infections in the MSM population. Although current treatment guidelines suggest the use of either azithromycin or doxycycline, several clinical studies reported on azithromycin treatment failures in the case of rectal C. trachomatis localizations. In this context, the biological reasons behind the lack of azithromycin efficacy for C. trachomatis infections at the rectal level are still poorly understood.
OBJECTIVES:
To evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, mimicking the urogenital localization and the rectal site of infection.
METHODS:
The susceptibility to macrolides (i.e. azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G), both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines.
RESULTS:
For all the C. trachomatis strains, regardless of the genovar, macrolides showed higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Moreover, azithromycin MICs were significantly higher with increasing levels of C. trachomatis elementary bodies on Caco-2 cells.
CONCLUSIONS:
The higher azithromycin MICs observed in colorectal cells, together with the positive correlation between MICs and C. trachomatis loads found, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site
Tools and Avenues for Nanotechnology-based Vectors Exploitation for Biomarker Signature and Therapeutical Drug Delivery
No full textNanomedicine main goal is to ameliorate biodistribution and side effects of therapeutics. Proper Micro (MP) and Nanoparticles (NP) can i) protect therapeutic molecules from reticuloendothelial clearance, ii) transport them to the site of action minimizing their influence on normal tissues and iii) enhance drug concentration and effects in target tissues and cells, allowing the use of lower doses. However a single component nanovector cannot accomplish all those duties; therefore, our efforts are focused on the optimization and performance evaluation of multi-component drug-delivering nanosystems, made of silica, silicon, polymers, lipids and proteins from synthetic and biological sources. In recent years, the groups in Naples and in Houston in cooperation have studied many different strategies which permit MP and NP to overcome one or more biological barriers (BB) that hamper the delivery of therapeutics to the desired site of action. The two research groups have produced a series of results which will be briefly summarized in this report and then discussed in greater detail in the talk.
1) Diverse types of Multistage vector (MSV) were produced and used to enhance the delivery of free and encapsulated drugs in cellular and mice models of cancer. MSV is constituted by a silicon mesoporous microparticle which protect nanoparticles or free drugs from reticuloendothelial clearance, superbly marginates toward vessel walls and preferentially accumulates on inflamed endothelium [1-3].
2) A Biomimetic drug delivery platform was produced with a nanoporous silicon core and and a shell derived from the leukocyte cell membrane. This Leukolike vector (LLV) and it’s therapeutic potentialities were extensively investigated in vitro and in vivo, from a biological and biochemical point of view. LLV shows the ability to: 1) evade the immune system; 2) circulate longer in the blood stream; 3) communicate with endothelial cells through receptor–ligand interactions increasing endothelium permeability; 4) transport and release a payload across inflamed endothelium; 5) accumulate in a tumour [4-7].
3) Innovative microfluidic systems were developed to mimick blood capillary circulation, in order to study and to predict the in flow dynamics and the margination tendency of different types of MP, also in presence of blood cells, using real human blood as circulating solution [8].
4) Enzyme-functionalized silica NPs were conceived and produced to digest tumor extracellular matrix, in vitro and in vivo achieving a better penetration in the tumoral tissue [9].
5) pH-responsive hybrid nanoparticles (HNP) were conceived, produced, characterized and successfully used to achieve efficient siRNA delivery in cell culture and in mice models of human breast cancer. HNP are constituted by a shell of cationic hydrogel able to electrostatically bind siRNA and by a supporting nanostructured core of silica that provides mechanical stability to the system. HNP are able to escape from endolysosomal compartment through a proton sponge effect [5,10].
In the light of what has been briefly outlined, the following avenues will be pursued in the near future and will be discussed:
(i) Production and evaluation of different types of MSV for cancer therapy
(ii) Protein and peptide functionalization of NPs to enhance tumor recognition and penetration
(iii) Production and evaluation of different types of NP for siRNA delivery
(iv) Production of Organ-on-chip microfluidic devices able to study and predict the fluidic and biological performance of MP and NP.
(v) Characterization of plasma Protein Corona (PC) [11] of MP and NP by proteomic approaches and correlations between PC composition and targeting of specific tissues.
References
[1] Tasciotti E, Liu X, Bhavane R, Plant K, Leonard AD, Price BK, Cheng MM, Decuzzi P, Tour JM, Robertson F, Ferrari M. Mesoporous silicon particles as a multistage delivery system for imaging and therapeutic applications. Nat Nanotechnol 3, 151-7 (2008).
[2] Martinez JO, Evangelopoulos M, Karun V, Shegog E, Wang JA, Boada C, Liu X, Ferrari M, Tasciotti E. The effect of multistage nanovector targeting of VEGFR2 positive tumor endothelia on cell adhesion and local payload accumulation. Biomaterials 35, 9824-32 (2014).
[3] Martinez JO, Evangelopoulos M, Bhavane R, Acciardo S, Salvatore F, Liu X, Ferrari M, Tasciotti E. Multistage Nanovectors Enhance the Delivery of Free and Encapsulated Drugs. Drug Targets [Epub ahead of print] (2014).
[4] Parodi A, Quattrocchi N, van de Ven AL, Chiappini C, Evangelopoulos M, Martinez JO, Brown BS, Khaled SZ, Yazdi IK, Enzo MV, Isenhart L, Ferrari M, Tasciotti E. Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions. Nat Nanotechnol 8,61-8 (2013).
[5] Parodi A, Corbo C, Cevenini A, Molinaro R, Palomba R, Pandolfi L, Agostini M, Salvatore F, Tasciotti E. Enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. Nanomedicine UK 10,1923-40 (2015).
[6] Corbo C, Parodi A, Evangelopoulos M, Engler DA, Matsunami RK, Engler AC, Molinaro R, Scaria S, Salvatore F, Tasciotti E. Proteomic profiling of a biomimetic drug delivery platform. Curr Drug Targets [Epub ahead of print] (2014).
[7] Palomba R, Parodi A, Evangelopoulos M, Acciardo S, Corbo C, De Rosa E, Yazdi I, Scaria S, Salvatore F, Tasciotti E. A leukolike vector increase the permeability of tumor vasculature. Submitted.
[8] D'Apolito R, Tomaiuolo G, Taraballi F, Minardi S, Kirui D, Liu X, Cevenini A, Palomba R, Ferrari M, Salvatore F, Tasciotti E, Guido S. Red blood cells affect the margination of microparticles in synthetic microcapillaries and intravital microcirculation as a function of their size and shape. J Control Release [Epub ahead of print] (2015).
[9] Parodi A, Haddix SG, Taghipour N, Scaria S, Taraballi F, Cevenini A, Yazdi IK, Corbo C, Palomba R, Khaled SZ, Martinez JO, Brown BS, Isenhart L, Tasciotti E. Bromelain surface modification increases the diffusion of silica nanoparticles in the tumor extracellular matrix. ACS Nano 8,9874-83 (2014).
[10] Khaled SZ, Cevenini A, Yazdi IK, Parodi A, Evangelopoulos M, Corbo C, Scaria S, Hu Y, Chiappini C, Haddix SG, Corradetti B, Salvatore F, Tasciotti E. One-pot synthesis of pH-responsive hybrid nanogel particles for the intracellular delivery of small interfering RNA. Under review for pubblication in Biomaterials.
[11] Corbo C, Molinaro R, Parodi A, Toldeno Furman NE, Salvatore F, Tasciotti E. The impact of the protein corona on nanoparticles and implications for toxicity, immunotoxicity and target drug delivery. Under review for publication in Nanomedicine UK
Metalloproteinases gene expression in a model of genital Chlamydia infection in female BALB/c mice
No full textObjectives.
Chlamydia trachomatis genital infections can cause long-term complications in female reproductive tract, even in absence of acute symptoms. The most common complication is pelvic inflammatory disease (PID), that can lead to tubal infertility and extra-uterine pregnancies. Although the pathologic consequences of Chlamydia genital infection are well-established, the mechanisms leading to tissue damage are not completely understood. In this study we analyzed gene expression of metalloproteinases (MMP-2 and MMP-9) in genital organs obtained from female mice infected by C. muridarum, closely mimicking human infections in the mouse model.
Methods.
All the experiments were approved by the Ethical Committee of the University of Bologna. Animals used were 30 female Balb/c mice, 6-8 weeks old. All the animals received medroxyprogesterone acetate 9 and 2 days prior the infection. Twelve mice were infected by placing 15 μl of sucrose-phosphate-glutamic acid (SPG) buffer containing 106 inclusion forming units (IFUs) of C. muridarum into the vaginal vault. Twelve animals were treated with 15 μl of SPG containing heat-inactivated 106 IFUs of C. muridarum. As controls of inflammation, 6 animals were challenged with 15 μl of SPG. At 3, 10, and 20 days post-infection 4 infected animals, 4 animals inoculated with heat-inactivated bacteria and 2 controls were sacrificed. Genital tracts were divided into the cervical-vaginal region, uterine horns, and oviducts. A part of uterine horns, oviducts and vagina were stored in formalin and later processed for histological examinations. The remaining parts of the organs were used for RNA extraction, by using Trizol Reagent (Invitrogen), in combination
with RNeasy Mini Kit (Qiagen). cDNA was synthesized with 500 ng of total RNA and SuperScript III RT (Invitrogen). Real-time RT-PCR was performed with SYBR Green Fast start kit (Roche Diagnostics). Primers used in Real-time RT-PCR to assess GAPDH, MMP-2, and MMP-9 levels were from SuperArray (SABiosciences Corporation).
Results.
At histological examination no controls showed inflammation. On the contrary, scores of inflammation in all the organs from infected animals peaked at day 10, whereas only a single animal inoculated with inactivated bacteria showed a very mild inflammation at day 10 in its right uterine horn. At day 10, organs from infected animals showed significantly higher gene expression of MMP-2 and MMP-9 than the respective organs obtained from non-infected mice.
Conclusions.
Our study showed statistically significant higher MMPs gene expression in infected animals compared to both controls and animals injected with inactivated chlamydiae. These results confirm the pivotal role of MMPs in the development of tissue damage observed during Chlamydia genital infection
Ventilator setting dependence of parameters for detecting expiratory flow limitation in mechanical ventilation: a simulation study
No full text- …
