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Objectives. 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.

Ulcerative colitis (UC) is a chronic inflammatory condition of the rectum and colon. The conventional medical treatment of inflammatory bowel disease (IBD) involves anti-inflammatory, immunosuppressive drugs and antibiotics.1 Recently, considerable attention has been given to the identification of naturally occurring substances, particularly those present in dietary and medicinal plants as a useful tool in the prevention and treatment of many pathological conditions. Turmeric (Curcuma longa L.) is extensively used in Ayurvedic medicine as a treatment for several inflammatory and functional conditions of the gastrointestinal tract such as diarrhea. Since intestinal motility has been reported to be impaired in IBD patients, our investigation was aimed at evaluating whether turmeric, besides its anti-inflammatory properties, exerts also a myorelaxant effect on the intestine. Acute and chronic colitis were induced in Balb/c mice by Dextran Sulphate Sodium (DSS) administration (5% and 2.5%,w/v, respectively) and either Curcuma extract (200 mg/kg/day) or placebo was thereafter administered for 7 and 21 days respectively. Similarly Curcuma extract was administered to healthy mice and in vitro evaluated in healthy intestine. The biological activity of Curcuma extract was evaluated towards Carbachol induced contraction in isolated ileum and colon in healthy animals, acute and chronic colitis animals and after administration of Curcuma.3 Curcuma extract reduced the spontaneous contractions in the ileum and colon both in the healthy intestine and in the pathological intestinal strips. In addition, it inhibited the maximal response to Carbachol in a non-competitive and reversible manner. Similar results were observed in ileum and colon from Curcuma fed mice. The administration of DSS reduced the motility, mainly in the colon; Curcuma almost restored both the spontaneous contractions and the response to Carbachol after 14 days assumption, compared to standard diet; however a prolonged administration of Curcuma, once the mucosal damage of the intestine was repaired, reduced the spontaneous and Carbachol-induced contractions.3 Curcuma extract has been shown to exert a direct and indirect myorelaxant effect on mouse ileum and colon, independent of the anti-inflammatory effect. The effect is reversible and non-competitive and it is present also in the normal intestine. These results suggest the use of curcuma extract as a spasmolytic agent 3 not only in IBD, but also as a useful tool in the management of functional disorders References 1. Bossa, F.; Colombo, E.; Andriulli, A.; Annese, V. Treatment of steroid-naive ulcerative colitis. Expert Opin Pharmacother, 2009, 10, 1449-1460. 2. Jurenka, J.S. Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: a review of preclinical and clinical research. Altern Med Rev, 2009, 14, 141-153. 3. Aldini, R.; Budriesi, R.; Roda, G.; Micucci, M.; Ioan, P.; D’Errico-Grigioni, A.; Sartini, A.; Guidetti, E.; Marocchi, M.; Cevenini, M.; Rosini, F.; Montagnani, M.; Chiarini, A.; Mazzella, G. Effect of Curcuma longa extract on the intestinal motility in acute and chronic DSS induced colitis in mice. Plose One, 2012, 7, e44650.

Background. Although the pathologic consequences of Chlamydia genital infection are well-established, the mechanisms leading to tissue damage are not completely understood. Methods. All the experiments were approved by the Ethical Committee of the University of Bologna. Animals used were 24 female Balb/c mice, 7 weeks old. All 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. Nine animals were treated with 15 µl of SPG containing heat-inactivated 106 IFUs of C. muridarum. As controls of inflammation, 3 animals were challenged with 15 µl of SPG. At 3, 10, and 20 days post-infection 4 infected animals, 3 animals inoculated with heat-inactivated bacteria and 1 control were sacrificed. Genital tracts were divided into the cervical-vaginal region, uterine horns, and oviducts. Right uterine horns and oviducts 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 INF-γ, TNF-α, MMP-2, MMP-9 and GAPDH 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 uterus. At day 10, organs from infected animals showed significantly higher gene expression for MMP-2 and MMP-9 than the respective organs obtained from non-infected mice. Conclusions. Our study confirms the pivotal role of MMPs in the development of tissue damage.

Usefulness 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...

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. Here we report about a comparison between Positron Emitting Tomography and traditional histology, in a mouse model of genital C. muridarum infection. METHODS Animals used were 22 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. Eighteen mice were challenged intravaginally with 10^6 IFUs of C. muridarum under Ketamine anaesthesia. As control, 4 animals were challenged with sucrose phosphate buffer. At 5, 12 and 18 days after challenge 9 infected mice and 1 control underwent a 11C-Choline PET. Each animal was anaesthetised and injected with approximately 20 MBq of 11C-Choline. Images were acquired with a small animal PET tomograph for 20 min. Standard uptake value (SUV) was calculated measuring concentration of labelled tracer in the region of interest and correcting it for body weight and injected dose. Twelve infected mice were sacrificed to study histology of genital tract at 5, 12, 18 days after infection (3 infected animals, plus 1 control at each point). Sections of hysterectomy specimen were preserved in 10% neutral buffered formalin for 48h 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. RESULTS At 5 days, mean vaginal SUV (±SD) of cases was 0.83±0.39, whereas the control showed a value of 0.278. At 12 and 18 days, SUV of cases increased to 0.94±0.25 and 1.17±0.21, respectively, whereas SUV of the control was 0.299 and 0.302, respectively. All sections of hysterectomy of infected mice showed an intense grade of inflammation, characterized by an acute infiltrate in the initial phase, and by a prevalent chronic infiltrate in the successive period. Tissues collected from the 3 control mice showed only mild proliferation of endometrium. CONCLUSION 11C-Choline PET seems to be a promising diagnostic technique to assess inflammation due to Chlamydia genital infection.

Different techniques were used to assess gastric emptying (GE) in small animals; most of them require sophisticated equipment, animal sacrifice and are expensive. In the present investigation a simple, non-invasive method based on bioluminescence imaging (BLI) is reported to study GE, using light-emitting Escherichia coli cells as a marker of the gastric content.A new thermostable red-emitting luciferase was chosen as reporter gene to transform E. coli cells. Bioluminescent (BL) bacteria were administered to fasting mice, after a solid meal, and in response to different doses of metoclopramide (MET) and hyoscine butylbromide (HY). Bioluminescence imaging allowed to evaluate the real time 2D spatial and temporal distribution of bacteria along the gastrointestinal tract in animals and to calculate GE rate in basal conditions and following pharmacological stimulation. The administered BL bacteria were easily imaged and localized in the stomach and subsequently followed in the duodenum and upper intestine allowing to accurately calculate GE. Gastric emptying after the test meal was significantly slower (T(1/2) 16 ± 3 min) than that obtained in fasting conditions (T(1/2) 2 ± 1 min); administration of HY (1 mg kg(-1) b.w.) significantly (P 0.05) increased T(1/2) that was delayed up to 25 ± 4 min; MET (1 mg kg(-1) b.w.) significantly (P 0.05) accelerated T(1/2), that was achieved within 8 ± 2 min.The reported model is simple, inexpensive, reliable, sensitive and accurate; it can detect both acceleration and slowdown of GE. The model is useful in the investigation of new drug-induced alterations of gastric motility allowing to reduce the number of experimental animals.

The nuclear receptor PPAR-alpha (Peroxisome proliferator-activated receptor-alpha) regulates the transcription of genes involved in fatty acid and bile acid metabolism and transport. PPAR-alpha is activated following binding with natural (fatty acids) or exogenous (fibrates) ligands. A common polymorphism in the PPAR-alpha gene (PPARA) is a functional missense mutation which causes a leucine to valine substitution (L162V). Another frequent polymorphism is a G to C transversion in intron 7. In order to test the etiopathogenic relevance of PPARA polymorphisms in gallstone disease, we investigated the prevalence of exon 5 and intron 7 variants, either isolated or in combination, in gallstone patients and in a control population. One hundred and twenty seven gallstone patients (56 males, 71 females) admitted to the hospital for either elective cholecystectomy or endoscopic treatment of choledocolithiasis, were screened for the presence of PPARA polymorphisms. Control population was composed of 110 age matched hospital workers and university students (53 males, 57 females). After DNA extraction from peripheral blood leukocytes, PCR amplification of exon 5 and intron 7 was performed. Denaturing-high performance liquid chromatography (D-HPLC) was then used to screen the PCR products for the presence of polymorphisms. Automated sequencing was employed to confirm and characterize the polymorphisms. Statistical analysis was performed by the chi-square test. The overall carrier frequency of exon 5 L162V polymorphism was 11% in the control population and 13.4% in the gallstone group (p=NS). The total carrier frequency for intron 7 polymorphism was 25% in the control population, while in male and female lithiasic patients it was 43% and 37%, respectively (p= 0.03). The haplotype characterized by exon 5 L162V, in the absence of intron 7 polymorphism, was not found in males with gallstones, while a 11% frequency was observed in male controls (p=0.02). These data demonstrate a significant higher prevalence of PPARA intron 7 polymorphism in gallstone patients, mainly due to its frequency in the male subgroup. Furthermore, the haplotype characterized by exon 5 L162V, in the absence of an associated intron 7 polymorphism, seems to confer protection from gallstones in males but not in females, supporting an hormone modulation on PPAR-alpha polymorphic variants.

Stimulation of isolated rat Kupffer cells by viable Leptospira interrogans, Treponema pallidum and Borrelia garinii elicited cellular response resulting in the release of different tumor necrosis factor alpha (TNF-) levels, depending on the spirochetes. L. interrogans induced TNF- levels higher than those achieved with, in the order, B. garinii and T.pallidum, but lower than the levels achieved with lipopolysaccharide (LPS). In contrast with L. interrogans, pretreatment of borreliae and treponemes with polymyxin B did not substantially diminish the ability of B. garinii and T. pallidum to stimulate Kupffer cells. Purified T. pallidum lipoproteins TpN47, TmpA, TpN15- TpN17, and B. gariniii OspA induced TNF- response comparable to that achieved by LPS. This response was almost insensitive to the action of polymyxin B.