Experimental toxoplasmosis in Balb/c mice. Prevention of vertical disease transmission by treatment and reproductive failure in chronic infection

In a study of congenital transmission during acute infection of Toxoplasma gondii, 23 pregnant Balb/c mice were inoculated orally with two cysts each of the P strain. Eight mice were inoculated 6-11 days after becoming pregnant (Group 1). Eight mice inoculated on the 10th-15th day of pregnancy (Group 2) were treated with 100 mg/kg/day of minocycline 48 h after inoculation. Seven mice inoculated on the 10th-15th day of pregnancy were not treated and served as a control (Group 3). Congenital transmission was evaluated through direct examination of the brains of the pups or by bioassay and serologic tests. Congenital transmission was observed in 20 (60.6%) of the 33 pups of Group 1, in one (3.6%) of the 28 pups of Group 2, and in 13 (54.2%) of the 24 pups of Group 3. Forty-nine Balb/c mice were examined in the study of congenital transmission of T. gondii during chronic infection. The females showed reproductive problems during this phase of infection. It was observed accentuated hypertrophy of the endometrium and myometrium. Only two of the females gave birth. Our results demonstrate that Balb/c mice with acute toxoplasmosis can be used as a model for studies of congenital T. gondii infection. Our observations indicate the potential of this model for testing new chemotherapeutic agents against congenital toxoplasmosis.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

In vivo synergism of ceftobiprole and vancomycin against experimental endocarditis due to vancomycin-intermediate Staphylococcus aureus.

The efficacy of ceftobiprole combined with vancomycin was tested against two vancomycin-intermediate Staphylococcus aureus (VISA) strains, PC3 and Mu50, in rats with experimental endocarditis. Animals with infected aortic vegetations were treated for 3 days with doses simulating the kinetics after intravenous administration in humans of (i) the standard dose of ceftobiprole of 500 mg every 12 h (b.i.d.) (SD-ceftobiprole), (ii) a low dose of ceftobiprole of 250 mg b.i.d. (LD-ceftobiprole), (iii) a very low dose of ceftobiprole of 125 mg b.i.d. (VLD-ceftobiprole), (iv) SD-vancomycin of 1 g b.i.d., or (v) LD- or VLD-ceftobiprole combined with SD-vancomycin. Low dosages of ceftobiprole were purposely used to highlight positive drug interactions. Treatment with SD-ceftobiprole sterilized 12 of 14 (86%) and 10 of 13 (77%) vegetations infected with PC3 and Mu50, respectively (P < 0.001 versus controls). In comparison, LD-ceftobiprole sterilized 10 of 11 (91%) vegetations infected with PC3 (P < 0.01 versus controls) but only 3 of 12 (25%) vegetations infected with Mu50 (P > 0.05 versus controls). VLD-ceftobiprole and SD-vancomycin alone were ineffective against both strains (≤8% sterile vegetations). In contrast, the combination of VLD-ceftobiprole and SD-vancomycin sterilized 7 of 9 (78%) and 6 of 14 (43%) vegetations infected with PC3 and Mu50, respectively, and the combination of LD-ceftobiprole and SD-vancomycin sterilized 5 of 6 (83%) vegetations infected with Mu50 (P < 0.05 versus controls and monotherapy). Thus, ceftobiprole monotherapy simulating standard therapeutic doses was active against VISA experimental endocarditis. Moreover, subtherapeutic LD- and VLD-ceftobiprole synergized with ineffective vancomycin to restore efficacy. Hence, combining ceftobiprole with vancomycin broadens the therapeutic margin of these two compounds against VISA infections.

Aotus infulatus monkey is susceptible to Plasmodium falciparum infection and may constitute an alternative experimental model for malaria

Aotus is one of the WHO-recommended primate models for studies in malaria, and several species can be infected with Plasmodium falciparum or P. vivax. Here we describe the successful infection of the species A. infulatus from eastern Amazon with blood stages of P. falciparum. Both intact and splenectomized animals were susceptible to infection; the intact ones were able to keep parasitemias at lower levels for several days, but developed complications such as severe anemia; splenectomized monkeys developed higher parasitemias but no major complications. We conclude that A. infulatus is susceptible to P. falciparum infection and may represent an alternative model for studies in malaria.

Action of pentoxifylline on experimental cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis

In the animal model of leishmaniasis caused by Leishmania (Leishmania) amazonensis there is a complex mechanism of the host-parasite interaction. The present study was performed to interfere with the inflammatory reaction to the parasites, through immune modulation. Female C5BL/6 isogenic mice were used, some of which were inoculated on the right ear and others on the right footpad with 3.10(6) stationary phase promastigotes of the MHOM/BR/PH8 strain of L. (L.) amazonensis, and were allocated in three groups: the first received pentoxifylline 8mg/kg every 12 h, since the first day; the second one received the same dose since the 40th day of infection and a control group that did not receive any treatment. All the ears excised were analyzed to determine the variation in weight between both ears and for histopathological analyses. A quantification of the parasites was done using the limiting dilution assay. A significant reduction of the number of parasites, was observed among the animals treated which had an accordingly significant reduction on the weight of the ears. Pentoxifylline reduced the macrophages propensity to vacuolation and induced a more effective destruction of the parasites by these cells. Moreover, the group that began the treatment later did not show the same effectiveness.

Jornada de camp a la Unitat Experimental de la Terra Alta (UETA)

La Unitat Experimental de la Terra Alta (IRTA – INCAVI) acull diverses parcel·les experimentals basades en els tres conreus més importants de la Terra Alta: vinya, ametller i olivera. La jornada que us proposem enguany fa un èmfasi especial en els dos conreus típics de la fructicultura mediterrània (olivera i ametller), comptant amb la presència dels investigadors de l’IRTA: Joan Tous (nous models de producció en olivera) i Francisco Vargas (varietats d’ametller IRTA). Finalment, els assistents que així ho desitgin podran visitar, com cada any, els diferents assajos que tenim establerts en els tres conreus assenyalats.

Life-history traits of Fossaria cubensis (Gastropoda: Lymnaeidae) under experimental exposure to Fasciola hepatica (Trematoda: Digenea)

The effect of exposing the lymnaeid snail Fossaria cubensis to the trematode Fasciola hepatica on the snail population's life-history traits was studied under laboratory conditions. Exposed individuals showed a lower survival rate than control snails, although from week 7 onward a slower decrease of this parameter in relation to the control group was observed. There were higher values of fecundity rate for the controls compared to the exposed group except during weeks 9, 10, 11 and 12, which was the time that followed the period when almost all of the infected snails died. Both the intrinsic and finite rates of natural increase were significantly higher for the control group, but exposed snails still attained a lower mean generation time. Age-specific trade-offs were found, mainly for the weekly increase in size versus the number of eggs per mass, the weekly increase in size versus the number of viable eggs per mass, the number of masses versus the hatching probability and the number of eggs versus the hatching probability. All these negative associations were significant for juveniles of both control and exposed snails and not for adults; however, exposed young individuals exhibited much higher values of the correlation coefficient than control animals.

Characterization of Nedd4-2 ubiquitin ligase expression in experimental neuropathic pain

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs). The ubiquitin ligase Nedd4-2 regulates sodium channels and we have previously demonstrated in expression systems that this protein decreases the Nav1.7 current. Nav1.7 is the most abundant VGSC in dorsal root ganglion (DRG) and is a major contributor to pain perception. We hypothesize that Nedd4-2 modulates Nav1.7 channel density at the neuronal cell membrane and the goal of this present experiment is to characterize Nav1.7 and Nedd4-2 expression in the context of neuropathic pain. Methods: Biotinylation, Western Blot and Immunohistochemistry experiments for Nav1.7 and Nedd4-2 were performed in HEK transfected cells or in rodent DRGs 7 days after SNI surgery. We used antibodies against Nedd4-2 and Nav1.7 and several comarkers of DRG neurons (Peripherin for nociceptors, NF-200 for large myelinated cells, ATF3 for injured neurons). Data are expressed in proportion of positive cells (%) and protein signal ratio } SEM, n = 3-4 in each condition. Results: In HEK293 cells, upon co-expression of Nedd4-2, a decrease of 50% of Nav1.7 signal at the membrane is demonstrated (p ≤0.005). Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 } 1.2%) versus sham group (43.4 } 3.5%) (p <0.005). Nedd4-2 is mainly colocalized with markers of small neurons and almost absent in large neurons. In addition, Nedd4-2 is predominantly decreased in injured ATF3 positive cells. Conclusion: Our results indicate that Nedd4-2 decreases Nav1.7 channels and currents at the cell membrane and that it is mainly expressed in nociceptors and downregulated after nerve injury. Taken together, our data suggest that the reduction of Nedd4-2, after nerve injury, modulates Nav1.7 activity and can contribute to neuropathic pain. We will further try to restore a normal level of Nedd4.2 via a gene therapy approach with viral vectors in order to soothe symptoms of neuropathic pain.

Experimental peripheral arterial disease: new insights into muscle glucose uptake, macrophage, and T-cell polarization during early and late stages.

Peripheral arterial disease (PAD) is a common disease with increasing prevalence, presenting with impaired walking ability affecting patient's quality of life. PAD epidemiology is known, however, mechanisms underlying functional muscle impairment remain unclear. Using a mouse PAD model, aim of this study was to assess muscle adaptive responses during early (1 week) and late (5 weeks) disease stages. Unilateral hindlimb ischemia was induced in ApoE(-/-) mice by iliac artery ligation. Ischemic limb perfusion and oxygenation (Laser Doppler imaging, transcutaneous oxygen pressure assessments) significantly decreased during early and late stage compared to pre-ischemia, however, values were significantly higher during late versus early phase. Number of arterioles and arteriogenesis-linked gene expression increased at later stage. Walking ability, evaluated by forced and voluntary walking tests, remained significantly decreased both at early and late phase without any significant improvement. Muscle glucose uptake ([18F]fluorodeoxyglucose positron emission tomography) significantly increased during early ischemia decreasing at later stage. Gene expression analysis showed significant shift in muscle M1/M2 macrophages and Th1/Th2 T cells balance toward pro-inflammatory phenotype during early ischemia; later, inflammatory state returned to neutrality. Muscular M1/M2 shift inhibition by a statin prevented impaired walking ability in early ischemia. High-energy phosphate metabolism remained unchanged (31-Phosphorus magnetic resonance spectroscopy). Results show that rapid transient muscular inflammation contributes to impaired walking capacity while increased glucose uptake may be a compensatory mechanisms preserving immediate limb viability during early ischemia in a mouse PAD model. With time, increased ischemic limb perfusion and oxygenation assure muscle viability although not sufficiently to improve walking impairment. Subsequent decreased muscle glucose uptake may partly contribute to chronic walking impairment. Early inflammation inhibition and/or late muscle glucose impairment prevention are promising strategies for PAD management.

Experimental evolution.

Experimental evolution is the study of evolutionary processes occurring in experimental populations in response to conditions imposed by the experimenter. This research approach is increasingly used to study adaptation, estimate evolutionary parameters, and test diverse evolutionary hypotheses. Long applied in vaccine development, experimental evolution also finds new applications in biotechnology. Recent technological developments provide a path towards detailed understanding of the genomic and molecular basis of experimental evolutionary change, while new findings raise new questions that can be addressed with this approach. However, experimental evolution has important limitations, and the interpretation of results is subject to caveats resulting from small population sizes, limited timescales, the simplified nature of laboratory environments, and, in some cases, the potential to misinterpret the selective forces and other processes at work.

Experimental evaluation of an electromechanical artificial urinary sphincter in an animal model.

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The AMS 800 urinary control system is the gold standard for the treatment of urinary incontinence due to sphincter insufficiency. Despite excellent functional outcome and latest technological improvements, the revision rate remains significant. To overcome the shortcomings of the current device, we developed a modern electromechanical artificial urinary sphincter. The results demonstrated that this new sphincter is effective and well tolerated up to 3 months. This preliminary study represents a first step in the clinical application of novel technologies and an alternative compression mechanism to the urethra. OBJECTIVES: To evaluate the effectiveness in continence achievement of a new electromechanical artificial urinary sphincter (emAUS) in an animal model. To assess urethral response and animal general response to short-term and mid-term activation of the emAUS. MATERIALS AND METHODS: The principle of the emAUS is electromechanical induction of alternating compression of successive segments of the urethra by a series of cuffs activated by artificial muscles. Between February 2009 and May 2010 the emAUS was implanted in 17 sheep divided into three groups. The first phase aimed to measure bladder leak point pressure during the activation of the device. The second and third phases aimed to assess tissue response to the presence of the device after 2-9 weeks and after 3 months respectively. Histopathological and immunohistochemistry evaluation of the urethra was performed. RESULTS: Bladder leak point pressure was measured at levels between 1091 ± 30.6 cmH2 O and 1244.1 ± 99 cmH2 O (mean ± standard deviation) depending on the number of cuffs used. At gross examination, the explanted urethra showed no sign of infection, atrophy or stricture. On microscopic examination no significant difference in structure was found between urethral structure surrounded by a cuff and control urethra. In the peripheral tissues, the implanted material elicited a chronic foreign body reaction. Apart from one case, specimens did not show significant presence of lymphocytes, polymorphonuclear leucocytes, necrosis or cell degeneration. Immunohistochemistry confirmed the absence of macrophages in the samples. CONCLUSIONS: This animal study shows that the emAUS can provide continence. This new electronic controlled sequential alternating compression mechanism can avoid damage to urethral vascularity, at least up to 3 months after implantation. After this positive proof of concept, long-term studies are needed before clinical application could be considered.

Effect of interferon-alpha on experimental septal fibrosis of the liver - study with a new model

Interferon-alpha is used in antiviral therapy in humans, mainly for viral hepatitis B and C. An anti-fibrotic effect of interferon has been postulated even in the absence of anti-viral response, which suggests that interferon directly inhibits fibrogenesis. Rats infected with the helminth Capillaria hepatica regularly develop diffuse septal fibrosis of the liver, which terminates in cirrhosis 40 days after inoculation. The aim of this study was to test the anti-fibrotic effect of interferon in this experimental model. Evaluation of fibrosis was made by three separate methods: semi-quantitative histology, computerized morphometry and hydroxyproline measurements. Treatment with interferon-alpha proved to inhibit the development of fibrosis in this model, especially when doses of 500,000 and 800,000 IU were used for 60 days. Besides confirming the anti-fibrotic potential of interferon-alpha on a non-viral new experimental model of hepatic fibrosis, a clear-cut dose-dependent effect was observed.

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.

Resistance to starvation of Rhodnius neivai Lent, 1953 (Hemiptera: Reduviidae: Triatominae) under experimental conditions

The period of resistance to starvation and the loss of weight until death of Rhodnius neivai in all stages of development were studied. Work was based on experiments conducted under controlled laboratory conditions. One hundred specimens of each nymphal instar were observed: 50 were fed on chicken and 50 on rabbit. Adult females and males were kept together and fed on each host. All bugs were weighed weekly until death. Laid eggs were collected weekly and observed during five weeks to obtain hatchability. Resistance to starvation was similar with both hosts and increased with the evolutionary stage, excepting the 5th nymphal instar and adults. With both hosts, loss of weight was abrupt in the first week and steady in the following weeks. In adults, on the first weeks after eating, there was little or no mortality, after which mortality increased rapidly with the starving time. Reproductive output was higher in the bugs fed on rabbit. R. neivai is among the least resistant triatomine species.