Surgical treatment of scoliosis in a rare disease: arthrogryposis

Background

The reported incidence of scoliosis in arthrogryposis varies from 30% to 67% and, in most cases, the curves progress rapidly and become stiff from early age. The authors report six cases of scoliosis in arthrogryposis to assess the role of surgical treatment.

Methods

Six cases (3 males, 3 females; mean age at surgery 13.2 years) with arthrogryposis multiplex congenita associated with the characteristic amyoplasia were reviewed: they were operated on for scoliosis at the authors' Spine Surgery Department between 1987 and 2008. Surgery was performed using the Harrington-Luque instrumentation (2 cases), the Luque system (1), a hybrid segmental technique with hooks and screws (1) and spinal anchoring with pedicle screws (2).

Results

The patients were clinically and radiologically reviewed at a mean follow-up of 4.2 years, ± 2.7 (range, 1 to 9 years). Three minor postoperative complications were encountered; a long-term pulmonary complication was seen in one case after reintervention and was successfully resolved after 10 days. Surgery was successful in the other 5 cases, where solid arthrodesis was achieved and no significant curve progression was observed at follow-up.

Conclusions

The experience acquired with the present case series leads the authors to assert that prompt action should be taken when treating such aggressive forms of scoliosis. In case of mild spinal deformities in arthrogryposis, brace treatment should be attempted, the evolution of the curves being unpredictable; however, when the curve exceeds 40° and presents with marked hyperkyphosis, hyperlordosis or pelvic obliquity, surgery should not be delayed.     

Itraconazole affects Toxoplasma gondii endodyogeny

The antifungal agent itraconazole is an effective drug against systemic mycoses inhibiting cytochrome P-450-mediated ergosterol synthesis, essential for fungal survival. In this work, we show the activity of this azole as a potential agent against Toxoplasma gondii, the causative agent of toxoplasmosis. Monolayers of LLC-MK2 epithelial cells infected with tachyzoites of RH strain were incubated with different concentrations of itraconazole for 24 and 48 h. The IC(50) values obtained were 114.0 and 53.6 nM for 24 and 48 h, respectively. Transmission electron microscopy (TEM) analysis of itraconazole-treated intracellular tachyzoites showed endoplasmic reticulum and nuclear envelope swelling. The drug also caused rupture of the parasite's surface membrane and affected the parasite's division by endodyogeny. This observation was confirmed both by fluorescence microscopy of cells labeled with diamidino-2-phenylindole and by three-dimensional reconstruction of serial thin sections analyzed by TEM. The treatment with itraconazole led to the formation of a mass of daughter cells, suggesting the interruption of the scission process during the parasite's cell division.     

Thiolactomycin analogues as potential anti-Toxoplasma gondii agents

The discovery of new compounds active against Toxoplasma gondii is extremely important due to the severe disease caused by this pathogen in immunocompromised hosts and to congenital infection. Type II fatty acid biosynthesis has shown to be a promising target for drug intervention in toxoplasmosis. Here we describe the inhibitory effect of 8 thiolactomycin (TLM) analogues against tachyzoite-infected LLC-MK₂ cells. The TLM analogues demonstrated anti-T. gondii activity, arresting tachyzoite proliferation with IC₅₀ values in the micromolar level after 24 h and 48 h of treatment. Metabolic labelling of extracellular parasites treated with TLM analogues using [³H]acetate demonstrated that these drugs affected acylglycerol synthesis. The rapid reduction of parasite load suggests that these compounds have selective cytotoxic effects against T. gondii. Transmission electron microscopy demonstrated that TLM analogues interfered with membrane-bounded organelles and parasite division and this in turn affected parasite development and survival.     

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK₂ epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK₂ cells after 4 days of infection, when 72.3 ± 15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.     

Activation of the P2X7 receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages

Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which is widespread throughout the world. After active penetration, the parasite is enclosed within a parasitophorous vacuole and survives in the host cell by avoiding, among other mechanisms, lysosomal degradation. A large number of studies have demonstrated the importance of ATP signalling via the P2X₇ receptor, as a component of the inflammatory response against intracellular pathogens. Here we evaluate the effects of extracellular ATP on T. gondii infection of macrophages. ATP treatment inhibits the parasite load and the appearance of large vacuoles in the cytoplasm of intracellular parasites. ROS and NO assays showed that only ROS production is involved with the ATP effects. Immunofluorescence showed colocalization of Lamp1 and SAG1 only after ATP treatment, suggesting the formation of phagolysosomes. The involvement of P2X₇ receptors in T. gondii clearance was confirmed by the use of P2X₇ agonists and antagonists, and by infecting macrophages from P2X₇ receptor-deficient mice. We conclude that parasite elimination might occur following P2X₇ signalling and that novel therapies against intracellular pathogens could take advantage of activation of purinergic signalling.     

Bacteroides fragilis adherence to Caco-2 cells

The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.     

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.     

Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids

Solanum nudum Dunal (Solanaceae) is a plant used in traditional medicine in Colombian Pacific Coast, from which five steroids denominated SNs have been isolated. The SNs compounds have antiplasmodial activity against asexual blood stages of Plasmodium falciparum strain 7G8 with an IC₅₀ between 20–87 µM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We found that trophozoite and schizont stages were the most sensitive to SNs. By Giemsa-stained smears, induction of crisis forms was observed. Transmission electron microscopy of treated parasites showed morphological abnormalities such as a cytoplasm rich in vesicles and myelinic figures. The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment.     

Toxoplasma gondii: Fluconazole and itraconazole activity against toxoplasmosis in a murine model

Toxoplasma gondii is an important opportunistic pathogen affecting immunocompromised patients with AIDS. Toxoplasmic encephalitis is responsible for high morbidity and mortality. In this study, we investigated the activity of the antifungals fluconazole (FLZ) and itraconazole (ITZ) against T. gondii in mice infected with the Me49 strain. As previously reported for ITZ, FLZ also demonstrated a selective effect against T. gondii in vitro; the IC₅₀ values obtained for FLZ were 8.9 μM and 3.1 μM after 24 h and 48 h of treatment, respectively. A 10-day treatment of mice with orally or intraperitoneally administered 20 mg/kg/day FLZ showed a significant survival difference compared to untreated mice. The administration of 20 mg/kg/day ITZ significantly reduced the brain cyst burden compared to untreated mice but did not exert significant protection against death. The results obtained in this work are rather promising as ITZ and FLZ are safe and low-cost drugs available on the market.     

Bacteroides fragilis interferes with iNOS activity and leads to pore formation in macrophage surface

Bacteroides fragilis is the anaerobe most commonly recoverable from clinical specimens. The wide genetic diversity of this bacterium related with virulence potential is still an open question. In this study, we analyzed the morphological aspects and microbicide action of M? during interactions with B. fragilis. A filamentous cytoplasm content release and a different actin organization colocalized with iNOS were detected. It was also possible to observe the reduction of NO production in the same conditions. The scanning electron microscopy showed the formation of pore-like structures in the surface of macrophages in the bacterial presence and by transmission electron microscopy we could observe the extrusion of cytoplasm contents as well as the condensation of chromatin in the nucleus periphery. These data suggest the existence of an inhibitory mechanism developed by B. fragilis strains for one of the macrophage microbicide actions.