Treatment of large bone defects with the Ilizarov technique.

Between 1985 and 1990 we treated 11 large segmental bone defects (average 6.7 cm) in ten patients with the Ilizarov technique. Open fractures, type III according to Gustilo, represented the largest group (8 of 11 cases). The average delay before the Ilizarov technique was initiated was 8.9 months. The external fixator was usually maintained for 1 year. Bone regeneration was obtained in every case. Consolidation was not fulfilled with this technique in three cases. The complications observed were one refracture, four leg-length discrepancies (average 1.5 cm), and five axial deformities exceeding 5 degrees. No pin-track infection was observed. In our limited series of four type IIIC open fractures treated by the Ilizarov technique, no patients required amputation. The Ilizarov technique is particularly useful in the treatment of large bone defects, without major complications, especially if there is an adequate initial debridement.

Imaging Properties of MS Lesions Correlate with Attention Deficits in Early Multiple Sclerosis

Introduction: Cognitive impairment affects 40-65% of multiple sclerosis (MS) patients, often since early stages of the disease (relapsing remitting MS, RRMS). Frequently affected functions are memory, attention or executive abilities but the most sensitive measure of cognitive deficits in early MS is the information processing speed (Amato, 2008). MRI has been extensively exploited to investigate the substrate of cognitive dysfunction in MS but the underlying physiopathological mechanisms remain unclear. White matter lesion load, whole-brain atrophy and cortical lesions' number play a role but correlations are in some cases modest (Rovaris, 2006; Calabrese, 2009). In this study, we aimed at characterizing and correlating the T1 relaxation times of cortical and sub-cortical lesions with cognitive deficits detected by neuropsychological tests in a group of very early RR MS patients. Methods: Ten female patients with very early RRMS (age: 31.6 ±4.7y; disease duration: 3.8 ±1.9y; EDSS disability score: 1.8 ±0.4) and 10 age- and gender-matched healthy volunteers (mean age: 31.2 ±5.8y) were included in the study. All participants underwent the following neuropsychological tests: Rao's Brief Repeatable Battery of Neuropsychological tests (BRB-N), Stockings of Cambridge, Trail Making Test (TMT, part A and B), Boston Naming Test, Hooper Visual Organization Test and copy of the Rey-Osterrieth Complex Figure. Within 2 weeks from neuropsychological assessment, participants underwent brain MRI at 3T (Magnetom Trio a Tim System, Siemens, Germany) using a 32-channel head coil. The imaging protocol included 3D sequences with 1x1x1.2 mm3 resolution and 256x256x160 matrix, except for axial 2D-FLAIR: -DIR (T2-weighted, suppressing both WM and CSF; Pouwels, 2006) -MPRAGE (T1-weighted; Mugler, 1991) -MP2RAGE (T1-weighted with T1 maps; Marques, 2010) -FLAIR SPACE (only for patient 4-10, T2-weighted; Mugler, 2001) -2D Axial FLAIR (0.9x0.9x2.5 mm3, 256x256x44 matrix). Lesions were identified by one experienced neurologist and radiologist using all contrasts, manually contoured and assigned to regional locations (cortical or sub-cortical). Lesion number, volume and T1 relaxation time were calculated for lesions in each contrast and in a merged mask representing the union of the lesions from all contrasts. T1 relaxation times of lesions were normalized with the mean T1 value in corresponding control regions of the healthy subjects. Statistical analysis was performed using GraphPad InStat software. Cognitive scores were compared between patients and controls with paired t-tests; p values ≤ 0.05 were considered significant. Spearmann correlation tests were performed between the cognitive tests, which differed significantly between patients and controls, and lesions' i) number ii) volume iii) T1 relaxation time iv) disease duration and v) years of study. Results: Cortical and sub-cortical lesions count, T1 values and volume are reported in Table 1 (A and B). All early RRMS patients showed cortical lesions (CLs) and the majority consisted of CLs type I (lesions with a cortical component extending to the sub-cortical tissue). The rest of cortical lesions were characterized as type II (intra-cortical lesions). No type III/IV lesions (large sub-pial lesions) were detected. RRMS patients were slightly less educated (13.5±2.5y vs. 16.3±1.8y of study, p=0.02) than the controls. Signs of cortical dysfunction (i.e. impaired learning, language, visuo-spatial skills or gnosis) were rare in all patients. However, patients showed on average lower scores on measures of visual attention and information processing speed (TMT-part A: p=0.01; TMT-part B: p=0.006; PASAT-included in the BRB-N: p=0.04). The T1 relaxation values of CLs type I negatively correlated with the TMT-part A score (r=0.78, p<0.01). The correlations of TMT-part B score and PASAT score with T1 relaxation time of lesions as well and the correlation between TMT-part A, TMT-part B and PASAT score with lesions' i) number ii) volume iii) disease duration and iv) years of study did not reach significance. In order to preclude possible influences from partial volume effects on the T1 values, the correlation between lesion volume and T1 value of CLs type I was calculated; no correlation was found, suggesting that partial volume effects did not affect the statistics. Conclusions: The present pilot study reports for the first time the presence and the T1 characteristics at 3 T of cortical lesions in very early RRMS (< 6 y disease duration). It also shows that CLS type I represents the most frequent cortical lesion type in this cohort of RRMS patients. In addition, it reveals a negative correlation between the attentional test TMT-part A and the T1 properties of cortical lesions type I. In other words, lower attention deficits are concomitant with longer T1-relaxation time in cortical lesions. In respect to this last finding, it could be speculated that long relaxation time correspond to a certain degree of tissue loss that is enough to stimulate compensatory mechanisms. This hypothesis is in line with previous fMRI studies showing functional compensatory mechanisms to help maintaining normal or sub-normal attention performances in RR MS patients (Penner, 2003).

Detection of hilA gene sequences in serovars of Salmonella enterica sufigbspecies enterica

hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has been found in Salmonella serovar Typhimurium, being important for the regulation of type III secretion apparatus genes. We detected hilA gene sequences in Salmonella serovars Typhi, Enteritidis, Choleraesuis, Paratyphi A and B, and Pullorum, by polymerase chain reaction (PCR) and hybridization techniques. The primers to carry out PCR were designed according to hilA sequence. A low stringency hybridization with the probe pVV441 (hilA open-reading-frame plasmid) was carried out. To find hilA gene sequences in other Salmonella sp. suggest that these serovars could have similar sequences of this kind of virulence genes.

Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion.

Cell invasion targets specific tissues in physiological placental implantation and pathological metastasis, which raises questions about how this process is controlled. We compare dermis and endometrium capacities to support trophoblast invasion, using matching sets of human primary fibroblasts in a coculture assay with human placental explants. Substituting endometrium, the natural trophoblast target, with dermis dramatically reduces trophoblast interstitial invasion. Our data reveal that endometrium expresses a higher rate of the fibronectin (FN) extra type III domain A+ (EDA+) splicing isoform, which displays stronger matrix incorporation capacity. We demonstrate that the high FN content of the endometrium matrix, and not specifically the EDA domain, supports trophoblast invasion by showing that forced incorporation of plasma FN (EDA-) promotes efficient trophoblast invasion. We further show that the serine/arginine-rich protein serine/arginine-rich splicing factor 1 (SRSF1) is more highly expressed in endometrium and, using RNA interference, that it is involved in the higher EDA exon inclusion rate in endometrium. Our data therefore show a mechanism by which tissues can be distinguished, for their capacity to support invasion, by their different rates of EDA inclusion, linked to their SRSF1 protein levels. In the broader context of cancer pathology, the results suggest that SRSF1 might play a central role not only in the tumor cells, but also in the surrounding stroma.

Infection of Calomys callosus (Rodentia Cricetidae) with strains of different Trypanosoma cruzi biodemes: pathogenicity, histotropism, and fibrosis induction

The influence of different Trypanosoma cruzi biodemes on the evolution of the infection and on the histopathological lesions of the heart and skeletal muscles, during the experimental infection of Calomys callosus, was investigated. Three groups of C. callosus were infected, respectively, with parasite strains representative of three different Biodemes: Type I (Y strain), Type II (21 SF strain), and Type III (Colombian strain). For each group, normal C. callosus were also used as controls. Marked differences have been detected in the responses of C. callosus to the infection with the three strains in this model. The strains Types I and II (Y and 21 SF) determined moderate lesions, mostly in the myocardium, with low parasitism, a rapid course, and total regression of the lesions by the 60th day of infection. Differently, Type III strain (Colombian), was more pathogenic for C. callosus and induced necrotic-inflammatory lesions in skeletal muscles and myocardium, in correspondence to intracellular parasitism. Proliferation of fibroblasts and amorphous matrix deposits, followed by interstitial fibrosis were present. Progressive regression of the inflammatory changes and collagen deposits occurred spontaneously. The progression and regression of both inflammation and fibrosis induced by the Colombian strain were further submitted to quantitative evaluation by morphometry. Results of the morphometric studies presented good correlation with the histopathological findings. The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.

Distribution and pathogenicity of Trypanosoma cruzi isolated from peridomestic populations of Triatoma infestans and Triatoma guasayana from rural Western Argentina

We assessed the distribution of Trypanosoma cruzi infection in peridomestic triatomines collected manually at a district-wide scale in rural villages around Olta, Western Argentina, and typed the isolated strains according to their pathogenicity to laboratory mice. Of 1623 triatomines examined, only 14 (0.9%) were infected with T. cruzi based on microscopical examination of feces. The prevalence of T. cruzi infection was 0.8% in Triatoma infestans, 2.3% in T. guasayana, and nil in T. garciabesi, T. platensis, and T. eratyrusiformis. Local transmission occurred in kitchens, store-rooms and goat corrals or nearby, though at very low levels. T. cruzi was detected by at least one parasitological method in 11 (79%) of 14 microscope-positive bugs. Hemoculture was the most sensitive method (67%) followed by culture of organ homogenates, histopathology or xenodiagnosis of inoculated suckling mice (55-58%), and culture of microscope-positive bug feces (46%). The evidence suggests that most of the isolated T. cruzi strains would be myotropic type III. Our study establishes for the first time that peridomestic, microscope-positive T. guasayana nymphs were actually infected with T. cruzi, and may be implicated as a putative secondary vector of T. cruzi in domestic or peridomestic sites.

Participation of cytokines in the necrotic-inflammatory lesions in the heart and skeletal muscles of Calomys callosus infected with Trypanosoma cruzi

Calomys callosus, a sylvatic reservoir of Trypanosoma cruzi, when infected with the Colombian strain (Biodeme Type III, T. cruzi I ) develops necrotic-inflammatory lesions and intense early fibrogenesis in the heart and skeletal muscles, that spontaneously regress. Participation of pro-inflammatory and pro-fibrogenic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma) , and tumor growth factor-beta (TGF-beta), in the pathogenesis of the lesions is herein studied. Eighty C. callosus weighing 20 to 30 g were used. Seventy of them were inoculated with the Colombian strain (10(5) blood forms) and 10 were maintained as intact non-infected controls. After infection, C. callosus were sacrificed at different time-points from 15 to 70 days. The heart and skeletal muscle were processed for histopathology and cryopreserved for immunohistochemistry. Early necrotic lesions of parasitized skeletal muscle and myocardium with intense inflammatory lesions were present. Search for the in situ presence of TNF-alpha and IFN-gamma, was performed using rat-IgG anti-mouse antibodies against these cytokines. For the in situ search of TGF-beta, rabbit IgG anti-mouse antibodies were used. Immunolabeling of the cytokines in tissues of infected C. callosus was successful. The cytokines TNF-alpha, IFN-gamma , and TGF-beta were detected in the cytoplasm of macrophages and in the necrotic material from 15 to 45 days post-infection, decreasing their intensity until complete disappearance by the 65th day, which correlated with subsiding histopathological lesions. These findings suggest the participation of these cytokines in the control of parasite multiplication, in the development of an early fibrogenesis and in the regression of fibrotic-inflammatory lesions observed in C. callosus.

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Oximetry alone versus portable polygraphy for sleep apnea screening before bariatric surger

Rapport de SynthèseIntroductionLa recherche des apnées du sommeil est recommandée dans la prise en charge préopératoire des patients obèses chez qui une chirurgie bariatrique est envisagée. Toutefois le type d'examen nécessaire pour la détection des apnées dans cette population reste encore discuté. L'objectif de cette étude était de comparer la sensibilité de l'oxymétrie par rapport à la polygraphie lors du screening préopératoire de l'apnée obstructive du sommeil.MéthodeNous avons analysé rétrospectivement les données d'enregistrement de la polygraphie (moniteur portable de type III) et de l'oxymétrie de 68 patients consécutifs adressés au Centre du sommeil dans le cadre de leur bilan avant chirurgie bariatrique.Nous avons comparé la sensibilité de l'index de désaturation 3% ou 4% (à partir de l'oxymétrie seule) avec l'index d'apnée-hypopnée (à partir de la polygraphie) pour diagnostiquer l'apnée obstructive du sommeil. Les patients ont été réparti en 3 groupes selon la sévérité de leur atteinte: normale (< 10 événements/heure), faible à moyenne (10-30 événements/heure), sévère (>30 événements/heure).RésultatsSi l'on considère l'index d'apnée-hypopnée (polygraphie), la prévalence de l'apnée obstructive du sommeil avec un index d'apnée-hypopnée supérieur à 10 événements par heure était de 57,4% : 16,2% des patients étaient classés comme sévèrement atteints, 41,2% comme faiblement à moyennement atteints et 42.6% comme normaux.Si l'on considère l'index de désaturation 3%, 22,1% des patients étaient classés commes sévères , 47,1% comme faiblement à moyennement atteints et 30,9% comme normaux.Avec un index de désaturation de 4%, 17,6% étaient classés comme sévères, 32,4 % comme faiblement à moyennement atteints et 50% comme normaux.En comparant l'index de désaturation 3% à l'index d'apnée-hypopnée (>10 événements/heure), nous avons obtenu une valeur prédictive négative de 95% pour exclure une apnée obstructive du sommeil et une sensibilité de 100% dans la détection des cas sévères d'apnées obstructives du sommeil (index apnée-hypopnée >30 événements/heure). Le coefficient de concordance entre l'index d'apnée-hypopnée et l'index de désaturation 3% était de 0,759 alors qu'il était de 0,856 entre l'index d'apnée-hypopnée et l'index de désaturation de 4%.

A thirty years follow-up study on Schistosomiasis mansoni in a community of Minas Gerais, Brazil

During thirty years - 1973-2003 - a group of individuals infected by Schistosoma mansoni in Capitão Andrade, Rio Doce Valley, Minas Gerais, Brazil, was evaluated by the same authors, being one of the longest follow-up studies on schistosomiasisis mansoni in an endemic area. The diagnosis of S. mansoni was based on parasitological stool tests. In the clinical classification, three groups were considered: type I - schistosomiasis-infection, type II - hepatointestinal form, and type III- hepatosplenic form. The prevalence of infection were 60.8% in 1973, 36.2% in 1984, 27.3% in 1994, and 19.4% in 2003, while the index of hepatosplenomegaly were respectively 5.8%, 2.8%, 2.3% and 1.3%. The maintenance of high prevalence and severity of clinical forms are probably related to reinfection.

Genetic analysis of caveolin-1 and eNOS genes in colorectal cancer.

Caveolae are involved in physical compartmentalization between different groups of signaling events. Its main component, CAV1, modulates different pathways in cellular physiology. The emerging evidence pointing to the role of CAV1 in cancer led us to study whether different alleles of this gene are associated with colorectal cancer (CRC). Since one of the most characterized enzymes regulated by CAV1 is eNOS, we decided to include both genes in this study. We analyzed five SNPs in 360 unrelated CRC patients and 550 controls from the general population. Two of these SNPs were located within eNOS and three within the CAV1 gene. Although haplotype distribution was not associated with CRC, haplotype TiA (CAV1) was associated with familiar forms of CRC (p<0.05). This was especially evident in CRC antecedents and nuclear forms of CRC. If both CG (eNOS) and TiA (CAV1) haplotypes were taken together, this association increased in significance. Thus, we propose that CAV1, either alone or together with eNOS alleles, might modify CRC heritability.

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.