Toughening robocast chitosan/biphasic calcium phosphate composite scaffolds with silk fibroin: Tuning printable inks and scaffold structure for bone regeneration.

The present work aims the production of composite bioceramic scaffolds by robocasting suppressing sintering as post printing process. To achieve this purpose, extrudable ink compositions containing a high concentration of bioceramic powders (hydroxyapatite and ß-tricalcium phosphate) embedded in aqueous polymeric solutions of chitosan and silk fibroin were fine-tuned. Polymeric solutions of chitosan/silk fibroin with different ratios were tested, maintaining the total amount of bioceramic solids at 30 vol%. The inks were characterized by rheological studies in viscometry and oscillatory modes, being the printable ones selected to produce scaffolds with different macropore sizes (300 µm and 500 µm). The scaffolds were characterized by mechanical properties (dry and wet conditions) and morphological features, as well as its degradability. In vitro studies were also evaluated in the scaffolds that presented the best structural performance. The addition of 2 wt% silk fibroin to a 5 wt% chitosan matrix allows to significantly improve the mechanical performance of the printed composite scaffolds, reflected in high values for Young's modulus and maximum compressive strength. This trend was continued in wet scaffolds with a concomitant reduction of mechanical properties. Regarding degradability, the scaffolds in general presented a weight loss in the range of 14-18% after 28 days incubation in HEPES solution at two different pH values at 37 °C, with an associated release of calcium and phosphorus ions. The scaffold with 300 µm porosity comprising the both polymers in its composition presented the less rate degradation when compared to the scaffolds with similar porosity and containing only chitosan as base matrix. Moreover, the combined natural polymers gave rise to a significant increase in the metabolic activity of human osteoblasts grown on the scaffolds with both macropore' size, being in line with the full cellular filling of their surfaces, demonstrated by SEM and confocal imaging. The advances presented in this work are a promising path in the ink's development for extrusion-based additive manufacturing techniques and subsequent biomaterials, encompassing suitable physical and chemical characteristics with high potential to be used as bone substitutes.

Effect of orthodontic force associated with cigarette smoke inhalation in healthy and diseased periodontium. A histometric and immunohistochemistry analysis in rats.

BACKGROUND: The aim of the present study is to evaluate the effect of orthodontic forces in healthy or diseased periodontium of rats submitted/not submitted to cigarette smoke inhalation. MATERIAL AND METHODS: Fifty-six male Wistar rats were allocated into two groups of conditions: smoking and non-smoking. Each group was divided into the following subgroups: control (C), orthodontic tooth movement (OTM), ligature-induced periodontitis (P) and P+OTM (POTM), with n = 14 each. Periodontitis was induced in the lower first molar by cotton ligature, and a 4 mm closed stainless steel spring was used for orthodontic movement. Animals were exposed to the smoke of 10 cigarettes for 8 minutes, 3 times a day for 60 days before P induction and OTM. Evaluation parameters were macroscopic analysis of dental movement, bone loss and bone density. In addition, the receptor activator of nuclear factor-kappaB (RANK) immunostaining and RANK ligand/osteoprotegerin ratio in the furcation region were assessed. RESULTS: There was no statistically significant difference between groups, ie, smoking and non-smoking conditions (P = .338). Bone loss intragroup analysis between the P and POTM groups was not significant in smoking (P = 1) and non-smoking (P = .5) conditions; both were different from OTM and C in each condition. Regarding bone density, POTM and P were significant to C (P < .05). The POTM group was significant to the P and C (P = .001) regarding dental movement. The RANK ligand/osteoprotegerin ratio in the non-smoking condition was higher in P and POTM compared to C and OTM and to P and POTM in the smoking condition. RANK immunostaining was significant in the smoking condition for the P and POTM groups (P < .05). CONCLUSION: Within the limitations of the present study, it was concluded that cigarette smoke inhalation had no influence on the evaluated groups, even with the presence of low levels of nicotine, carbon monoxide and tar. The POTM groups did not present greater bone loss compared to P groups, thus periodontal disease is essential for bone loss.

Vaccine effectiveness and use of collar impregnated with insecticide for reducing incidence of Leishmania infection in dogs in an endemic region for visceral leishmaniasis, in Brazil.

Although a national programme for control of visceral leishmaniosis (VL) is being run in Brazil, the disease continues to spread. This programme is essentially based on culling infected dogs from endemic regions. Thus, there is an urgent need to develop other control measures against VL to deter its advance. Here, a subunit vaccine, a recombinant vaccine, an insecticide-impregnated collar and the associations between these measures were evaluated for reducing the incidence of Leishmania infection in dogs. This was through a cohort study conducted in an endemic region of Brazil, considering the incidence and time of total exposure over a period of 1 year. The incidence of VL was estimated by means of serological and molecular diagnostic tests, 180 and 360 days after the application of the control measures. The estimates of the effectiveness (EF) were not significant in any cohort. The EF of the subunit vaccine, the recombinant vaccine and the collar were 26.4%, 32.8% and 57.7% and the upper limit of the 95% confidence interval for EF were 63.7%, 67.9% and 82.5%, respectively. In conclusion, under the conditions of this study, none of the immunogens for VL control was sufficiently effective to protect dogs against infection. On the other hand, use of collars impregnated with insecticide seems to constitute a method with better prognosis, corroborating other studies in this field.

Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

Serological and molecular diagnostic tests for canine visceral leishmaniasis in Brazilian endemic area: one out of five seronegative dogs are infected.

Euthanasia of infected dogs is one of the measures adopted in Brazil to control visceral leishmaniasis (VL) in endemic areas. To detect infected dogs, animals are screened with the rapid test DPP® Visceral Canine Leishmaniasis for detection of antibodies against K26/K39 fusion antigens of amastigotes (DPP). DPP-positives are confirmed with an immunoenzymatic assay probing soluble antigens of promastigotes (ELISA), while DPP-negatives are considered free of infection. Here, 975 dogs from an endemic region were surveyed by using DPP, ELISA and real-time PCR (qPCR) for the diagnosis of VL. When DPP-negative dogs were tested by qPCR applied in blood and lymph node aspirates, 174/887 (19·6%) were positive in at least one sample. In a second sampling using 115 cases, the DPP-negative dogs were tested by qPCR in blood, lymph node and conjunctival swab samples, and 36/79 (45·6%) were positive in at least one sample. Low-to-moderate pairwise agreement was observed between all possible pair of tests. In conclusion, the official diagnosis of VL in dogs in Brazilian endemic areas failed to accuse an expressive number of infected animals and the impact of the low accuracy of serological tests in the success of euthanasia-based measure for VL control need to be assessed.

Correlations between peripheral parasite load and common clinical and laboratory alterations in dogs with visceral leishmaniasis.

Intensity of peripheral parasite infection has an important role in the transmission of Leishmania spp. from one host to another. As parasite load quantification is still an expensive procedure to be used routinely in epidemiological surveillance, the use of surrogate predictors may be an important asset in the identification of dogs with high transmitting ability. The present study examined whether common clinical and laboratory alterations can serve as predictors of peripheral parasitism in dogs naturally infected with Leishmania spp. Thirty-seven dogs were examined in order to establish correlations between parasite load (PL) in multiple peripheral tissues and common clinical and laboratory findings in canine visceral leishmaniasis (CVL). Quantitative polymerase chain reaction was employed to determine PL in conjunctival swabs, ear skin, peripheral blood and buffy coat. Additionally, a series of hematological, biochemical and oxidative stress markers were quantified. Correlations between net peripheral infection and severity of clinical alterations and variation in laboratory parameters were assessed through a new analytical approach, namely Compressed Parasite Load Data (CPLD), which uses dimension reduction techniques from multivariate statistics to summarize PL across tissues into a single variable. The analysis revealed that elevation in PL is positively correlated with severity of clinical sings commonly observed in CVL, such as skin lesions, ophthalmic alterations, onycogriphosis, popliteal lymphadenomegaly and low body mass. Furthermore, increase in PL was found to be followed by intensification of non-regenerative anemia, neutrophilia, eosinopenia, hepatic injury and oxidative imbalance. These results suggest that routinely used clinical and laboratory exams can be predictive of intensity of peripheral parasite infection, which has an important implication in the identification of dogs with high transmitting ability.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction.

The aim of this study was to detect cross infections by Leishmania spp. and Trypanosoma spp. using enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Thus, 408 blood samples were collected from dogs domiciled in Araçatuba Municipality, São Paulo State, Brazil; the dogs were of both sexes, of several breeds and aged 6 months. For Leishmania spp., 14.95% (61 out of 408) of dogs were reactive using IFAT. Positivity was 20.10% (82 out of 408) using ELISA and 29.66% (121 out of 408) using PCR, with significant differences for the sex and age of these animals (p < 0.05). For Trypanosoma spp., antibody occurrence using ELISA was 10.54% (43 out of 408), while PCR indicated 2.45% (10 out of 408) positive dogs. Using IFAT, 10.29% (42 out of 408) of animals were considered positive and only sex showed a significant difference (p < 0.05). In this study, 10.54% (43 out of 408) of animals were seropositive according to ELISA for Trypanosoma spp., of which 79.07% (34 out of 43) showed positive results in the molecular diagnosis for Leishmania spp., while of the 10.29% (42 out of 408) positive dogs according to IFAT, 95.24 % (40 out of 42) had confirmed infection by this parasite. The obtained results demonstrate evidence of cross infections by both protozoa in the animals analysed in this study.

Targeted nucleotide exchange in bovine myostatin gene.

The myostatin gene, known as Growth Differentiation Factor 8 (GDF8), located at chromosome 2 (BTA2) in cattle, is specifically expressed during embryo development and in the adult skeletal muscle. Molecular analysis of this gene reveals the presence of three exons and two introns. Several cattle breeds, such as Piedmontese, Belgian Blue, Blond'Aquitaine, among others, show polymorphisms in this gene, which are directly related to double muscling phenotype. Piedmontese cattle shows a nucleotide transition G --> A (G938A) at exon 3, resulting in the substitution of cysteine to tyrosine, leading to a protein structure change, which determines myostatin inactivation and consequent muscular hypertrophy. The objective of this work was to implant the polymorphism G938A, naturally existent in Piedmontese breed, into in vitro propagated foetal myoblasts, from Nellore cattle. Single strand DNA (ssDNA) oligonucleotides were used to direct the same nucleotidic transition (G938A) to exon 3. Two transfection protocols (cationic lipid solution and electroporation) were tested and, 48 hours after transfection, RNA and DNA were extracted from myoblasts. Reverse transcription and polymerase chain reaction (PCR) were performed, using primers flanking the mutation region. The PCR products were cloned and analyzed by DNA sequencing, and it was possible to detect the nucleotidic CT transition at position 938, in the electroporated myoblasts. The existence of a positive signal in the transfection indicates that ssDNA oligonucleotides can be used to introduce this point mutation in specific functional gene sites.

Should an iridectomy be routinely performed as a part of trabeculectomy? Two surgeons' clinical experience.

PURPOSE: To investigate the effects of performing peripheral iridectomy on the outcome of trabeculectomy. METHODS: Retrospective chart review of the medical records of 75 patients (75 eyes) who underwent trabeculectomy surgery, with or without peripheral iridectomy, who had been followed for more than 1 year. Data were collected preoperatively, 1 day postoperatively, on days 30-90 postoperatively, and 1-3 years postoperatively. The collected data included visual acuity, intraocular pressure, bleb development, postoperative inflammation, and complications. Thirty-six eyes (48%) had cataract extraction at the time of trabeculectomy. A peripheral iridectomy was performed in 43 cases (57%). Student's t-test was used for the statistical analyses. RESULTS: Patients having peripheral iridectomy had more inflammation on days 30-90 than those who did not have peripheral iridectomy performed (in patients having cataract extraction with trabeculectomy (P=0.018) and those not having cataract extraction (P=0.038)). There was no statistically significant difference in intraocular pressure in eyes with or without iridectomy. Postoperative complications were rare in both groups but greater in number in the eyes with peripheral iridectomy. CONCLUSIONS: Trabeculectomy performed without peripheral iridectomy appears to be as effective in lowering intraocular pressure as when performed with peripheral iridectomy, but it is a safer procedure, with a lower incidence of postoperative inflammation. It may be an advantage to avoid performing peripheral iridectomy during trabeculectomy in eyes that are not predisposed to postoperative shallowing of the anterior chamber or pupillary block.

Correlation of Disc Damage Likelihood Scale, visual field, and Heidelberg Retina Tomograph II in patients with glaucoma.

PURPOSE: To correlate the analysis of the optic nerve head (ONH) obtained with Disc Damage Likelihood Scale (DDLS), Heidelberg Retina Tomograph II (HRT II), and visual field (VF) parameters (Aulhorn scale, mean deviation [MD], and pattern standard deviation [PSD]) in patients with glaucoma. METHODS: Sixty-five eyes were evaluated using the DDLS classification. Fifty-seven eyes had stable primary open-angle glaucoma (POAG) and eight eyes were normal (control group). Classification of the ONH was performed using the HRT II, with Moorfields Regression Analysis (MRA) and Cup Shape Measure (CSM) indexes. The VF was examined by Full Threshold test (30-2) using the Humphrey Field Analyzer (HFA) perimeter and evaluated by three METHODS: Aulhorn's scale of 0 to 6, and two global indices, MD and PSD. RESULTS: The amount of ONH damage as estimated by the DDLS correlated well with topographic damage determined by HRT II (r=0.923 with MRA and r=0.807 with CSM) and with visual field loss as measured by the MD (r=-0.792) and PSD (r=0.718) of the HFA and Aulhorn field staging system (r=0.849). CONCLUSIONS: This study shows that evaluation of the ONH using the DDLS provides information that correlates well with VF and with data obtained from HRT II.

Comparative evaluation of an indirect ELISA test for diagnosis of swine cysticercosis employing antigen from Taenia solium and Taenia crassiceps metacestodes.

Taenia solium cysticercosis is still a serious public health problem in several countries where poverty and lack of hygiene favor transmission. Because pigs are the primary intermediate hosts, prevalence of porcine cysticercosis is a reliable indicator of active transmission zones. Serological diagnostic methods are important tools for epidemiological studies since they can be applied to living animals on a large scale. Four antigen preparations (cyst fluid and crude) from T. solium and T. crassiceps metacestodes were compared for swine cysticercosis diagnosis by indirect ELISA (IE). Twenty-eight serum samples from swine naturally and experimentally infected by cysticerci of T. solium and 56 serum samples from swine reared in commercial herds were tested. Best results of overall sensitivity were obtained by the use of cyst fluid and crude antigen of T. crassiceps metacestode (100 and 96.4%, respectively). Using homologous antigen preparations we have observed higher specificity percentage (98.2% for cyst fluid and 96. 4% for crude metacestode T. solium antigen). We concluded that sensitivity is of far more importance than specificity for identification of endemic areas in order to prevent transmission to man. We conclude, therefore, that IE performed with cyst fluid antigen of T. crassiceps metacestode is a better tool for that purpose.

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism.

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.

[Presence of larva migrans in sand boxes of public elementary schools, Araçatuba, Brazil]. / Ocorrência de larva migrans na areia de áreas de lazer das escolas municipais de ensino infantil, Araçatuba, SP, Brasil.

There are sandboxes in public elementary school playground areas in Brazil, which can be harmful to children. They are at risk of cutaneous and visceral larva migrans infection caused by Ancylostoma spp. and Toxocara spp., respectively. The study was designed to investigate contamination by Toxocara spp. and/or their eggs and Ancylostoma spp. larvae in sand samples collected from the schools' sandboxes. Five hundred and thirty-five sand samples from 28 public elementary schools were collected during summer and winter and analyzed by both Baerman's method and centrifugal flotation technique. Ancylostoma spp. larvae were found in 35.7% (10/28) schools in summer time and in 46.4% (13/28) schools in the winter time. Eggs of Toxocara spp. could not be recovered from the samples analyzed and eggs from Ancylostoma spp. were seen in 0.56% (3/535) of the samples.

Toxocariasis: serological diagnosis by indirect antibody competition ELISA.

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

Detection of leptospires in bovine semen by polymerase chain reaction.

OBJECTIVE: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. DESIGN: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. RESULT: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. CONCLUSION: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

Detection of Leptospira spp. from pure cultures and from experimentally contaminated bovine semen by polymerase chain reaction

Due to the high importance of the venereal transmission of bovine leptospirosis, this study aimed to test the ability of PCR to detect Leptospira interrogans serovar hardjo DNA in experimentally contaminated bovine semen. Employing primers directed to the 16S rRNA gene, 10 bacteria/ml of semen could be detected by PCR. Results achieved in this work show that PCR can have a great potential to detect Leptospira spp. in insemination centers

Immunoblot with cerebrospinal fluid from patients with neurocysticercosis using antigen from cysticerci of Taenia solium and Taenia crassiceps.

A comparative study was conducted on membrane (M) and vesicular fluid (VF) from cysticerci of Taenia solium (Tso) obtained from naturally infected swine and the Taenia crassiceps ORF strain (Tc) maintained by experimental infection of female BALB/c mice. The study was carried out using immunoblotting to detect antibodies in cerebrospinal fluid (CSF) from patients with neurocysticercosis. No reactivity was observed in the 32 samples from a control group. Of the 23 CSF fluid samples from patients with neurocysticercosis, 22 (95.6%) were reactive in the M-Tso blot and 21 (91.3%) were reactive in the other three blots (VF-Tso, M-Tc, and VF-Tc). Immunodominant peptides in each antigen were 98-92 kD, 56-52 kD, and 72-68 kD in M-Tso; 72-68 kD, 120 kD, 155 kD, 98-94 kD, 76 kD, and 115-108 kD in VF-Tso: 72 kD, 62 kD, and 42 kD in M-Tc; and 72-68 kD and 95-92 kD in VF-Tc. The cross-reactivity observed in the immunoblots performed on CSF samples from patients with neurocysticercosis indicates that the parasites share important epitopes present at sufficient concentrations for use in immunologic tests.

[Evaluation of the dog population in an urban area of southeastern Brazil]. / Avaliação da população canina da zona urbana do município de Araçatuba, São Paulo, SP, Brasil.

Planning control programs, for diseases such as rabies requires information on the size and structure of the dog and cat population. In order to evaluate the dog population of the urban area of Araçatuba city, S. Paulo State, Brazil, a survey was conducted using a questionnaire to interview members of house-holds. Eighty-eight districts were visited (37,778 houses) and the interview was possible at 77.93% of these. Human population size evaluated was 113,157 inhabitants. Houses that owned animals represented 55.2%, 26,926 of the animals concerned were dogs and 5,755 were cats. Of the dogs, 56.64% were 1-4 year olds and males represented 56.2% of the total population. Dog: person ratio was estimated at 2.8 dogs to every 10 persons, almost 3 times the ratio hitherto estimated and used in the planning of rabies vaccination campaigns.