High-Throughput IgG Epitope Mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design.

Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in TeNT have been described using various approaches, a comprehensive list of its antigenic determinants that are involved with immunity has not been elucidated. To this end, a high-resolution analysis of the linear B-cell epitopes in TeNT was performed using antibodies generated in vaccinated children. Two hundred sixty-four peptides that cover the entire coding sequence of the TeNT protein were prepared in situ on a cellulose membrane through SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes, which were further characterized and validated using immunoassays. Forty-four IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as multiple antigen peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the efficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-41/G and TT-43/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines.

B-Cell Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT that is responsible for protection remains to be clarified. A library of one-hundred, twenty-two 15-mer peptides covering the entire sequence of the three chains of the CT protein (CTP) was prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice vaccinated with an oral inactivated vaccine (Schankol™) allowed the mapping of continuous B-cell epitopes, topological studies, multi-antigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three V. cholera specific epitopes, Vc/TxA-3, Vc/TxB-11, and Vc/TxP-16, were synthesized as MAP4 and used to coat ELISA plates in order to screen immunized mouse sera. Sensitivities and specificities of 100% were obtained with the MAP4s of Vc/TxA-3 and Vc/TxB-11. The results revealed a set of peptides whose immunoreactivity reflects the immune response to vaccination. The array of peptide data can be applied to develop improved serological tests in order to detect cholera toxin exposure, as well as next generation vaccines to induce more specific antibodies against the cholera toxin.

Canine papillomavirus type 16 associated to squamous cell carcinoma in a dog: virological and pathological findings.

Papillomaviruses (PVs) are circular double-stranded DNA virus belonging to Papillomaviridae family. During the infection cycle, PVs translate proteins that can influence cell growth and differentiation, leading to epidermal hyperplasia and papillomas (warts) or malignant neoplasms. Canis familiaris papillomaviruses (CPVs) have been associated with different lesions, such as oral and cutaneous papillomatosis, pigmented plaques, and squamous cell carcinomas (SCCs). Here, we report a clinical case of a mixed bred female dog with pigmented plaques induced by CPV16 (Chipapillomavirus 2) that progressed to in situ and invasive SCCs. Gross and histological findings were characterized, and the lesions were mainly observed in ventral abdominal region and medial face of the limbs. In situ hybridization (ISH) revealed strong nuclear hybridization signals in the neoplastic epithelial cells, as well as in the keratinocytes and koilocytes of the pigmented viral plaques. The full genome of the CPV16 recovered directly from the lesions was characterized, and the phylogenetic relationships were determined. The identification of oncoprotein genes (E5, E6, and E7) by high throughput sequencing (HTS) and their expected domains are suggestive of the malignant transformation by CPV16.

Bovine papillomavirus 24: a novel member of the genus Xipapillomavirus detected in the Amazon region.

Bovine papillomaviruses (BPVs) have been described as etiologic agents of cutaneous and mucosal papillomas in cattle. In the present study, we describe a new BPV that was detected in a cutaneous papilloma from a cow. Phylogenetic analysis suggests that this virus belong to the genus Xipapillomavirus, and we refer to it here as BPV type 24 (BPV24). Coinfection with members of the genera Epsilonpapillomavirus and Deltapapillomavirus in a cutaneous papilloma from a different animal was also detected, and the full genomes of these viruses were sequenced. Both papillomas were from cattle within Acre State in the Amazon region of Brazil. The data presented here demonstrate the utility of using high-throughput methods to indentify coinfections and allow the characterization of new genomes.

Clinical outcomes of arthroscopic rotator cuff repair: correlation between the University of California, Los Angeles (UCLA) and American Shoulder and Elbow Surgeons (ASES) scores.

BACKGROUND: There are more than 40 outcome scores for evaluating shoulder pain and function. Some studies have correlated the results obtained using different scales, but none has compared the results obtained by the University of California, Los Angeles (UCLA) and American Shoulder and Elbow Surgeons (ASES) scores. METHODS: We performed a retrospective study to evaluate patients who underwent arthroscopic rotator cuff repair with 2 years' follow-up. The patients were evaluated by the UCLA and ASES scores preoperatively and at 6, 12, and 24 months after surgery. The Pearson correlation coefficient (r) was calculated to measure the degree of correlation between the 2 outcome scores. RESULTS: We evaluated 143 patients. At 24 months postoperatively, the UCLA and ASES scores were 30.4 ± 5.8 and 81.2 ± 20.8, respectively (P < .001). The UCLA and ASES scores showed a very high correlation (r = 0.91, P < .001). In all the postoperative clinical evaluations, the scores obtained from the 2 scales were highly or very highly correlated (r = 0.87-0.92, P < .001). For the preoperative scores, the correlation was moderate (r = 0.67, P < .001). CONCLUSION: The UCLA and ASES scores presented a very high correlation in the evaluation of surgical treatment of rotator cuff tear. In the preoperative period, the correlation was moderate.

Genome characterization of a bovine papillomavirus type 5 from cattle in the Amazon region, Brazil.

Papillomaviruses are small and complex viruses with circular DNA genome that belongs to the Papillomavirus family, which comprises at least 39 genera. The bovine papillomavirus (BPV) causes an infectious disease that is characterized by chronic and proliferative benign tumors that affect cattle worldwide. In the present work, the full genome sequence of BPV type 5, an Epsilonpapillomavirus, is reported. The genome was recovered from papillomatous lesions excised from cattle raised in the Amazon region, Northern Brazil. The genome comprises 7836 base pairs and exhibits the archetypal organization of the Papillomaviridae. This is of significance for the study of BPV biology, since currently available full BPV genome sequences are scarce. The availability of genomic information of BPVs can provide better understanding of the differences in genetics and biology of papillomaviruses.

How many papillomavirus species can go undetected in papilloma lesions?

A co-infection comprising to at least seven papillomavirus (PV) types was detected by next generation sequencing (NGS) of randomly primed rolling circle amplification (RCA) products of a bovine (Bos taurus) papilloma lesion from the Brazilian Amazon region. Six putative new PV types that could not be detected by commonly used PCR protocols were identified. Their overall L1 nucleotide identities were less than 90% compared to described PV species and types. L1 nucleotide BLAST sequence hits showed that each new type was related to Beta, Gamma, Dyokappa, Dyoeta, and Xipapillomavirus, as well as two likely new unclassified genera. Our results show that the employment of NGS is relevant to the detection and characterization of distantly related PV and is of major importance in co-infection studies. This knowledge will help us understand the biology and pathogenesis of PV, as well as contribute to disease control. Moreover, we can also conclude that there are many unknown circulating PVs.

Novel Bovine Papillomavirus Type Discovered by Rolling-Circle Amplification Coupled with Next-Generation Sequencing.

Currently, fifteen bovine papillomavirus (BPV) types have been identified and classified into four genera: Deltapapillomavirus, Epsilonpapillomavirus, Dyoxipapillomavirus, and Xipapillomavirus. Here, the complete genome sequence of a new BPV type (BPV 04AC14) recovered from a papillomatous lesion is reported. The genome is 7,282 bp in length and exhibits the classic genetic organization and motifs of the members of Papillomaviridae. Maximum likelihood phylogenetic analyses revealed that BPV 04AC14 clusters with members of the Xipapillomavirus genus. The nucleotide sequence of the L1 capsid protein of the novel BPV is closely related to its counterpart, BPV3, with which it shares 79% similarity. These findings suggest that this virus is a new BPV type of the Xipapillomavirus genus.

Complete genome sequence of Deltapapillomavirus 4 (bovine papillomavirus 2) from a bovine papillomavirus lesion in Amazon Region, Brazil.

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2,E4, E5, E6,E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available.

Genetic characterization of Amazonian bovine papillomavirus reveals the existence of four new putative types.

Papillomaviruses are small and complex viruses that belong to the Papillomaviridae family, which comprises 39 genera. The bovine papillomavirus (BPV) causes an infectious disease that is characterized by chronic and proliferative benign tumors that affect cattle worldwide. Different genotypes of BPVs can cause distinct skin and mucosal lesions and the immunity they raise has low cross-protection. This report aimed to genotype BPVs in cattle from Northern Brazil based on nucleotide partial sequences of the L1 ORF. Skin wart samples from 39 bovines clinically and histopathologically diagnosed as cutaneous papillomatosis from Acre and Rondônia States were analyzed. The results revealed four already reported BPV types (BPVs 1, 2, 11, and 13), nine putative new BPV subtypes and four putative new BPV types as well as two putative new BPV types that were already reported. To our knowledge, this is the first record of BPVs from the Brazilian Amazon region that identified new possible BPV types and subtypes circulating in this population. These findings point to the great genetic diversity of BPVs that are present in this region and highlight the importance of this knowledge before further studies about vaccination are attempted.

Identification of linear B epitopes of pertactin of Bordetella pertussis induced by immunization with whole and acellular vaccine.

Pertussis is a serious infectious disease of the respiratory tract caused by the gram-negative bacteria Bordetella pertussis. There has been a reemergence of this disease within the population of several countries that have well established vaccination programs. Analyzes of clinical isolates suggest an antigenic divergence between the vaccine-based strains to the circulating strains. Although antibodies against P.69 are involved in the observed protective immunity, the sequences recognized as antigenic determinants in P.133, the precursor for P.69, P.3.4 and P.30, have not be determined. Here, the precise mapping of linear B-cell epitopes within the predicted P.133 pertactin sequences was accomplished using the SPOT-synthesis of peptide arrays onto cellulose membranes and screening with murine sera generated by vaccination with either the Pertussis cellular (miPc) or Pertussis acellular (miPa) vaccine. A total of 23 major epitopes were identified by sera from miPc vaccinated mice, while thirteen were identified by sera from miPa vaccinated mice. Of these epitopes, 12 epitopes were specifically identified by antibodies produced in response to the miPc vaccine and two were specific to the miPa vaccine. These epitopes were distributed throughout the pertactin sequence but a significant number were concentrated to the P.30 Prn segment. An analysis of the epitope correlation homologies indicated that the variations from the observed mutations in pertactin would not constitute a problem using these vaccines. In addition, the mapping of epitopes demonstrated a higher number of linear B-cell epitopes immunized with the Pc vaccine than the Pa vaccine.

Tratamento endodôntico convencional de dente invaginado com periodontite apical / Conventional endodontic treatment of a dens invaginatus with apical periodontitis

Dente invaginado é uma anomalia do desenvolvimento caracterizada por invaginação dos tecidos dentários que varia de ligeiro aumento da fosseta do cíngulo a um profundo sulco que pode se estender até o ápice dentário. O objetivo deste trabalho foi discutir um caso clínico de dente invaginado tipo II em incisivo lateral superior em que foi realizado o tratamento endodôntico convencional. Posterior ao acompanhamento de 11 anos e frente aos exames clínicos e imaginológicos observou-se tratamento endodôntico satisfatório e regressão da rarefação óssea. Baseado nisso, foi possível concluir que o tratamento endodôntico convencional é uma alternativa viável para tratamento de dente invaginado tipo II.

Dens invaginatus is a developmental anomaly characterized by invagination of the dental tissues ranging from slight rise of the pit to a depth of the cingulate sulcus and can be extended to the apical portion of the tooth. The aim of this study was to relate a case of dens invaginatus type II in upper lateral incisor in which a conventional treatment was realized. After eleven years-follow up and based on clinical and images exams, it was observed that the endodontic treatment was efficient and the bone rarefaction reduced. Based on this, it was possible to conclude that conventional endodontic treatment is a viable alternative for type II dens invaginatus.

A novel three-dimensional dynamic anorectal ultrasonography technique (echodefecography) to assess obstructed defecation, a comparison with defecography.

AIM: To test the effectiveness of echodefecography, the dynamic 3D anorectal ultrasonography technique -(EDF). To assess women with obstructed defecation (OD), as compared with conventional defecography (DF). METHODS: A prospective study was carried out with 30 women with OD symptoms, the mean validated Wexner constipation score was 14 (range 7-25) and the mean age 47.7 years. All patients were submitted to DF followed by EDF and the results compared. RESULTS: Six patients were normal at DF and five were normal at EDF. Defecography identified grade I rectocele in five patients (average size: 1.8 cm), grade II in seven (average size: 2.9 cm) and grade III in 12 (average size: 4.6 cm). Different sizes of anorectocele were also observed at EDF and quantified according to DF classification (grade I: 1.3 cm). Significant differences were observed between anorectocele sizes (p < 0.05) and between normal patients and grade I (p < 0.001). The level of agreement between the techniques was high (kappa = 0.902), with only one normal case wrongly identified as anorectocele III at EDF. Rectal intussusception was identified in five patients at DF; EDF confirmed these cases and revealed seven others, demonstrating moderate agreement (kappa = 0.462). Anismus was identified in nine patients in DF and in eight in EDF (kappa = 0.901). CONCLUSION: Echodefecography may be used as an alternative method to assess patients with OD as it has been shown to detect the same anorectal dysfunctions observed in DF. It is minimally invasive, well tolerated, inexpensive, avoids exposure to radiation, and clearly demonstrates all the anatomic structures involved with defecation.

Anal canal anatomy showed by three-dimensional anorectal ultrasonography.

BACKGROUND: Demonstrate precisely the anatomic configuration of the anal canal and the length and thickness of the anal sphincters using three-dimensional (3-D) anorectal ultra-sonography in both genders. METHODS: Twelve normal volunteer males and 14 females, with a mean age of 52.4 and 50.3 years, respectively, were prospectively enrolled in this study. All individuals from both groups were submitted to anorectal ultra-sonography. The anal canal was analyzed, measuring the length and thickness of the external anal sphincter (EAE), internal anal sphincter (IAS), puborectalis muscle (PR) and the gap (distance from the anterior EAS to the anorectal junction) in the midline longitudinal (ML) and transverse (MT) planes, and the results were compared between quadrants and genders. RESULTS: The distribution of sphincter muscles is asymmetric in both genders. The anterior upper anal canal is an extension of the rectal wall with all layers clearly identified. The anterior IAS is formed in the distal upper anal canal and is significantly shorter in female than in male in all quadrants. The anterior IAS length is shorter than the posterior and lateral in both genders. The anterior EAS length is significantly shorter (2.2 cm) and the gap is longer (1.2 cm) in female than in male (3.4 cm) (0.7 cm) (p < 0.05), respectively. The posterior and lateral EAS-PR is significant longer in males (3.6 cm) (3.9 cm) than in females (3.2 cm) (3.5 cm) (p < 0.05), respectively. The lateral EAS-PR is significant longer than the posterior part in both genders. The anterior IAS is significantly thicker in males (0.19 cm) than in females (0.12 cm) (p = 0.04). CONCLUSION: 3-D anal endosonography enabled measurement of the different anatomical structures of the anal canal and demonstrated its asymmetrical configuration. The shorter anterior EAS and IAS associated with a longer gap could justify the higher incidence of pelvic floor dysfunction in females, especially fecal incontinence and anorectocele with rectal intussusception.

Ecodefecografia tridimensional dinâmica. Nova técnica para avaliação da Síndrome da Defecação Obstruída (SDO) / Dynamic three-dimensional echodefecography. New technique for evaluation of the Syndrome of Obstructed Defecation

O objetivo deste estudo é apresentar novas técnicas para avaliação da SDO,utilizando a ultra-sonografia endorretal tridimensional dinâmica e comparando os resultados com a defecografia.Foram incluídas neste estudo 25 mulheres adultas,distribuídas em 2 grupos.Grupo I:15 mulheres normais,idade média de 52,4anos(23-76)e todas se submeteram ao exame proctológico completo e à ultra-sonografia anorretal tri-dimensional dinâmica para se estabelecer os padrões de normalidade do canal anal e reto.Grupo II:10 pacientes mulheres com evacuação obstruída,idade média de 47,8anos(33-65)apresentando como principais sintomas a sensação de evacuação incompleta, disquezia e digitação vaginal ou perineal.Submeteram-se...A ecodefecografia...O tamanho médio do ângulo formado pelo músculo PR no repouso foi 87,13º(variação 78,9-90,8º)(+-1,01)e no esforço evacuatório 99,22º(variação 84,9-114,5º)(+-1,84)nas mulheres normais(grupo I).Houve elevação do ângulo em todas as pacientes normais,significando relaxamento normal do PR durante o esforço evacuatório.Com relação à avaliação da anoretocele,a parede posterior da vagina se manteve na posição horizontal durante todo o esforço evacuatório,exceto nas portadoras de anoretocele.Foram diagnosticadas anoretocele(grau I=1,grau II=5,grau III=4) em todas as pacientes do grupo II pelo exame clínico e defecografia.Todos os casos foram confirmados pela ecodefecografia.A partir dos resultados,foram estabelecidos valores para classificar a anoretocele de acordo com a ecodefecografia(grau I-distância entre as posições da parede vaginal até 5,0mm,grau II de 6,0 a 12,0mm,grau III além de 12,0mm).Foi identificado anismus em uma paciente com anoretcele grau II e em outra com grau III na defecografia e confirmado na ecodefecografia pela redução no ângulo formado pelo PR ao comparar as posições em repouso e durante o esforço evacuatório.A defecografia demonstrou também 4 casos de intussuscepção enquanto a ecodefecografia confirmou estes casos e identificou