Dental pain in Brazilian preschool children: association with the severity of dental caries and impact on oral health-related quality of life.

PURPOSE: This study aimed to verify the association between dental pain and severity of dental caries (caries morbidity stages) and the impact on oral health-related quality of life (OHRQoL) in preschool children. METHODS: A cross-sectional study with 199 children (2-5 years old) enrolled at preschools in Capão do Leão-RS, Brazil. The self-report of mothers of children with a history of dental pain in the last 6 months and perception of their child's OHRQoL (ECOHIS) were obtained through a structured questionnaire. This questionnaire also collected independent variables. Children's oral examination was performed using the CAST instrument to determine caries morbidity stage. Crude and adjusted Poisson regression analysis was performed. RESULTS: The prevalence of dental pain was 14.57%. The chance of the occurrence of dental pain was higher among children diagnosed in morbidity [Prevalence ratio-PR: 5.29 (95% confidence interval-95% CI 1.91-14.61); p = 0.001] and severe morbidity [RP = 6.12 (95 CI% 2.25-16.64); p < 0.001] stages. Children with dental pain presented higher scores in the total ECOHIS [rate ratio = 7.11 (95% CI 4.55-11.09); p < 0.001] and in all of the domains of this instrument. Furthermore, children with a history of dental trauma [PR = 2.41 (95% CI 1.15-5.04); p < 0.001] and those whose reason for last visit to the dental office was for restorative/endodontic/extraction treatment [PR = 1.29 (95% CI 1.01-6.19); p = 0.049] had a higher prevalence of dental pain. CONCLUSION: A substantial prevalence of dental pain in the last 6 months and negative impact on children's OHRQoL was identified in this sample. Children diagnosed with carious dentin lesions and abscess and fistula were more likely to have dental pain.

Omentum as non-strangled inguino scrotal hernia content in stallion: case report / [Omento como conteúdo de hérnia inguino-escrotal não estrangulada em garanhão: relato de caso]

Non-strangulated acquired hernias in stallions are rare, especially when the herniated content is not intestinal loops. Thus, the aim of the current study is to describe a case of acquired non-strangulated inguinoscrotal hernia in a stallion, whose herniated content was the omentum. The patient was a Criollo stallion with history of rhabdomyolysis, laminitis and bilateral scrotal volume increase observed in the left scrotal region. The animal presented pain in both thoracic limbs, abnormal blood test, especially hypoproteinemia, and mild pain during palpation in the left inguinal ring region. Hydrocele secondary to hypoproteinemia was suspected. After admission, the animal showed signs of acute abdomen, which were clinically reversed. With this, the animal was subjected to ultrasound examination of the scrotal region, whose findings suggested non-strangulated inguinal hernia, although the content could not be identified. Surgical treatment was chosen in order to identify the herniated content and remove the left testicle. Access to the affected scrotum was performed, in which the presence of fluid and a portion of the omentum was observed surrounding the testis and adhering to it. The animal was discharged after he recovered from the surgery and from laminitis. During the breeding season, the stallion remained with a herd of mares for natural mating. After 15 months of surgery, the animal was reassessed and showed no active signs of inflammatory and degenerative processes in the remaining testis. On this occasion, a pregnancy diagnosis was also performed, and all the mares were pregnant. It is concluded that the presence of omentum as a herniated content does not represent a surgical emergency but can make the reproductive prognosis reserved. In addition, removal of the affected testicle can benefit the spermatogenesis of the remaining testicle.(AU)

As hérnias adquiridas não estranguladas em garanhões são raras, principalmente quando o conteúdo herniado não é de alças intestinais. Assim, o objetivo deste trabalho foi descrever um caso de hérnia inguino escrotal adquirida e não estrangulada em garanhão, cujo conteúdo herniado era composto pelo omento. Foi atendido um garanhão da raça Crioula com histórico de rabdomiólise, laminite e aumento de volume escrotal bilateral, evidenciado na região escrotal esquerda. O animal apresentava dor nos membros torácicos, alterações na avaliação sanguínea, destacando-se a hipoproteinemia, e demonstrava dor leve à palpação na região do anel inguinal esquerdo. Suspeitou-se de hidrocele secundária a hipoproteinemia. Após a internação, o animal apresentou sinais de abdômen agudo, revertidos clinicamente. Com isso, realizou-se ultrassonografia da região escrotal, cujos achados sugeriram hérnia inguinal não estrangulada, sem que o conteúdo pudesse ser identificado. Optou-se pelo tratamento cirúrgico, com o intuito de identificação do conteúdo herniado e remoção do testículo esquerdo. Foi realizado acesso à bolsa escrotal afetada, na qual se observou presença de líquido e de uma porção do omento envolvendo o testículo e aderido a ele. O animal recebeu alta após restabelecimento da cirurgia e da laminite. Na propriedade, durante temporada reprodutiva, o garanhão permaneceu com uma manada de éguas para realização de monta natural. Passados 15 meses da cirurgia, o animal foi reavaliado e não demonstrou sinais ativos de processo inflamatório e degenerativos no testículo remanescente. Nessa ocasião, também foi realizado diagnóstico de gestação e todas as éguas encontravam-se prenhes. Conclui-se que a presença de omento como conteúdo herniado não representa uma emergência cirúrgica, mas pode tornar o prognóstico reprodutivo reservado. Ainda, a remoção do testículo afetado pode trazer benefícios à espermatogênese do remanescente.(AU)

Evaluation of oral sugar test response for detection of equine metabolic syndrome in obese Crioulo horses.

Owing to the high prevalence of obesity in Crioulo horses, information allowing early diagnosis of equine metabolic syndrome (EMS) and prevention of the associated laminitis is of great value. The aim of this study was to evaluate the occurrence of EMS and the response to an oral sugar test (OST) in obese Crioulo horses. Twenty-two Crioulo horses were allocated into 3 groups according to their body condition score (BCS out of 9) and presence or absence of laminitis as follows: CON (6/22), BCS < 7; OB (8/22), BCS ≥ 7; and LAM (8/22), BCS ≥7 with clinical and/or radiographic signs of laminitis. A complete clinical history was obtained, followed by a physical examination, morphometric measurements, radiographic evaluation of front feet, and ultrasonography measurements of subcutaneous body fat. For the OST, animals were fasted overnight, and blood samples were collected for glucose and insulin concentration before and after sugar administration. Morphometric and metabolic differences (P < 0.05) were observed between CON animals and obese ones, with horses from the LAM group presenting the highest morphometric measurements and insulin plasma concentrations. A delayed peak glucose response for OST was observed in the majority of obese animals, indicating that sampling between 60 and 90 min after sugar administration without glycemic curve follow-up, as previously used for hyperinsulinism detection, can be inadequate. The observed delay in the return to glucose baseline levels, combined with high insulin levels, supports the diagnosis of insulin dysregulation. These results indicate that there are clear obesity-related differences in the glucose and insulin responses of Crioulo horses to an OST.

Response of three digital anesthetic technics in horses with forelimb hoof lameness / Resposta de três técnicas anestésicas digitais em equinos com claudicação dos membros anteriores ligada ao casco

The presented study aimed to assess objectively the response of distal interphalangeal joint (DIJ), navicular bursa (NB) and deep digital flexor tendon sheath (DDFTS) anesthesia in horses with forelimb hoof lameness; and evaluate if the presence of radiographic abnormalities on navicular bone could interfere on blocks' results. Fifteen horses with lameness improvement above 70% after palmar digital nerve (PDN) block were selected for this study. Blocks were assessed separately on five consecutive trials at seven different time-points. The fifth trial was performed to evaluate the influence of exercise on preexisting lameness. Most of horses (73.33%) presented pain related to the podotrochlear apparatus based on clinical and lameness exam and blocks' responses. NB and DIJ anesthesia differed on the frequency of horses with lameness improvement above 70% only at 10min (p=0.03), and both differed from DDFTS block until 30'(p<0.05). The blocks' response was variable along the time and the highest means for NB, DIJ and DDFTS were observed at 5-10 minutes ('), 15-20' and 10-15' respectively.Exercise had low interference on lameness intensity since no improvement above 50% was observed and an increase on lameness intensity over time was identified in seven horses. Variable grades of navicular bone radiographic lesions were observed in 14 horses, although these lesions had no interference on blocks' response (p>0.05). The NB and DIJ blocks had similar responses and both were superior to DDFTS anesthesia, coincident with a major prevalence of podotroclear apparatus abnormalities in this equine population.(AU)

O presente estudo avaliou, de forma objetiva, as respostas do bloqueio da articulação interfalangeana distal (AID), da bursa do navicular (BN) e da bainha do tendão flexor digital profundo (BTFDP) em equinos com claudicação ligada ao casco nos membros torácicos; além de analisar a influência das alterações radiográficas do osso navicular no resultado dos bloqueios. Quinze cavalos, que apresentaram uma melhora da claudicação acima de 70% após o bloqueio do nervo digital palmar, foram selecionados para este estudo. Os bloqueios foram avaliados separadamente em cinco turnos consecutivos e em sete tempos diferentes. O quinto turno foi utilizado para analisar a influência do exercício sobre a claudicação preexistente. A maioria dos cavalos (73,33%) apresentou dor relacionada à porção palmar do casco, com base nos achados do exame clínico em movimento e nas respostas dos bloqueios. As anestesias da BN e da AID apresentaram diferença quanto à frequência de cavalos com melhora da claudicação acima de 70% apenas aos 10min (p=0.03), e ambos diferiram do bloqueio da BTFDP até os 30min (p<0.05). A resposta dos bloqueios foi variada ao longo do tempo, e as maiores médias de melhora da claudicação para os bloqueios BN, AID e BTFDP foram observadas aos 5-10min, 15-20min e 10-15min, respectivamente. O exercício teve pequena interferência na intensidade da claudicação, uma vez que nenhuma melhora acima de 50% foi observada e sete cavalos aumentaram a intensidade da claudicação ao longo do tempo. A presença de diferentes graus de lesão radiográfica do osso navicular foi observada em 14 cavalos, porém essas lesões não interferiram na resposta dos bloqueios (p>0,05). Os bloqueios da BN e da AID apresentaram respostas semelhantes, e ambos foram superiores ao bloqueio da BTFDP, coincidindo com uma marcada prevalência de doença do aparato podotroclear nesta população de equinos.(AU)

Lactococcus lactis carrying a DNA vaccine coding for the ESAT-6 antigen increases IL-17 cytokine secretion and boosts the BCG vaccine immune response.

AIMS: A regimen utilizing Bacille Calmette-Guerin (BCG) and another vaccine system as a booster may represent a promising strategy for the development of an efficient tuberculosis vaccine for adults. In a previous work, we confirmed the ability of Lactococcus lactis fibronectin-binding protein A (FnBPA+) (pValac:ESAT-6), a live mucosal DNA vaccine, to produce a specific immune response in mice after oral immunization. In this study, we examined the immunogenicity of this strain as a booster for the BCG vaccine in mice. METHODS AND RESULTS: After immunization, cytokine and immunoglobulin profiles were measured. The BCG prime L. lactis FnBPA+ (pValac:ESAT-6) boost group was the most responsive group, with a significant increase in splenic pro-inflammatory cytokines IL-17, IFN-γ, IL-6 and TNF-α compared with the negative control. CONCLUSIONS: Based on the results obtained here, we demonstrated that L. lactis FnBPA+ (pValac:ESAT-6) was able to increase the BCG vaccine general immune response. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is of great scientific and social importance because it represents the first step towards the development of a booster to the BCG vaccine using L. lactis as a DNA delivery system.

Impact or push-off lameness presentation is not altered by the type of track surface where horses are trotted / Claudicação de impacto ou elevação não é alterada pelo tipo de superfície onde os cavalos são troteados

This study aimed to evaluate the influence of the track surface on which horses are examined, regarding the phase of lameness presentation. Ten horses with lameness in at least one limb were evaluated with wireless inertial sensors on three track surfaces (concrete, loose sand and grass). Six crossover track sequences were established. The variables vector sum, maximum and minimum height of the head and pelvis, variation coefficient of the maximum and minimum height of the head and pelvis were analyzed using ANOVA, followed by Tukey test to compare means between track surface and sequence, at 5% significance level. The lameness phase (impact or pushoff) was analyzed considering the proportion of affected animals. There were no differences on vector sum, maximum and minimum height or variation coefficient of head and pelvis. Difference was observed on the number of strides registered on sand compared to grass and concrete (p <0.0001) for fore and hindlimbs. Impact lameness on forelimbs was presented by a larger number of animals on the concrete surface; pushoff lameness was more evident on the grass surface. In the hindlimbs, impact lameness was more evident on the grass surface, while pushoff lameness was in greater number of animals on concrete surfaces. The track sequence on which horses were trotted during evaluation does not seem to be a factor, but the number of lame horses and the phase of lameness manifestation can vary between track surfaces, as some horses showed impact lameness on soft ground and elevation lameness on hard ground.

Este estudo teve como objetivo avaliar a influência do tipo de superfície em que os cavalos são examinados em relação à fase de apresentação da claudicação. Dez cavalos com claudicação em pelo menos um dos membros foram avaliados com sensores inerciais sem fio em três tipos de superfície (concreto, areia e grama). Seis sequências de cruzamento de tipo de superfície foram estabelecidas. As variáveis soma vetorial, altura máxima e mínima da cabeça e da pélvis, o coeficiente de variação da altura máxima e mínima da cabeça e da pélvis foram analisadas utilizando uma Análise de Variância, seguida do teste de Tukey para comparação das médias entre tipos de superfície e sequências, a um nível de significância de 5%. A fase da claudicação (impacto ou elevação) foi analisada considerando a proporção de animais afetados. Não houve diferença na soma vetorial, altura máxima ou mínima e coeficiente de variação da altura máxima e mínima da cabeça e pélvis. Foi observada diferença no número de passos registrados na areia em comparação com grama e concreto (p <0,0001) para membros torácicos e pélvicos. Claudicação de impacto nos membros torácicos foi apresentada em um número maior de animais na superfície de concreto, já claudicação de elevação foi mais evidente na superfície de grama. Em membros pélvicos, a claudicação de impacto foi mais evidente na superfície de grama, enquanto claudicação de elevação esteve em maior número de animais na superfície de concreto. A sequência de superfícies em que os cavalos foram troteados durante a avaliação não foi um fator importante na manifestação da claudicação, mas o número de cavalos claudicantes e a fase de manifestação da claudicação podem variar entre as superfícies, visto que alguns cavalos mostraram claudicação de impacto em solo macio e claudicação de elevação em solo duro.

Maternal attitudes towards tooth decay in children aged 12-18 months in Pelotas, Brazil.

AIM: This was to assess mothers' attitudes towards dental caries in children aged 12-18 months. METHODS: This study targeted mothers of children aged 12-18 months. Data about demographic and socioeconomic status were collected by interviews with each mother. In addition, the mother was asked about her attitudes regarding caries in her child's primary teeth. A dental examination of each child was also conducted. Chi-square, bivariate, and multiple logistic regression analyses were performed. RESULTS: A total of 262 mother-child pairs were included, and 18.7 % of the children had dental caries. If a child presented with dental caries in their primary teeth, 93.5 % of the mothers reported that they would take the child to a dentist. Mothers who had only one child and those who had children with dental caries were more likely to report that they did not expect primary dental caries treatment by the dentist. CONCLUSION: Most mothers reported that they would take their children to a dentist when they presented with dental caries. Despite this positive result, educational measures should continue to be emphasised, especially among mothers of children at a higher risk of caries and among first-time mothers.

Prospective uses of recombinant Lactococcus lactis expressing both listeriolysin O and mutated internalin A from Listeria monocytogenes as a tool for DNA vaccination.

In this study, Lactococcus lactis was engineered to express mutated internalin A on its surface and to secrete large amounts of listeriolysin O (LLO) in order to improve its potential as a vehicle for DNA vaccination. Western blotting experiments demonstrated that the bacterium expressed LLO in both the cytoplasmic and extracellular compartments, with higher quantities found in the culture supernatants. A hemolytic assay showed that the recombinant strain secreted 250 ng active LLO/mg total protein. This mInlA/LLO-producing strain of L. lactis may be used as an alternative tool in DNA vaccination against a number of infectious diseases or in cancer therapy.

Correlation between the cariogenic response in biofilms generated from saliva of mother/child pairs.

This study aimed to correlate the cariogenic responsiveness of biofilms generated from the saliva of mothers and children. The mother-child pairs were classified according to the children's caries levels: caries-free, early childhood caries (ECC) or severe ECC. Microcosm biofilms were grown on enamel discs for 10 days. Factors under evaluation were caries experience levels, inoculum source (mothers and children) and growth conditions including cariogenic challenge (growth medium provided with and without sucrose) and no cariogenic challenge (growth medium sucrose-free). Statistical analysis was performed with ANOVA and Tukey's test, and the Spearman correlation test. Regular sucrose exposure resulted in a higher surface hardness change (%SHC). The correlation between biofilms formed from saliva of mother-child pairs was significant regarding pH, total aciduric microorganisms and lactobacilli counts under cariogenic challenge. Biofilm growth originating from mother-child pairs under regular sucrose exposure promoted the same cariogenic response independently of caries experience and the microbiological profile of the donors.

Neonatal handling alters the structure of maternal behavior and affects mother-pup bonding.

During early life, a mother and her pups establish a very close relationship, and the olfactory learning of the nest odor is very important for the bond formation. The olfactory bulb (OB) is a structure that plays a fundamental role in the olfactory learning (OL) mechanism that also involves maternal behavior (licking and contact). We hypothesized that handling the pups would alter the structure of the maternal behavior, affect OL, and alter mother-pup relationships. Moreover, changes in the cyclic AMP-response element binding protein phosphorylation (CREB) and neurotrophic factors could be a part of the mechanism of these changes. This study aimed to analyze the effects of neonatal handling, 1 min per day from postpartum day 1 to 10 (PPD 1 to PPD 10), on the maternal behavior and pups' preference for the nest odor in a Y maze (PPD 11). We also tested CREB's phosphorylation and BDNF signaling in the OB of the pups (PPD 7) by Western blot analysis. The results showed that handling alters mother-pups interaction by decreasing mother-pups contact and changing the temporal pattern of all components of the maternal behavior especially the daily licking and nest-building. We found sex-dependent changes in the nest odor preference, CREB and BDNF levels in pups OB. Male pups were more affected by alterations in the licking pattern, and female pups were more affected by changes in the mother-pup contact (the time spent outside the nest and nursing).

Effect of yogurt containing Bifidobacterium animalis subsp . lactis DN-173010 probiotic on dental plaque and saliva in orthodontic patients.

AIM: To assess how consumption of yogurt containing Bifidobacterium animalis subsp. lactis DN-173010 probiotic for a period of 2 weeks affects salivary and dental plaque levels of mutans streptococci and lactobacilli in patients undergoing orthodontic treatment. METHODS: A crossover, double-blind, randomized and placebo-controlled clinical trial was performed with 26 volunteers. The study was divided into four periods. During periods 2 and 4, the volunteers ingested yogurt containing probiotic or control yogurt daily for 2 weeks. Periods 1 and 3 were a 1-week run-in period and 4-week washout period, respectively. Saliva and dental plaque samples were collected from each participant at the end of each period. Mutans streptococci, lactobacilli, and total cultivable microorganisms were counted. Values were compared between groups and across periods with the Wilcoxon's test. RESULTS: There was no difference between the yogurt containing probiotic and the control yogurt for any of the studied variables (all p > 0.05). A reduction in counts of total cultivable microorganisms was observed in dental plaque samples after ingestion of either yogurts (both p < 0.05 vs. baseline), but not in saliva (p < 0.05). CONCLUSION: Daily ingestion of yogurt with or without B. animalis subsp. lactis for a period of 2 weeks was beneficial in reducing total microbial counts in dental plaque. Therefore, no additional benefits were achieved by the use of the tested probiotic strain.

Immunotherapy of allergic diseases using probiotics or recombinant probiotics.

Allergic diseases affect up to 30% of the western population, and their prevalence is increasing. Probiotics are able to modulate the mucosal immune response, and clinical trials demonstrated that specific strains, especially lactic acid bacteria (LAB) ones, reduce allergic symptoms. Moreover, the use of recombinant probiotics has been evaluated as possible strategies for the immunotherapy of allergic diseases. The production and delivery of allergens by recombinant LAB in concert with their ability to induce a Th1-type immune response have been shown to be a promising mucosal vaccination strategy in mouse model. The aim of this article is to review the applications of probiotics in allergy immunotherapy with a special focus on recombinant LAB delivering proteins or DNA.

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis.

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U.S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Lactobacillus acidophilus and Lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus.

Probiotic lactic acid bacteria (LAB) have been shown to alleviate inflammation, enhance the immunogenicity of rotavirus vaccines, or reduce the severity of rotavirus diarrhoea. Although the mechanisms are not clear, the differential Th1/Th2/Th3-driving capacities and modulating effects on cytokine production of different LAB strains may be the key. Our goal was to delineate the influence of combining two probiotic strains of Lactobacillus acidophilus and Lactobacillus reuteri on the development of cytokine responses in neonatal gnotobiotic pigs infected with human rotavirus (HRV). We demonstrated that HRV alone, or HRV plus LAB, but not LAB alone, initiated serum cytokine responses, as indicated by significantly higher concentrations of IFN-α, IFN-γ, IL-12, and IL-10 at postinoculation day (PID) 2 in the HRV only and LAB+HRV+ pigs compared to LAB only and LAB-HRV- pigs. Peak cytokine responses coincided with the peak of HRV replication. LAB further enhanced the Th1 and Th2 cytokine responses to HRV infection as indicated by significantly higher concentrations of IL-12, IFN-γ, IL-4 and IL-10 in the LAB+HRV+ pigs compared to the LAB-HRV+ pigs. The LAB+HRV+ pigs maintained relatively constant concentrations of TGF-ß compared to the HRV only group which had a significant increase at PID 2 and decrease at PID 7, suggesting a regulatory role of LAB in maintaining gut homeostasis. At PID 28, cytokine secreting cell (CSC) responses, measured by ELISpot, showed increased Th1 (IL-12, IFN-γ) CSC numbers in the LAB+HRV+ and LAB-HRV+ groups compared to LAB only and LAB-HRV- pigs, with significantly increased IL-12 CSCs in spleen and PBMCs and IFN-γ CSCs in spleen of the LAB+HRV+ group. Thus, HRV infection alone, but not LAB alone was effective in inducing cytokine responses but LAB significantly enhanced both Th1 and Th2 cytokines in HRV-infected pigs. LAB may also help to maintain immunological homeostasis during HRV infection by regulating TGF-ß production.

An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies.

Microcosm biofilms formed in microplates have demonstrated complex community dynamics similar to natural dental biofilm. No simplified microcosm models to evaluate enamel demineralization and dose-response effect to anticariogenic therapies have yet been established, thus this study was designed to develop a pre-clinical model fulfilling this purpose. Experiments were carried out to establish the time of biofilm formation and the sucrose concentration and exposure regimen. Biofilms were initiated from saliva and grown for up to 10 days on bovine enamel discs in 24-well plates, with a saliva analogue medium. Data were collected as pH readings and the percentage enamel surface hardness change. A dose-response evaluation was performed with chlorhexidine, which significantly affected the pH and mineral loss. Overall, the established model parameters, 5 days of biofilm growth with intermittent 1% sucrose exposure of 6 h per day, was suitable as a pre-clinical model for enamel demineralization and dose-response studies.

Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges.

OBJECTIVES: The source of saliva inocula and the individual characteristics of saliva donors could affect the cariogenic activity of in vitro biofilms, but this could also be modulated by environmental determinants, such as the frequency of sugar consumption. Therefore, the aim of this study was to compare the cariogenicity of microcosm biofilm growths from the saliva of caries-free (CF) children, children with early childhood caries (ECC) and with severe ECC (S-ECC), under regular sucrose exposure. METHODS: Microcosm plaque biofilms were initiated from the saliva of CF, ECC and S-ECC children. Biofilms were grown in 24-well microplates on bovine enamel discs for up to 10 days in artificial saliva, which was replaced daily. Growth conditions comprised cariogenic challenge (artificial saliva supplemented with 1% sucrose 6 h/day) or no cariogenic challenge. Daily pH was obtained from the artificial saliva, and after the experimental period, the biofilm formed on the enamel discs was collected for microbiological analyses. Mineral loss in enamel discs was estimated by percentage of surface hardness change. RESULTS: Overall, no statistically significant differences were found among saliva sources (p > 0.05). Streptococcus mutans and lactobacilli counts increased in the biofilms grown under cariogenic challenge (p < 0.05), while a substantial decrease in the artificial saliva pH was detected under the same condition (p < 0.001). Higher demineralization (p < 0.001) was observed under sucrose exposure regardless of caries experience of children. CONCLUSIONS: While the sucrose exposure determined the cariogenicity of the biofilms, the caries experience of children who provided the inocula did not affect mineral loss associated with these biofilms.

Pathogenesis and immune responses in gnotobiotic calves after infection with the genogroup II.4-HS66 strain of human norovirus.

We previously characterized the pathogenesis of two host-specific bovine enteric caliciviruses (BEC), the GIII.2 norovirus (NoV) strain CV186-OH and the phylogenetically unassigned NB strain, in gnotobiotic (Gn) calves. In this study we evaluated the Gn calf as an alternative animal model to study the pathogenesis and host immune responses to the human norovirus (HuNoV) strain GII.4-HS66. The HuNoV HS66 strain caused diarrhea (five/five calves) and intestinal lesions (one/two calves tested) in the proximal small intestine (duodenum and jejunum) of Gn calves, with lesions similar to, but less severe than, those described for the Newbury agent 2 (NA-2) and NB BEC. Viral capsid antigen was also detected in the jejunum of the proximal small intestine of one of two calves tested by immunohistochemistry. All inoculated calves shed virus in feces (five/five calves), and one/five had viremia. Antibodies and cytokine (proinflammatory, tumor necrosis factor alpha [TNF-alpha]; Th1, interleukin-12 [IL-12] and gamma interferon [IFN-gamma]; Th2, IL-4; Th2/T-regulatory, IL-10) profiles were determined in serum, feces, and intestinal contents (IC) of the HuNoV-HS66-inoculated calves (n = 5) and controls (n = 4) by enzyme-linked immunosorbent assay in the acute (postinoculation day 3 [PID 3]) and convalescent (PID 28) stages of infection. The HuNoV-HS66-specific antibody and cytokine-secreting cells (CSCs) were quantitated by ELISPOT in mononuclear cells of local and systemic tissues at PID 28. Sixty-seven percent of the HuNoV-HS66-inoculated calves seroconverted, and 100% coproconverted with immunoglobulin A (IgA) and/or IgG antibodies to HuNoV-HS66, at low titers. The highest numbers of antibody-secreting cells (ASC), both IgA and IgG, were detected locally in intestine, but systemic IgA and IgG ASC responses also occurred in the HuNoV-HS66-inoculated calves. In serum, HuNoV-HS66 induced higher peaks of TNF-alpha and IFN-gamma at PIDs 2, 7, and 10; of IL-4 and IL-10 at PID 4; and of IL-12 at PIDs 7 and 10, compared to controls. In feces, cytokines increased earlier (PID 1) than in serum and TNF-alpha and IL-10 were elevated acutely in the IC of the HS66-inoculated calves. Compared to controls, at PID 28 higher numbers of IFN-gamma and TNF-alpha CSCs were detected in mesenteric lymph nodes (MLN) or spleen and Th2 (IL-4) CSCs were elevated in intestine; IL-10 CSCs were highest in spleen. Our study provides new data confirming HuNoV-HS66 replication and enteropathogenicity in Gn calves and reveals important and comprehensive aspects of the host's local (intestine and MLN) and systemic (spleen and blood) immune responses to HuNoV-HS66.

Cytokine and antibody responses in gnotobiotic pigs after infection with human norovirus genogroup II.4 (HS66 strain).

A human norovirus genogroup II.4 strain HS66 (HuNoV-HS66) infects and causes mild diarrhea in gnotobiotic (Gn) pigs (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). In this study we evaluated systemic and intestinal humoral and cellular immune responses to HuNoV-HS66 in orally inoculated pigs. Antibodies and type I interferon (IFN-I or IFN-alpha), proinflammatory interleukin-6 (IL-6), Th1 (IL-12 and IFN-gamma), Th2 (IL-4), and Th2/regulatory T ([T(reg)] IL-10) cytokine profiles in serum and intestinal contents (IC) of the HuNoV-HS66-inoculated pigs and controls were assessed by enzyme-linked immunosorbent assay at selected postinoculation days (0 to 28). Using an enzyme-linked immunospot assay, we evaluated immunoglobulin M (IgM), IgA, and IgG antibody-secreting cells (ASC) and cytokine-secreting cells (CSC) in intestine, spleen, and blood. In the HuNoV-inoculated pigs, antibody titers in serum and IC were generally low, and 65% seroconverted. Pigs with higher diarrhea scores were more likely to seroconvert and developed higher intestinal IgA and IgG antibody titers. The numbers of IgA and IgG ASC were higher systemically than in the gut. In serum, HuNoV induced persistently higher Th1 (low transient IFN-gamma and high IL-12) than the other cytokines, but also low Th2 (IL-4) and Th2/T(reg) (IL-10) levels; low, transient proinflammatory (IL-6) cytokines; and, notably, a delayed IFN-alpha response. In contrast, intestinal innate (IFN-alpha early and late) and Th1 (IL-12 late) cytokines were significantly elevated postinfection. HuNoV-HS66 also elicited higher numbers of Th1 (IL-12 and IFN-gamma) CSC than Th2 (IL-4) and proinflammatory (IL-6) CSC, with the latter responses low in blood and intestine, reflecting low intestinal inflammation in the absence of gut lesions. These data provide insights into the kinetics of cytokine secretion in serum and IC of HuNoV-inoculated Gn pigs and new information on intestinal humoral and cellular immune responses to HuNoV that are difficult to assess in human volunteers.

Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus.

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.

Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus.

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.