Fast acetone tissue processing of human organs provides tissue characteristics equal to conventional processing.

Routine preparation of paraffin embedded tissue for histopathological diagnosis, here termed conventional histological technique (CT), whether performed manually or using an automated system, requires approximately 12 h. We developed earlier a rapid acetone dehydration technique (AT) for processing biopsies of nervous tissue that meets requirements for preserving tissue morphology and staining properties, and reduces processing time to 3.3 h. We compared the morphology and staining properties of human organ biopsies including adrenal gland, liver, ovary, pancreas, prostate, testis and thyroid prepared using both AT and CT. Following fixation with 10% formaldehyde and processing by either AT or CT, sections were stained using routine and special staining, and immunohistochemical methods. We evaluated nuclear and cytoplasmic staining, staining intensity, sharpness of images and presence of artifacts such as cracking and folding. AT preserved the morphology and staining properties of the tissues as well as CT. Consequently, the rapid AT procedure is a promising alternative technique for tissue processing.

Diagnóstico de la higiene bucal en pacientes con aparatos de ortodoncia

Fundamento:una adecuada higiene bucal es necesaria en los pacientes que portan aparatos de ortodoncia.Objetivo:identificar conocimientos, actitudes, prácticas e higiene bucal en pacientes con aparatos de ortodoncia.Métodos:se realizó un estudio con diseño no experimental, descriptivo y transversal en la Clínica Estomatológica Docente Provincial de Sancti Spíritus en el período entre septiembre de 2018 y julio de 2019. Se utilizaron métodos del nivel teórico, empírico (encuesta, entrevista, observación y estadístico). La población estuvo constituida por 30 pacientes de esta institución con tratamiento de ortodoncia. Se midieron las variables: edad y sexo del paciente, tipo de aparato de ortodoncia, nivel de conocimiento sobre salud bucodental, actitud y prácticas del paciente de higiene bucal, frecuencia, forma y eficiencia del cepillado dental, así como el cepillado o no después de la ingestión de alimentos azucarados.Resultados:el 100 % de los pacientes presentó conocimientos deficientes sobre salud bucal y prácticas desfavorables, aunque se constató actitudes favorables en el 63,3 % de los pacientes. Predominó una higiene bucal regular en la población estudiada antes de iniciar el tratamiento y después de instalar los aparatos, de manera similar en los grupos con aparatos removibles, funcionales y fijos.Conclusiones:los pacientes de la población estudiada necesitan educación para apropiarse de conocimientos suficientes para mantener actitudes, prácticas e higiene bucal saludables.[AU]

Management of the oral hemangiomas in infants and children: Scoping review.

BACKGROUND: Pediatric oral hemangiomas are benign vascular tumors that can be seen from birth, particularly in females. Hemangiomas are most frequent located in the lips and usually regress spontaneously, thus they do not require any type of treatment in most cases. The present scoping review pretended to synthesize the most relevant and currently available information from the international dental literature published in the last 25 years, regarding the management of pediatric oral hemangiomas. MATERIAL AND METHODS: An exhaustive literature search was performed in four electronic databases (PubMed, Embase, Google Scholar, and Cochrane). Initially, 241 related titles and abstracts were found. After the duplication removal, screening, and assessment processes, 37 records were included for full-text reading. Finally, 20 articles in the English language were included in the scoping review for data extraction and assessment. RESULTS: We identified and subsequently discussed three fundamental issues associated to the management of pediatric oral hemangiomas: (i) clinical characteristics, differential diagnosis, and histopathological findings; (ii) evolution and complications; and (iii) current available treatment modalities. CONCLUSIONS: Although these like-tumor lesions are uncommon, pediatric dentistry practitioners must be familiar with the inherent clinical characteristics, diagnosis approaches, and currently available treatment options. Nowadays, surgical removal and non-invasive medical/pharmacologic therapies are the best management modalities for pediatric oral hemangiomas.

Variation of the CD4, CD8, and MHC II cell population in granulomas of immunocompetent and immunosuppressed rabbits in Encephalitozoon cuniculi infection.

Encephalitozoon cuniculi (E. cuniculi) is a fungi-related, obligate, zoonotic, spore-forming intracellular eukaryotic microorganism. This emerging pathogen causes granulomas in brain and kidneys of infected individuals. The objective of this study was to detect the distribution of CD4, CD8 and MHCII-positive cells within granulomas in these organs in infected immunocompetent (group A) and infected immunosuppressed (group B) New Zealand white rabbits using immunohistochemistry. In brain, labeled CD4 immune cells were mainly located in the periphery of granulomas in group B. Kidneys of groups A and B, displayed CD4-positive in granulomas and were significant different when compared to brain. CD8 immune cells in brain and kidneys were disseminated in the granulomas in groups A and B; however, no significant difference was observed. MHCII-positive cells were more numerous in brain sections of group B and were significantly different when compared to kidney sections. Granulomas were not observed in control animals of group C and D. In conclusion, we identified CD4-positive cells in both the brain and kidneys of immunocompetent and immunosuppressed animals; CD8-positive cells were more numerous in brain of immunosuppressed rabbits and MHCII cells were more predominant in brain of immunocompetent rabbits. Apparently, the immunosuppression stimulated a change in the cellular phenotype of Th1- to Th2-like granulomas in brain and kidneys by an unknown mechanism. These results increase our understanding of CD4, CD8 and MHCII positive cells within the E. cuniculi granuloma microenvironment and will help in future microsporidian granulomas studies of both immunocompetent and immunosuppressed individuals.

Development of Lactococcus lactis encoding fluorescent proteins, GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system.

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.

Recombinant adenovirus delivery of calreticulin-ESAT-6 produces an antigen-specific immune response but no protection against a Mycobacterium tuberculosis challenge.

Bacillus Calmette-Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT-ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT-ESAT-6-CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.

Alternative life histories in Xiphophorus multilineatus: evidence for different ages at sexual maturity and growth responses in the wild.

In order to examine potential trade-offs in alternative life histories of the high-backed pygmy swordtail Xiphophorus multilineatus, otoliths were used from wild-caught males to determine if sneaker males had the advantage of maturing earlier in natural environments. The sneakers matured significantly earlier than courters, but there was no difference among the three courter variants. In addition, analyses suggested that the effect of the pituitary locus on size at sexual maturity and growth rates was a consequence of age at sexual maturity. Finally, one of the courter variants had a significantly different relationship between age and size at sexual maturity than the other variants, suggesting that in this variant, age at sexual maturity may be more closely related to size and therefore may be less plastic in its growth responses.

Efficient secretion of a modified E7 protein from human papilloma virus type-16 by Lactococcus lactis.

AIMS: To create and provide a strain of the food-grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type-16. METHODS AND RESULTS: We cloned the coding sequence of a modified E7 (E7m) from the HPV-16 in a plasmid regulated by the strong expression promoter p59. Secretion of the E7m was made by the signal peptide of the usp45 gene. The E7m was detected by Western blot in the cell-free-medium fraction, showing no degradation or aberrant forms. CONCLUSIONS: We constructed a strain of L. lactis able to secrete efficiently a HPV-16 E7 modified protein with diminished transforming activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Human papilloma virus infection is associated with more than 99% of cervical cancers. Immunotherapy targeting E7 to treat HPV-associated cervical malignancies has been demonstrated to be highly efficient. However, native E7 maintains transforming activity. We present this new strain of a food-grade bacterium able to efficiently secrete a HPV-16 E7-modified protein with diminished transforming activity. This new strain could be used as a live vaccine to deliver E7 at a mucosal level and generate antitumour immune responses against HPV-associated tumours.

Targeting and retention of HPV16 E7 to the endoplasmic reticulum enhances immune tumour protection.

The endoplasmic reticulum (ER) is where the major histocompatibility complex (MHC) class I molecules are loaded with epitopes to cause an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to amino acids and few epitopes reach the ER. Antigen targeting of this organelle by Calreticulin (CRT) fusion avoids this degradation and enhances the immune response. We constructed a recombinant adenovirus to express the E7 antigen with an ER-targeting signal peptide (SP) plus an ER retention signal (KDEL sequence). In cell-culture experiments we demonstrated that this new E7 antigen, SP-E7-KDEL, targeted the ER. Infection of mice with this recombinant adenovirus that expresses SP-E7-KDEL showed interferon induction and tumour-protection response, similar to that provided by an adenovirus expressing the E7 antigen fused to CRT. This work demonstrated that just by adding a SP and the KDEL sequence, antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumour protection. These results will have significant clinical applications.

Nuevas consideraciones sobre el control glicémico en pacientes con diabetes tipo 2 y sus implicancias sobre el riesgo cardiovascular / New considerations on glycemic control in patients with type 2 diabetes and its implications on cardiovascular risk

Los resultados de los últimos estudios utilizando una terapia hipoglicemiante intensiva en pacientes con diabetes mellitus tipo 2 han demostrado ser exitosos en el control glicémico de estos pacientes, sin embargo, no han logrado obtener una reducción significativa de la mortalidad por patología cardiovascular.

Ovarian response and embryo production in llamas treated with equine chorionic gonadotropin alone or with a progestin-releasing vaginal sponge at the time of follicular wave emergence.

The objective of the study was to compare the ovulatory response and embryo production in llamas (Lama glama) treated with a single dose of equine chorionic gonadotropin (eCG) alone or combined with intravaginal medroxyprogesterone acetate (MPA) at the time of follicular wave emergence. Llamas with a growing follicle >or=7 mm in diameter were assigned to one of the following groups: (1) Control (n=28): Nonstimulated llamas were mated and embryos were collected 7 d after mating. (2) eCG (n=32): Llamas were given 5mg luteinizing hormone (LH) (Day 0) to induce ovulation, 1000 IU eCG on Day 2, a luteolytic dose of prostaglandin F(2alpha) on Day 6, mating on Day 7, and embryo collection on Day 14. (3) eCG+MPA (n=34): Llamas were treated as those in the eCG group, but a sponge containing 60 mg MPA was placed intravaginally from Days 2 to 6. Llamas that did not respond to synchronization or superstimulation were excluded, leaving data from n=26, 26, and 27 in the control, eCG, and eCG+MPA groups, respectively, for statistical analysis. The mean (+/-SD) number of follicles>7 mm at the time of mating was greatest in the eCG group, intermediate in the eCG+MPA group, and lowest in the control group (16.6+/-5.3, 12.9+/-3.7, and 1.0+/-0.0, respectively, P<0.001). The number of corpora lutea was similar between eCG and eCG+MPA groups (10.1+/-2.9 and 8.6+/-3.7, respectively); both were higher (P<0.001) than in controls (0.9+/-0.3). The number of embryos did not differ significantly between the eCG and eCG+MPA groups (4.8+/-2.8 and 3.5+/-3.0, respectively), but both were higher (P<0.001) than in the controls (0.7+/-0.4). In conclusion, eCG, with or without MPA effectively induced a superovulatory response and multiple embryo production in llamas.

Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected alpacas (Lama pacos), llamas (Lama glama) and vicunas (Lama vicugna) from Peru and Germany.

Sera of an experimentally Neospora caninum infected llama and a non-infected control llama were used to establish an immunoblot, an ELISA and an IFAT to detect antibodies against N. caninum tachyzoites. Subsequently, serum samples collected from a total of 871 South American Camelids (SAC: Lama glama, Lama pacos, Lama vicugna) of two farms in Peru and from 32 SAC of a farm in central Germany were examined for antibodies against N. caninum and Toxoplasma gondii. Based on the recognition of specific bands in the immunoblot, sera of SAC from Peru were differentiated into N. caninum-positive (n = 18) and T. gondii-positive (n = 30) samples and into samples negative or inconclusive for both parasites. Using the immunoblot results as the reference, a modified version of the p38-ELISA and the IFAT were evaluated for detecting N. caninum antibodies in SAC sera. Applying a cut-off as determined by two graph-receiver operating characteristic analysis both, the ELISA and the IFAT, exhibited a sensitivity and specificity of about 95% in the SAC sera from Peru. Serological testing confirmed that SAC may become infected with N. caninum under field conditions in Peru. In addition to alpacas and llamas also 114 wild living vicunas had been examined for antibodies against N. caninum. However, only the alpacas and llamas but no vicunas were found N. caninum-positive. In contrast, T. gondii-seropositive animals were detected in all three SAC species. The lack of N. caninum-seropositive vicunas indicates that in the study area in Peru wild canids might not serve as definitive hosts of N. caninum while for T. gondii a life cycle including wild felids is likely. On the German farm no N. caninum- but only T. gondii-seropositive SAC (n = 14) were detected. The seroprevalence of T. gondii infection was significantly higher in adult SAC (alpacas in Peru, llamas in Germany) than in crias (i.e. < 12 months old foals) indicating that the predominant route of infection is post natal. Since the present study was restricted to a few farms, the seroprevalences determined are not representative. However, our results confirm natural infections with N. caninum and T. gondii in SAC. Whether these infections are linked to any disease, e.g. reproductive losses, has to be clarified in further studies.

Selective agenesis of mesencephalic dopaminergic neurons in Nurr1-deficient mice.

Nurr1, a member of the nuclear receptor superfamily of transcription factors, has been found to be essential for the development of ventral midbrain dopamine (DA)ergic neurons. To study the regional selectivity and phenotypic specificity of regulation by Nurr1 of the genesis of DAergic neurons, we examined DAergic, serotonin (5-HT)ergic, norepinephrine (NE)ergic, cholinergic, glutamate (GLU)ergic, and gamma-aminobutyric acid (GABA)ergic neurons in the brains of Nurr1-deficient mice by immunohistochemistry and biochemistry. We demonstrated that in homozygous Nurr1-deficient mice (Nurr1-/-), DAergic neurons were totally absent in substantia nigra and ventral tegmental area, but preserved in other regions including diencephalon and hypothalamus, olfactory bulb (OB). Levels of DA in Nurr1-/- mice were decreased by 98% in striatum (Str) and 65% in OB. NEergic neurons in locus ceruleus, 5-HTergic neurons in raphe nuclei, and cholinergic neurons in basal forebrain and other regions were not changed. A 30% reduction of NE was found in the Str of Nurr1-/- mice. The levels of GLU and GABA and the activity of choline acetyl transferase in the brains of Nurr1-/- mice were not significantly altered. Our results demonstrate a selective and specific deficit of DA and absence of DAergic neurons in the mesencephalic structures of Nurr1-deficient mice, which resembles the pattern similar to that seen in patients with Parkinson's disease (PD). This model may contribute to our understanding of the mechanisms influencing DAergic cell survival in PD.

Restricted spatiotemporal expression of lactoferrin during murine embryonic development.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins. Lactoferrin is induced by estrogen in the mouse uterus during early pregnancy. However, the expression and function, if any, of lactoferrin in the preimplantation embryo during this developmental period has not been investigated. In the current study, the spatiotemporal expression of lactoferrin during murine embryogenesis was examined using in situ hybridization and immunohistochemical analyses. Lactoferrin expression was first detected in the 2-4 cell fertilized embryo and continued until the blastocyst stage of development. Interestingly, at the 16-cell stage, coinciding with the first major differentiation step in the embryo, lactoferrin messenger RNA (mRNA) is synthesized by the inner cells, whereas the protein is selectively taken up by the outside cells. This differential pattern of lactoferrin messenger RNA and protein localization continues until the blastocyst stage, with expression almost absent in the hatched blastocyst. Lactoferrin expression does not resume in the embryo until the latter half of gestation, where it is first detected in neutrophils of the fetal liver at embryonic day 11.5 and later in epithelial cells of the respiratory and digestive systems. Our results show that lactoferrin is expressed in a tightly regulated spatiotemporal manner during murine embryogenesis and suggest a novel paracrine role for this protein in the development of the trophoectodermal lineage during preimplantation development.

Restricted spatiotemporal expression of lactoferrin during murine embryogenesis.

Lactoferrin is a member of the transferrin family of iron-binding proteins to which several physiological functions have been ascribed. While there is a wealth of evidence about the distribution and function of this protein in the adult, the expression and function, if any, of lactoferrin during embryogenesis has not been investigated. In the current study, the spatiotemporal distribution of lactoferrin was analyzed during normal murine embryonic development. This analysis demonstrated that lactoferrin is expressed in three distinct patterns during embryogenesis. First, lactoferrin is expressed at the 2-cell stage in the preimplantation embryo where it continues to be expressed until the blastocyst stage when expression ceases. The second phase of lactoferrin expression is not detected until the latter half of gestation when the protein is detected in the myeloid cells, beginning in the fetal liver at embryonic day 11 and later in the spleen and bone marrow coinciding with the onset and diversification of myelopoiesis in these organs during embryogenesis. Finally, lactoferrin is detected in a variety of glandular epithelial cells and/or their secretions, including respiratory and oral epithelia which is consistent with the expression pattern observed for this protein in the adult where it plays an important role in host defense at the mucosal surface. Taken together, these analyses indicate that the role of lactoferrin in the developing embryo is restricted to the preimplantation stage and development of first and second line host defense systems.

Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons.

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the floor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.

Streptococcus pneumoniae bacteremia in a community hospital.

We reviewed 71 consecutive patients with Streptococcus pneumoniae bacteremia. The patients were analyzed by age, sex, ethnic background, and clinical presentation. Laboratory data reviewed included a CBC count, electrolyte levels, liver function studies, chest radiograph, HIV status, a sputum culture and Gram's stain, and sensitivities for the S pneumoniae isolated. Modalities of therapy, response to treatment, and ultimate outcome were examined. Many of the patients with pneumococcal bacteremia did not have cough, fever, or chills. HIV positivity was a risk factor for pneumococcal infection although it was not associated with increased mortality. Mortality correlated with elderly status, leukopenia, and lack of fever. Many patients had symptoms suggestive of atypical pneumonia including myalgia and mental status change. Hyponatremia and hyperbilirubinemia were commonly noted.

Cloning and structural organization of the gene encoding the murine nuclear receptor transcription factor, NURR1.

NURR1 is an immediate early gene product and a member of the nuclear receptor superfamily of transcription factors. Using the NURR1 cDNA as a probe, we isolated the genomic DNA encoding NURR1 from a mouse 129SvEv genomic library. The NURR1 gene is approximately 6.2 kb long and is organized into 7 exons separated by 6 introns. Structural analysis of the NURR1 reveals that this gene shares a similar structure with that of the nuclear receptor NUR77/NGF1-B. As in NUR77, the promoter region of NURR1 lacks an identifiable TATA box, but is GC-rich. The proximal promoter region also contains an ATF/CREB consensus binding site that may participate in cAMP-mediated induction of this immediate early gene product. Isolation and structural characterization of the NURR1 gene provides information for further developmental and transcriptional regulation studies of this gene.

Comparative distribution of NURR1 and NUR77 nuclear receptors in the mouse central nervous system.

NURR1 and NUR77 are members of the nuclear receptor superfamily of transcription factors. Both proteins can interact with common enhancer elements to regulate target gene expression. In order to establish whether both transcription factors are likely to regulate overlapping genes, we have used an in situ hybridization approach to relate the constitutive expression pattern of these mRNAs with functionally defined regions of the adult mouse brain. By Western analysis, NURR1 mRNA expressed by brain cells appeared to be translated. Here we show that both transcripts display a differential but partially overlapping pattern of expression within the central nervous system (CNS). The expression of NURR1 is more restricted than NUR77 and is localized predominantly in sensory neuronal structures associated with the limbic system and in the cerebellum. In contrast, the expression pattern of NUR77 is more widespread. Positively staining cells for NUR77 appear to overlap with NURR1-containing cells in the limbic system and cerebellum, suggesting overlapping roles for these proteins in mediating behavioral and cognitive function as well as equilibrium maintenance. However, the differential expression of NUR77 in motor areas of the cortex and basal ganglia suggest a selective role for this transcription factor in regulation of motor function at the constitutive level. Our data indicates that these nuclear receptors are likely to have both shared and independent gene regulatory roles in neuronal cells.