Energy requirements and efficiency of energy utilization in growing dairy goats of different sexes.

The aim of this study was to investigate the effects of sex on the requirements for maintenance and efficiency of energy utilization in growing Saanen goats. A database from 7 comparative slaughter studies that included 238 Saanen goats was gathered to provide information for the development of prediction equations of energy requirements for maintenance and efficiency of energy utilization. The experimental design provided different levels of metabolizable energy intake (MEI) and empty body weight (EBW). The data were analyzed so that sex (e.g., intact males, castrated males, and females; n = 98, 80, and 60, respectively) was a fixed effect, and blocks nested in the studies and goat sex were random effects. For the development of linear and nonlinear equations, we used the MIXED and NLMIXED procedures in SAS (SAS Institute Inc., Cary, NC). Nonlinear regression equations were developed to predict heat production (HP, kcal/kg0.75 of EBW; dependent variable) from MEI (kcal/kg0.75 of EBW; independent variable). Using the comparative slaughter technique, the net energy requirement for maintenance (NEM) was calculated as the value of HP at MEI equal to zero. Additionally, NEM was evaluated based on the degree of maturity. The metabolizable energy requirement for maintenance was calculated as the value at which HP is equal to MEI. Efficiency of ME utilization for maintenance (km) was calculated as the ratio between NEM and the metabolizable energy requirement for maintenance. Efficiency of energy utilization for growth (kg) was assumed to be the slope of the linear regression of retained energy (RE) on MEI above the maintenance stage (model intercept equal to 0). Efficiencies of RE as protein (kp) and as fat (kf) were calculated using the multiple linear regression of MEI above the maintenance (model intercept equal to 0) on RE as protein and as fat, respectively. Sex affected NEM (75.0 ± 1.76 kcal/kg0.75 of EBW for males and 63.6 ± 2.89 kcal/kg0.75 of EBW for females) and sex did not affect km (0.63). In contrast, sex no longer affected NEM when degree of maturity was considered on its estimation. The kg was different between sexes (0.31 for castrated males and females, and 0.26 for intact males), but kp (0.21) and kf (0.80) were similar between sexes. These results may be useful for improving robustness of the energy requirement recommendations for dairy goats.

HspBP1 and anti-HspBP1 levels in the serum of HIV-infected individuals are associated to the disease progression.

AIMS: The objective of this research was to quantify the levels of circulating HspBP1 and anti-HspBP1 IgG in HIV-infected individuals and to correlate them with CD4 T cell counts and viral load, as well as to determine the kinetics of those proteins during acute phase. METHODS AND RESULTS: Sixty serum samples from HIV-positive outpatients, thirty with high viral load and thirty with low viral load were analysed. The HspBP1 and anti-HspBP1 were quantified by ELISA. To investigate the kinetic of HspBP1 and anti-HspBp1 during the acute phase, these proteins and antibodies were quantified in samples of a commercial seroconverting HIV panel. All dosages were compared with the CD4 and CD8 T cell counts and HIV viral load. The results indicated that HIV positive outpatients presented significant increase in HspBP1 and anti-HspBP1 serum levels, compared with uninfected healthy. HspBP1 and anti-HspBP1 were negatively correlated with CD4 counts and CD4:CD8 ratio. In the acute phase, HspBP1 became significantly elevated 15 days after HIV infection. CONCLUSIONS: These results indicate that the quantification of HspBP1 can be associated to others well-established parameters of the HIV progression. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery that HspBp1 and anti-HspBp1 are associated with progression of HIV infection is new and corroborates to validate the quantification of these proteins as an additional strategy in the management of the HIV infection.

Association of CXCL13 serum level and ultrasonographic findings of joints in patients with systemic lupus erythematosus and Jaccoud's arthropathy.

Objectives The objective of this paper is to perform an ultrasonography (US) analysis of hands and wrists in two groups of patients with systemic lupus erythematosus (SLE), with and without Jaccoud's arthropathy, matched by age and disease duration and to correlate them with levels of CXCL13 clinical features, laboratory tests and disease activity score. Methods Sixty-four patients with SLE were enrolled, 32 with and 32 without Jaccoud's arthropathy. Each patient underwent physical examination, laboratory tests (including CXCL13 by ELISA) and bilateral US. Synovial hypertrophy, tenosynovitis and erosions were evaluated according to a semiquantitative grading system with a 0-3 rating. US findings were correlated with serum levels of CXCL13, other serological parameters and disease activity index. Results Synovitis was found in 25/64 patients (39%) and tenosynovitis in 14/64 (22%). These findings were more frequent in SLE patients with Jaccoud's arthropathy, particularly tenosynovitis ( p = 0.002) and synovitis ( p = 0.01). Median serum level of CXCL13 was 20.16 pg/ml in the whole population (23.21 pg/ml in the Jaccoud's arthropathy group and 11.48 pg/ml in the group without). There was an association between the presence of disease activity and high level of CXCL13 ( p = 0.004). However, no association was found between high levels of CXCL13 and "arthritis" in SLEDAI, swollen joints on physical examination or synovitis on US. Conclusions US findings in joints of SLE patients with Jaccoud's arthropathy confirm that synovitis and tenosynovitis are common in these patients. In addition, serum level of CXCL13 is associated with disease activity in SLE but does not seem to be a biomarker for arthritis in these patients.

iNKT cells are increased in children with severe therapy-resistant asthma.

BACKGROUND: Invariant natural killer T (iNKT) cells play complex functions in the immune system, releasing both Th1 and Th2 cytokines. The role of iNKT cells in human asthma is still controversial and never described in severe therapy-resistant asthma in children. The objective of this work was to analyse iNKT frequency in peripheral blood of children with severe therapy-resistant asthma (STRA), compared to children with milder asthma and healthy controls. METHODS: Children with asthma (n=136) (non-severe and STRA) from a referral centre and healthy controls (n=40) were recruited. Peripheral blood mononuclear cells were isolated, stained with anti-CD3 and anti-iNKT (Vα24Jα18), and analysed through flow cytometry. Atopic status was defined by measuring specific IgE in serum. Airway inflammation was assessed by induced sputum. RESULTS: Children with asthma presented an increased frequency of CD3+iNKT+ cells (median 0.38% IQR 0.18-1.9), compared to healthy controls (median 0.26% IQR 0.10-0.43) (p=0.025). Children with STRA also showed an increased frequency of iNKT cells (1.5% IQR 1.05-2.73) compared to healthy controls and non-severe asthmatic children (0.35% IQR 0.15-1.6; p=0.002). The frequency of iNKT cells was not different between atopic and non-atopic children. In addition, iNKT cells were not associated with any inflammatory pattern of induced sputum studied. CONCLUSION: Our data suggests that iNKT cells play a role in paediatric asthma, which is also associated with the severity of disease, but independent of the atopic status.

Cell wall changes during the formation of aerenchyma in sugarcane roots.

Background and Aims: Aerenchyma develops in different plant organs and leads to the formation of intercellular spaces that can be used by the plant to transport volatile substances. Little is known about the role of cell walls in this process, although the mechanism of aerenchyma formation is known to involve programmed cell death and some cell wall modifications. We assessed the role that cell wall-related mechanisms might play in the formation of aerenchyma in sugarcane roots. Methods: Sections of roots (5 cm) were subjected to microtomography analysis. These roots were divided into 1-cm segments and subjected to cell wall fractionation. We performed analyses of monosaccharides, oligosaccharides and lignin and glycome profiling. Sections were visualized by immunofluorescence and immunogold labelling using selected monoclonal antibodies against polysaccharide epitopes according to the glycome profiles. Key Results: During aerenchyma formation, gas spaces occupied up to 40 % of the cortex cross-section within the first 5 cm of the root. As some of the cortex cells underwent dissolution of the middle lamellae, leading to cell separation, cell expansion took place along with cell death. Mixed-linkage ß-glucan was degraded along with some homogalacturonan and galactan, culminating in the formation of cell wall composites made of xyloglucan, arabinoxylans, cellulose and possibly lignin. Conclusion: The composites formed seem to play a role in the physical-chemical properties of the gas chambers, providing mechanical resistance to forces acting upon the root and at the same time decreasing permeability to gases.

IL-8/IL-17 gene variations and the susceptibility to severe viral bronchiolitis.

Clinical manifestations of acute bronchiolitis (AB) vary from minimal disease to severe respiratory failure. The response to respiratory viral infections is possibly influenced by genetic polymorphisms linked to the regulation of the inflammatory response. In the present study, we investigated whether interleukin-8 (IL-8) and interleukin-17 (IL-17) genetic variants are associated with the severity of AB. A group of Brazilian infants hospitalized with AB and a control group (infants with no or mild AB, without hospitalization) were genotyped for four IL-8/IL-17 variations. For replication, we studied an Argentinean population sample of infants with mild and severe AB. IL-8 polymorphism (rs 2227543) and IL-17 (rs2275913) variants showed significant associations with the severity of AB. The effect of the IL-8 variation could be replicated in the Argentinean sample. This finding suggests that IL-8 variations may influence the severity of AB in young infants. Further genetic association studies in low- or middle-income populations are necessary with the aim of expanding knowledge in this area.

Sex effects on net protein and energy requirements for growth of Saanen goats.

Requirements for growth in the different sexes remain poorly quantified in goats. The objective of this study was to develop equations for estimating net protein (NPG) and net energy (NEG) for growth in Saanen goats of different sexes from 5 to 45 kg of body weight (BW). A data set from 7 comparative slaughter studies (238 individual records) of Saanen goats was used. Allometric equations were developed to determine body protein and energy contents in the empty BW (EBW) as dependent variables and EBW as the allometric predictor. Parameter estimates were obtained using a linearized (log-transformation) expression of the allometric equations using the MIXED procedure in SAS software (SAS Institute Inc., Cary, NC). The model included the random effect of the study and the fixed effects of sex (intact male, castrated male, and female; n = 94, 73, and 71, respectively), EBW, and their interactions. Net requirements for growth were estimated as the first partial derivative of the allometric equations with respect to EBW. Additionally, net requirements for growth were evaluated based on the degree of maturity. Monte Carlo techniques were used to estimate the uncertainty of the calculated net requirement values. Sex affected allometric relationships for protein and energy in Saanen goats. The allometric equation for protein content in the EBW of intact and castrated males was log10 protein (g) = 2.221 (±0.0224) + 1.015 (±0.0165) × log10 EBW (kg). For females, the relationship was log10 protein (g) = 2.277 (±0.0288) + 0.958 (±0.0218) × log10 EBW (kg). Therefore, NPG for males was greater than for females. The allometric equation for the energy content in the EBW of intact males was log10 energy (kcal) = 2.988 (±0.0323) + 1.240 (±0.0238) × log10 EBW (kg); of castrated males, log10 energy (kcal) = 2.873 (±0.0377) + 1.359 (±0.0283) × log10 EBW (kg); and of females, log10 energy (kcal) = 2.820 (±0.0377) + 1.442 (±0.0281) × log10 EBW (kg). The NEG of castrated males was greater than that of intact males and lower than that of females. Using degree of maturity for estimating NPG and NEG, we could remove the differences between sexes. These results indicate that NPG and NEG differ among sexes in growing Saanen goats, and this difference should be accounted for by feeding systems. Including the degree of maturity as predictor cancels out those differences across sexes in protein and energy requirements.

Immune response of toddlers with history of prematurity.

BACKGROUND: It is not quite well established how immune responses differ in term and preterm infants beyond the first year of life. This study aimed to evaluate aspects of the innate and adaptive immune responses in a group of preterm infants in comparison with their term peers. METHODS: In this cross-sectional study peripheral blood mononuclear cells (PBMC) were isolated from preterm and term children at age three years. Innate immune response was evaluated by the analysis of TLR receptors expression on CD11c+HLADRhigh cells and inflammatory cytokine production after PBMC stimulation with Toll like receptors (TLR) ligands. Adaptive immune response was evaluated by T cells' phenotyping and function after stimulation with polyclonal conventional T cell stimulus. CONCLUSION: We have found that the patterns of innate and adaptive immune responses at 3 years of age were not affected by the fact of the children having being born preterm or at term.

Polymorphisms in key bone modulator cytokines genes influence bisphosphonates therapy in postmenopausal women.

Osteoporosis is a multifactorial and debilitating disease resulting from decreased bone mineral density (BMD) and loss of tissue microarchitecture. Ineffective therapies may lead to bone fractures and subsequent death. Single nucleotide polymorphisms (SNPs) in key immune regulator genes have been associated with therapeutic response to bisphosphonates, which are the first therapeutic line of choice for osteoporosis. However, cytokine pathways and their relation with therapeutic adhesion remain to be fully elucidated. Aimed at better understanding these processes, we investigated the response to bisphosphonate therapy in postmenopausal women and four SNPs in key proinflammatory cytokines genes: IL23R +2284 (C>A) (rs10889677), IL17A +672 (G>A) (rs7747909), IL12B +1188 (T>G) (rs3212227) and INF-γ -1616 (G>A) (rs2069705). A total of 69 patients treated with bisphosphonate were followed for a period of 1 up to 4 years, genotyped and compared according to their changes in bone mineral density (BMD) and level of biochemical markers during their treatment. The INF-γ -1616 G/G associated with increased BMD values in femoral neck (GG/AA, p = 0.016) and decreased BMD values in total hip (GG/GA, p = 0.019; GG/AA, p = 0.011). In relation to biochemical markers, INF-γ -1616 SNP associated with increased alkaline phosphatase (GG/AA; p < 0.0001) and parathyroid hormone levels (AA/GA; p = 0.017). Vitamin D values changes were related to IL17A +672 (GG/GA, p = 0.034) and to IL12B +1188 (TT/TG, p = 0.046) SNPs. Besides, significant differences in changes of calcium levels correlated with IL23R +2284 (CC/CA, p = 0.016) genotypes. Altogether, we suggest that these polymorphisms may play an important role for therapeutic decisions in osteoporosis treatment.

Energy and protein requirements of weaned male and female Saanen goats.

The objective of this research was to estimate the energy and protein requirements for maintenance and growth in male (castrated and intact) and female Saanen goat kids between 15 and 30 kg BW. To determine the net energy requirements for maintenance (NEm ) and the net protein requirements for maintenance (NPm ), 75 goats (25 castrated and 26 intact males and 24 females) were used. Twenty-one goats (seven castrated and eight intact males and six females) were randomly assigned for slaughter to estimate the initial empty body composition. The 54 remaining animals (18 castrated and 18 intact males and 18 females) were randomly assigned in a split-plot design using a 3 × 3 factorial arrangement with three sexes and three levels of intake (ad libitum and restricted feed to 75% or 50% of the ad libitum intake). Within each sex, six blocks (three goats per block) were formed and one goat was randomly assigned to each level of intake. The 75% and the 50% of ad libitum rationing were determined daily, based on the DMI of the animal fed ad libitum on the previous day. All animals within block were slaughtered when the animal fed ad libitum reached 30 kg BW. The net energy requirements for gain (NEg ) and the net protein requirements for gain (NPg ) were obtained using 58 animals (20 castrated and 20 intact males and 18 females). The animals were fed ad libitum and slaughtered at targeted BW (15, 23 or 30 kg). Sex did not affect NEg and NPm (277.8 kJ/kg0.75  BW day and 2.98 g CP/kg0.75  BW day respectively), as well as NPg (180.9 ± 6.48 g/kg EBW gain) in Saanen goat kids. However, castrated males and females had similar NEg (varied from 12.6 ± 0.424 to 17.9 ± 1.38 MJ/kg EBW gain), greater than intact males (varied from 9.74 ± 0.420 to 10.7 ± 0.984 MJ/kg EBW gain), as the BW increased from 15 to 30 kg.

Respiratory syncytial virus induces phosphorylation of mTOR at ser2448 in CD8 T cells from nasal washes of infected infants.

Respiratory syncytial virus (RSV)-specific CD8(+) T cell responses do not protect against reinfection. Activation of mammalian target of rapamycin (mTOR) impairs memory CD8(+) T cell differentiation. Our hypothesis was that RSV inhibits the formation of CD8(+) T cells memory responses through mTOR activation. To explore this, human and mouse T cells were used. RSV induced mTOR phosphorylation at Ser2448 in CD8 T cells. mTOR activation by RSV was completely inhibited using rapamycin. RSV-infected children presented higher mTOR gene expression on nasal washes comparing to children infected with metapneumovirus and rhinovirus. In addition, RSV-infected infants presented a higher frequency of CD8(+) pmTORser2448(+) T cells in nasal washes compared to RSV-negative infants. Rapamycin treatment increased the frequency of mouse CD8 RSV-M282-90 pentamer-positive T cells and the frequency of RSV-specific memory T cells precursors. These data demonstrate that RSV is activating mTOR directly in CD8 T cells, indicating a role for mTOR during the course of RSV infection.

Isolation and characterization of microsatellite loci in Sisyrinchium (Iridaceae) and cross amplification in other genera.

Recent phylogenetic studies on Sisyrinchium strongly suggest that species classified in section Hydastylus and section Viperella belong to a single group of plants in recent adaptive radiation (Clade IV). These species neither present clear morphological differentiation among them nor show clear identification using DNA barcode markers. Thus, the main goal of this study was to develop a set of polymorphic microsatellite markers compatible for representative species of both sections to ensure variability that could be revealed by SSR markers. Therefore, microsatellite primers were isolated and characterized for Sisyrinchium palmifolium and S. marchioides. In addition, transferability of the developed primers was tested in Iridoideae, primarily in closely related species of Sisyrinchium. Sixteen microsatellite loci were developed from enriched genomic libraries, of which ten were polymorphic. GST values indicated higher differentiation among subpopulations of S. palmifolium than those from S. marchioides. Major transferability was obtained using primers isolated from S. marchioides. All primers exhibited higher rates of cross-amplification for species belonging to Clade IV of Sisyrinchium, as well as to the genera Calydorea and Herbertia. These developed microsatellite markers can be used as an efficient tool for characterization of genetic variability in species belonging to Iridoideae, as well as for studies on population dynamics, genetic structure, and mating system in other Sisyrinchium species.

Small-angle X-ray scattering and in silico modeling approaches for the accurate functional annotation of an LysR-type transcriptional regulator.

Xylella fastidiosa is a xylem-limited, Gram-negative phytopathogen responsible for economically relevant crop diseases. Its genome was thus sequenced in an effort to characterize and understand its metabolism and pathogenic mechanisms. However, the assignment of the proper functions to the identified open reading frames (ORFs) of this pathogen was impaired due to a lack of sequence similarity in the databases. In the present work, we used small-angle X-ray scattering and in silico modeling approaches to characterize and assign a function to a predicted LysR-type transcriptional regulator in the X. fastidiosa (XfLysRL) genome. XfLysRL was predicted to be a homologue of BenM, which is a transcriptional regulator involved in the degradation pathway of aromatic compounds. Further functional assays confirmed the structural prediction because we observed that XfLysRL interacts with benzoate and cis,cis-muconic acid (also known as 2E,4E-hexa-2,4-dienedioic acid; hereafter named muconate), both of which are co-factors of BenM. In addition, we showed that the XfLysRL protein is differentially expressed during the different stages of X. fastidiosa biofilm formation and planktonic cell growth, which indicates that its expression responds to a cellular signal that is likely related to the aromatic compound degradation pathway. The assignment of the proper function to a protein is a key step toward understanding the cellular metabolic pathways and pathogenic mechanisms. In the context of X. fastidiosa, the characterization of the predicted ORFs may lead to a better understanding of the cellular pathways that are linked to its bacterial pathogenicity.

Characterization of the human dynein light chain Rp3 and its use as a non-viral gene delivery vector.

Dynein light chains mediate the interaction between the cargo and the dynein motor complex during retrograde microtubule-mediated transport in eukaryotic cells. In this study, we expressed and characterized the recombinant human dynein light chain Rp3 and developed a modified variant harboring an N-terminal DNA-binding domain (Rp3-Db). Our approach aimed to explore the retrograde cell machinery based on dynein to enhance plasmid DNA (pDNA) traffic along the cytosol toward the nucleus. In the context of non-viral gene delivery, Rp3-Db is expected to simultaneously interact with DNA and dynein, thereby enabling a more rapid and efficient transport of the genetic material across the cytoplasm. We successfully purified recombinant Rp3 and obtained a low-resolution structural model using small-angle X-ray scattering. Additionally, we observed that Rp3 is a homodimer under reducing conditions and remains stable over a broad pH range. The ability of Rp3 to interact with the dynein intermediate chain in vitro was also observed, indicating that the recombinant Rp3 is correctly folded and functional. Finally, Rp3-Db was successfully expressed and purified and exhibited the ability to interact with pDNA and mediate the transfection of cultured HeLa cells. Rp3-Db was also capable of interacting in vitro with dynein intermediate chains, indicating that the addition of the N-terminal DNA-binding domain does not compromise its function. The transfection level observed for Rp3-Db is far superior than that reported for protamine and is comparable to that of the cationic lipid Lipofectamine™. This report presents an initial characterization of a non-viral delivery vector based on the dynein light chain Rp3 and demonstrates the potential use of modified human light chains as gene delivery vectors.

Unraveling the variability and genetic structure of barker frog Physalaemus cuvieri (Leiuperinae) populations from different regions of Brazil.

The barker frog Physalaemus cuvieri is widely distributed in South America and is found in all regions of Brazil. Significant intraspecific morphological variation in this species has been reported. To determine the genetic structure of the natural Brazilian populations of P. cuvieri, 10 different populations geographically separated by 99.41 to 2936.75 km were evaluated using 10 polymorphic microsatellite loci. In addition, mitochondrial DNA data were analyzed to determine genetic distance between the populations. The genetic variation was found to be significant in most of the populations (HE ranged from 0.40 to 0.59, and allelic richness ranged from 2.07 to 3.54). An FST value of 0.27 indicated that high genetic structure was present among the P. cuvieri populations. STRUCTURE analyses grouped the 10 populations into nine clusters and indicated that only two of the populations were not genetically differentiated. The genetic distance calculated from the mitochondrial DNA data showed values <0.03 for seven of the populations.

Microsatellite in Aeschynomene falcata (Leguminosae): diversity, cross-amplification, and chromosome localization.

Aeschynomene falcata is an important forage species; however, because of low seed production, it is underutilized as forage species. Aeschynomene is a polyphyletic genus with a challenging taxonomic position. Two subgenera have been proposed, and it is suggested that Aeschynomene can be split in 2 genera. Thus, new markers, such as microsatellite sequences, are desirable for improving breeding programs for A. falcata. Based on transferability and in situ localization, these microsatellite sequences can be applied as chromosome markers in the genus Aeschynomene and closely related genera. Here, we report the first microsatellite library developed for this genus; 11 microsatellites were characterized, with observed and expected heterozygosities ranging from 0.0000 to 0.7143 and from 0.1287 to 0.8360, respectively. Polymorphic information content varied from 0.1167 to 0.7786. The departure from Hardy-Weinberg equilibrium may have resulted from frequent autogamy, which is characteristic of A. falcata. Of the 11 microsatellites, 9 loci were cross-amplified in A. brevipes and A. paniculata and 7 in Dalbergia nigra and Machaerium vestitum. Five of these 7 cross-amplified microsatellites were applied as probes during the in situ hybridization assay and 2 showed clear signals on A. falcata chromosomes, ensuring their viability as chromosome markers.

New loci of Lychnophora ericoides and transferability to Lychnophora pinaster, endangered medicinal species from Brazil.

Lychnophora ericoides and Lychnophora pinaster are species used in popular medicine as analgesic or anti-inflammatory agents to treat contusions, rheumatism, and insect bites. In this study, 21 simple sequence repeat loci of L. ericoides were developed and transferred to L. pinaster. Three populations of L. ericoides and 2 populations of L. pinaster were evaluated; they were collected in the State of Minas Gerais. Population parameters were estimated, and the mean values of observed and expected heterozygosity were 0.297 and 0.408 (L. ericoides) and 0.228 and 0.310 (L. pinaster), respectively. Greater genetic variability was observed within populations than between populations of L. ericoides (62 and 37%) and L. pinaster (97 and 2.8%). These results provide information for genetic conservation and taxonomic studies of these endangered species.

Microsatellite markers for the Cabreúva tree, Myroxylon peruiferum (Fabaceae), an endangered medicinal species from the Brazilian Atlantic Forest.

The Cabreúva tree, Myroxylon peruiferum, is an endangered tropical species from Brazil used in forest restoration projects. It is known for its medicinal properties. Eleven microsatellite markers were developed for this species, from a microsatellite-enriched library. Nine of these markers, characterized in 30 individuals from a semideciduous forest remnant population in southeast Brazil, were polymorphic, with allele numbers ranging from 2 to 8 per locus; expected and observed heterozygosities ranged from 0.103 to 0.757 and 0.107 to 0.704, respectively. One locus (Mpe-C04) showed significant deviation from Hardy-Weinberg equilibrium, probably due to null alleles. Two other loci (Mpe-E09 and Mpe-H07) were monomorphic in this population. These microsatellite loci should be useful for future population genetic studies of this species.

Interleukin-21 expanded NKDC in vitro reduces the B16F10 tumor growth in vivo.

Innate immunity to tumors is mediated mainly by natural killer cells (NKs) and dendritic cells (DCs). The function of these cells is coordinated by cytokines produced during the inflammatory process. NK cells are highly active against tumors, being an important source of IFN-γ. Natural killer dendritic cells (NKDCs) were recently identified as a group of hybrid cells; some studies claim that they have lytic activity, produce IFN-γ and can also stimulate antigen-specific T cells. Interleukin 21 (IL-21) regulates the proliferation capacity and cytotoxicity of NK and T cells. The main objective of this study was to investigate if IL-21 influences the frequency of NKDCs in vitro as well as IFN-γ production and also to verify if these cells could enhance the antitumor activity against B16F10 tumor model in vivo. Splenocytes from C57BL/6 mice were isolated and the DC were enriched by immunomagnetic beads and cultured for four days with recombinant IL-21 (10, 20, 40 or 100 ng/ml). NKDC population was characterized as CD11clow/medB220+NK1.1+. Expanded cells were used to treat B16F10 tumor bearing mice and tumor growth was compared between the doses of IL-21 10 ng/ml and 20 ng/ml. The results indicate that IL-21 increases the expansion of splenic NKDCs in vitro in doses of 10 ng/ml and 20 ng/ml and these cells produce IFN-γ. In vivo, cells expanded with IL-21 and injected directly into the growing tumor efficiently reduced the tumor size. Together, these results showed for the first time that IL-21 influences the biology and the effector activity of NKDCs.

Effect of genetic polymorphisms in CA6 gene on the expression and catalytic activity of human salivary carbonic anhydrase VI.

Carbonic anhydrase isoenzyme VI (CA VI) plays an important role in the homeostasis of oral tissues participating in the processes of taste, protection of dental tissues against the loss of minerals, caries, and possibly in the formation of dental calculus in periodontal disease. This study aimed to verify the correlation between changes in the expression and activity of human salivary carbonic anhydrase VI and genetic polymorphisms in its gene (CA6). The study population consisted of 182 healthy volunteers (female and male, aged 18-22). Samples of total saliva were assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay. CA VI catalytic activity was detected by a modified protocol of Kotwica et al. [J Physiol Pharmacol 2006;57(suppl 8):107-123], adapted to CA VI in saliva. Samples of genomic DNA were genotyped for polymorphisms rs2274327 (C/T), rs2274328 (A/C) and rs2274333 (A/G) by TaqMan® SNP Genotyping Assays. The concentration and catalytic activity of the salivary CA VI obtained for the different genotypes were analyzed using the Kruskal-Wallis nonparametric test and the Dunn test. The results showed that individuals with TT genotype (rs2274327) had significantly lower CA VI concentrations than the individuals with genotypes CT or CC (p < 0.05). There was also an association between polymorphism rs2274333 and salivary CA VI concentrations. There were no associations between the three polymorphisms analyzed and variations in CA VI activity. Our results suggest that polymorphisms in the CA6 gene are associated with the concentrations of secreted CA VI.