Lipopolysaccharide Regulates Pro- and Anti-Inflammatory Cytokines, Corticosterone, and Melatonin in Toads.

Glucocorticoids and melatonin (MEL) show integrated and complex immunomodulatory effects, mostly described for endotherms, yet underexplored in amphibians. In this context, the RT-qPCR of molecules mediating inflammatory processes in amphibians is a valuable tool to explore the relationships among molecular biology, endocrine mediators, and immune response in these animals. In this study, toads (Rhinella diptycha) received an intraperitoneal saline injection or lipopolysaccharide (LPS; 2 mg/kg). Six hours post-injection, we analyzed plasma corticosterone (CORT) and MEL levels and pro- and anti-inflammatory molecules (IL-1ß, IL-6, IL-10, IFN-γ, and C1s). We found increased CORT and decreased MEL levels in response to LPS. Also, IL-1ß, IL-6, and IL-10 were upregulated in LPS-injected toads compared with saline-injected toads. Overall, our results demonstrate an LPS-induced inflammatory response with endocrine and immune modulation in R. diptycha toads, exhibiting expected patterns for an inflammatory stimulus within this time frame (6 h post-injection). Toads were responsive to LPS by secreting different cytokines, such as proinflammatory cytokines IL-1ß and IL-6, related to immune cell attraction to inflammatory sites and the anti-inflammatory cytokine IL-10, which limits the rate of leukocyte infiltration, inflammation, and downregulates the expression of proinflammatory cytokines. Increased circulating CORT levels are probably associated with the activation of the hypothalamus-pituitary-interrenal axis by the LPS and the endocrine actions of IL-6. Furthermore, decreased circulating MEL levels are likely due to inhibited MEL secretion by the pineal gland by inflammatory stimuli, indicating the activation/existence of the immune-pineal axis in amphibians.

Os glicocorticoides e a melatonina (MEL) apresentam efeitos imunomoduladores integrados e complexos, principalmente descritos para endotérmicos, porém pouco explorados em anfíbios. Nesse contexto, o RT-qPCR de moléculas mediadoras de processos inflamatórios em anfíbios é uma ferramenta valiosa para explorar as relações entre biologia molecular, mediadores endócrinos e resposta imune nesses animais. Neste estudo, sapos (Rhinella diptycha) receberam uma injeção intraperitoneal de solução salina ou lipopolissacarídeo (LPS; 2 mg/kg). Seis horas após a injeção, analisamos as concentrações plasmáticas de corticosterona (CORT) e MEL e moléculas pró e anti-inflamatórias (IL-1ß, IL-6, IL-10, IFN-γ e C1s). Encontramos aumento de CORT e diminuição da concentração de MEL em resposta ao LPS. Além disso, IL-1ß, IL-6 e IL-10 foram reguladas positivamente em sapos injetados com LPS em comparação com os sapos injetados com solução salina. No geral, nossos resultados demonstram uma resposta inflamatória induzida por LPS com modulação endócrina e imunológica em sapos R. diptycha, exibindo padrões esperados para um estímulo inflamatório dentro deste período (6 h pós-injeção). Os sapos foram responsivos ao LPS secretando diferentes citocinas, como as citocinas pró-inflamatórias IL-1ß e IL-6, relacionadas à atração de células imunes para os sítios inflamatórios e a citocina anti-inflamatória IL-10, que limita a taxa de infiltração leucocitária, inflamação, e diminui a expressão de citocinas pró-inflamatórias. As concentrações elevadas de CORT circulante estão provavelmente associados à ativação do eixo hipotálamo-hipófise-interrenal pelo LPS e às ações endócrinas da IL-6. Além disso, a diminuição da concentração circulante de MEL é provavelmente devida à inibição da secreção de MEL pela glândula pineal por estímulos inflamatórios, indicando a ativação/existência do eixo imune-pineal em anfíbios.

Evolution of morphology and locomotor performance in anurans: relationships with microhabitat diversification.

The relationships between morphology, performance, behavior and ecology provide evidence for multiple and complex phenotypic adaptations. The anuran body plan, for example, is evolutionarily conserved and shows clear specializations to jumping performance back at least to the early Jurassic. However, there are instances of more recent adaptation to habit diversity in the post-cranial skeleton, including relative limb length. The present study tested adaptive models of morphological evolution in anurans associated with the diversity of microhabitat use (semi-aquatic arboreal, fossorial, torrent, and terrestrial) in species of anuran amphibians from Brazil and Australia. We use phylogenetic comparative methods to determine which evolutionary models, including Brownian motion (BM) and Ornstein-Uhlenbeck (OU) are consistent with morphological variation observed across anuran species. Furthermore, this study investigated the relationship of maximum distance jumped as a function of components of morphological variables and microhabitat use. We found there are multiple optima of limb lengths associated to different microhabitats with a trend of increasing hindlimbs in torrent, arboreal, semi-aquatic whereas fossorial and terrestrial species evolve toward optima with shorter hindlimbs. Moreover, arboreal, semi-aquatic and torrent anurans have higher jumping performance and longer hindlimbs, when compared to terrestrial and fossorial species. We corroborate the hypothesis that evolutionary modifications of overall limb morphology have been important in the diversification of locomotor performance along the anuran phylogeny. Such evolutionary changes converged in different phylogenetic groups adapted to similar microhabitat use in two different zoogeographical regions.

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli.

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wild-type form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.

The evolution of jumping performance in anurans: morphological correlates and ecological implications.

We investigated the evolution of anuran locomotor performance and its morphological correlates as a function of habitat use and lifestyles. We reanalysed a subset of the data reported by Zug (Smithson. Contrib. Zool. 1978; 276: 1­31) employing phylogenetically explicit statistical methods (n = 56 species), and assembled morphological data on the ratio between hind-limb length and snout-vent length (SVL) from the literature and museum specimens for a large subgroup of the species from the original paper (n = 43 species). Analyses using independent contrasts revealed that classifying anurans into terrestrial, semi-aquatic, and arboreal categories cannot distinguish between the effects of phylogeny and ecological diversification in anuran locomotor performance. However, a more refined classification subdividing terrestrial species into 'fossorials' and 'non-fossorials', and arboreal species into 'open canopy', 'low canopy' and 'high canopy', suggests that part of the variation in locomotor performance and in hind-limb morphology can be attributed to ecological diversification. In particular, fossorial species had significantly lower jumping performances and shorter hind limbs than other species after controlling for SVL, illustrating how the trade-off between burrowing efficiency and jumping performance has resulted in morphological specialization in this group.

Efeito da Mannheimia granulomatis sobre cultivo de fibroblastos / Effect of Mannheimia granulomatis on fibroblastos cultures

Primary cultures of Mannheimia granulomatis were established in chicken embryos to assess their capacity to stimulate fibroblast proliferation. The capacity of the bacterium-activated macrophages to stimulate cytokine and enzyme proliferation was assessed in a mouse peritoneum macrophage culture. To evaluate the bacteria infection on fibroblasts and their growth within 48h in relation to the active macrophages, cultures were washed and trypsinized and the cells counted. Results showed no significant differences when the bacteria-infected fibroblasts were mixed with bacterial extract (P=0.9682). The treatment using just products of macrophages resulted similar to the negative control. Significant differences on cell proliferation were established (P=0,0039) when the products of M. granulomatis-activated macrophages were used, meaning that bacterial components were unable to promote fibroblast increase. Further research is needed to elucidate the effect of M. granulomatis on the macrophages.

[Tumor markers (advances and limits)]. / Marcadores tumorais (alcances e limites).

The author reviews and discusses the clinical utility of the major tumor markers. In spite of advances in the analytical systems, until recently there were no tumor markers that detect early stage cancer or that were absolutely specific or diagnostic for malignancy. Most of them are normal glycoproteins (that accompany the genetic deregulation of carcinogenesis) which are released into the blood and depend on liver function for their catabolism. Therefore such assays are of no use for screening healthy populations and a normal value does not exclude cancer. Their use may be of great help to the clinician in a number of ways: (1) primary staging of patients known to have cancer; (2) screening of high-risk individuals for the presence of some malignancies; (3) monitoring of the effectiveness of therapy; (4) patient's follow-up for early detection of relapses or metastases; (5) prognostic indexes; and (6) immunodetection of metastatic sites. Whatever the applications, a good clinical judgement is crucial for the use of these assays, with a critical look at the extent to which they contribute to a favorable outcome. At present, we think that their use in check-up studies should be avoided.