A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2K^b-restricted cytotoxic and interferon (IFN)γ-producing CD8⁺ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a⁺ CD8⁺ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ⁺ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas’ heart disease.     

New method to assess mitophagy flux by flow cytometry

Mitochondrial autophagy, also known as mitophagy, is an autophagosome-based mitochondrial degradation process that eliminates unwanted or damaged mitochondria after cell stress. Most studies dealing with mitophagy rely on the analysis by fluorescence microscopy of mitochondrial-autophagosome colocalization. However, given the fundamental role of mitophagy in the physiology and pathology of organisms, there is an urgent need for novel quantitative methods with which to study this process. Here, we describe a flow cytometry-based approach to determine mitophagy by using MitoTracker Deep Red, a widely used mitochondria-selective probe. Used in combination with selective inhibitors it may allow for the determination of mitophagy flux. Here, we test the validity of the use of this method in cell lines and in primary cell and tissue cultures.     

The plant cell inhibitor KRP6 is involved in multinucleation and cytokinesis disruption in giant-feeding cells induced by root-knot nematodes

The plant cell cycle inhibitor gene KRP6 has been investigated in roots infected by plant-parasitic root-knot nematodes (Meloidogyne spp.). Unexpectedly, KRP6 overexpressing lines revealed a distinct role for this specific KRP as an activator of the mitotic cell cycle. This function was confirmed in Arabidopsis thaliana suspension cultures ectopically expressing KRP6. A blockage in the mitotic exit was observed in cell suspensions and in giant cells resulted in the appearance of multi-nucleated cells. KRP6 expression during nematode infection and the similarity in phenotypes among KRP6 overexpressing cell cultures and giant-cell morphology strongly suggest that KRP6 is involved in multinucleation and acytokinesis occurring in giant-cells. Once again nematodes have been shown to manipulate the plant cell cycle machinery in order to promote gall establishment.     

Evolution of Skin Temperature after the Application of Compressive Forces on Tendon,Muscle and Myofascial Trigger Point

Some assessment and diagnosis methods require palpation or the application of certain forces on the skin, which affects the structures beneath, we highlight the importance of defining possible influences on skin temperature as a result of this physical contact. Thus, the aim of the present study is to determine the ideal time for performing thermographic examination after palpation based on the assessment of skin temperature evolution. Randomized and crossover study carried out with 15 computer-user volunteers of both genders, between 18 and 45 years of age, who were submitted to compressive forces of 0, 1, 2 and 3 kg/cm² for 30 seconds with a washout period of 48 hours using a portable digital dynamometer. Compressive forces were applied on the following spots on the dominant upper limb: myofascial trigger point in the levator scapulae, biceps brachii muscle and palmaris longus tendon. Volunteers were examined by means of infrared thermography before and after the application of compressive forces (15, 30, 45 and 60 minutes). In most comparisons made over time, a significant decrease was observed 30, 45 and 60 minutes after the application of compressive forces (p < 0.05) on the palmaris longus tendon and biceps brachii muscle. However, no difference was observed when comparing the different compressive forces (p > 0.05). In conclusion, infrared thermography can be used after assessment or diagnosis methods focused on the application of forces on tendons and muscles, provided the procedure is performed 15 minutes after contact with the skin. Regarding to the myofascial trigger point, the thermographic examination can be performed within 60 minutes after the contact with the skin.     

Targeting Ergosterol Biosynthesis in Leishmania donovani: Essentiality of Sterol 14alpha-demethylase

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.     

A single dose of S‐ketamine induces long‐term antidepressant effects and decreases oxidative stress in adulthood rats following maternal deprivation

Ketamine, an antagonist of N‐methyl‐d ‐aspartate receptors, has produced rapid antidepressant effects in patients with depression, as well as in animal models. However, the extent and duration of the antidepressant effect over longer periods of time has not been considered. This study evaluated the effects of single dose of ketamine on behavior and oxidative stress, which is related to depression, in the brains of adult rats subjected to maternal deprivation. Deprived and nondeprived Wistar rats were divided into four groups nondeprived + saline; nondeprived + S‐ketamine (15 mg/kg); deprived + saline; deprived + S‐ketamine (15 mg/kg). A single dose of ketamine or saline was administrated during the adult phase, and 14 days later depressive‐like behavior was assessed. In addition, lipid damage, protein damage, and antioxidant enzyme activities were evaluated in the rat brain. Maternal deprivation induces a depressive‐like behavior, as verified by an increase in immobility and anhedonic behavior. However, a single dose of ketamine was able to reverse these alterations, showing long‐term antidepressant effects. The brains of maternally deprived rats had an increase in protein oxidative damage and lipid peroxidation, but administration of a single dose of ketamine reversed this damage. The activities of antioxidant enzymes superoxide dismutase and catalase were reduced in the deprived rat brains. However, ketamine was also able to reverse these changes. In conclusion, these findings indicate that a single dose of ketamine is able to induce long‐term antidepressant effects and protect against neural damage caused by oxidative stress in adulthood rats following maternal deprivation. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1268–1281, 2015     

Correlation between Body Composition and Walking Capacity in Severe Obesity

Background

Obesity is associated with mobility reduction due to mechanical factors and excessive body fat. The six-minute walk test (6MWT) has been used to assess functional capacity in severe obesity.

Objective

To determine the association of BMI, total and segmental body composition with distance walked (6MWD) during the six-minute walk test (6MWT) according to gender and obesity grade.

Setting

University of São Paulo Medical School, Brazil; Public Practice.

Methods

Functional capacity was assessed by 6MWD and body composition (%) by bioelectrical impedance analysis in 90 patients.

Results

The mean 6MWD was 514.9 ± 50.3 m for both genders. The male group (M: 545.2 ± 46.9 m) showed a 6MWD higher (p = 0.002) than the female group (F: 505.6 ± 47.9 m). The morbid obese group (MO: 524.7 ± 44.0 m) also showed a 6MWD higher (p = 0.014) than the super obese group (SO: 494.2 ± 57.0 m). There was a positive relationship between 6MWD and fat free mass (FFM), FFM of upper limps (FFM_UL), trunk (FFM_TR) and lower limbs (FFM_LL). Female group presented a positive relationship between 6MWD and FFM, FFM_UL and FFM_LL and male group presented a positive relationship between 6MWD and FFM_TR. In morbid obese group there was a positive relationship between 6MWD with FFM, FFM_UL, FFM_TR and FFM_LL. The super obese group presented a positive relationship between 6MWD with FFM, FFM_TR and FFM_LL.

Conclusions

Total and segmental FFM is associated with a better walking capacity than BMI.     

Diet of the gracile mouse opossum Gracilinanus agilis (Didelphimorphia: Didelphidae) in a neotropical savanna: intraspecific variation and resource selection

Investigation of the effect of endogenous and exogenous factors on the diet of animals is necessary for a better understanding of their feeding habits. This approach can provide relevant information on the autoecology of a species and its ecological interactions. We investigated the composition and intraspecific variation in the diet of the marsupial Gracilinanus agilis in areas of dry woodland forests (i.e., cerradão) in the Cerrado of Central Brazil, taking into consideration the availability of prey (arthropods) in the environment. We found insects, spiders, birds, and fruits in the scats of G. agilis. Insects (orders Hymenoptera, Isoptera, Hemiptera, and Coleoptera) and fruits were the most frequently consumed resources. Males fed more heavily on insects than females did, whereas during the warm–wet season (October to April), the reproductive females fed on insects more than the nonreproductive females did. On the other hand, the consumption of fruits and vertebrates did not vary between seasons, sexes, or according to female reproductive condition. Moreover, reproductive females fed more frequently on ants and beetles than nonreproductive females did. We also detected both positive (for Isoptera and Hemiptera) and negative (for Hymenoptera) selection of insects during the cool–dry season, whereas in the warm–wet season, these resources were consumed according to their availability in the environment. Our study revealed that G. agilis is an insectivore–omnivore species, but fruits also are a relevant part of its diet. This marsupial seemed to select their prey qualitatively according to its energy demands and nutritional requirements.