The adequate management of parasite co-infections represents a challenge that has not yet been overcome, especially considering that the pathological outcomes and responses to treatment are poorly understood. Thus, this study aimed to evaluate the impact of Schistosoma mansoni infection on the efficacy of benznidazole (BZN)-based chemotherapy in Trypanosoma cruzi co-infected mice. BALB/c mice were maintained uninfected or co-infected with S. mansoni and T. cruzi, and were untreated or treated with BZN. Body weight, mortality, parasitemia, cardiac parasitism, circulating cytokines (Th1/Th2/Th17); as well as heart, liver and intestine microstructure were analyzed. The parasitemia peak was five times higher and myocarditis was more severe in co-infected than T. cruzi-infected mice. After reaching peak, parasitemia was effectively controlled in co-infected animals. BZN successfully controlled parasitemia in both co-infected and T. cruzi-infected mice and improved body mass, cardiac parasitism, myocarditis and survival in co-infected mice. Co-infection dampened the typical cytokine response to either parasite, and BZN reduced anti-inflammatory cytokines in co-infected mice. Despite BZN normalizing splenomegaly and liver cellular infiltration, it exacerbated hepatomegaly in co-infected mice. Co-infection or BZN exerted no effect on hepatic granulomas, but increased pulmonary and intestinal granulomas. Marked granulomatous inflammation was identified in the small intestine of all schistosomiasis groups. Taken together, our findings indicate that BZN retains its therapeutic efficacy against T. cruzi infection even in the presence of S. mansoni co-infection, but with organ-specific repercussions, especially in the liver.

Anabolic-androgenic steroids (AAS) abuse is often associated with metabolic disorders and infertility. However, the current evidence on AAS-induced reproductive toxicity is mainly based on male studies. Thus, AAS repercussions on female reproductive capacity remain poorly understood, despite scarce evidence that fertility determinants may be more severely impaired in females than males exposed to these drugs. Accordingly, this study used an integrated framework to investigate the impact of different testosterone 17β-cyclopentylpropionate (TC) doses on pain sensitivity, aggressiveness, anxiety, sexual behavior, ovarian, oviductal, uterine and reproductive morphofunctional and molecular outcomes. These parameters were used to explore the reproductive capacity in female mice exposed to this synthetic testosterone ester. The animals were untreated or intraperitoneally treated with 5, 10 and 20 mg/kg TC every 48 h for 12 weeks. Our findings indicated that testosterone was upregulated while the hormones luteinizing, follicle-stimulating, estrogen and progesterone were down-regulated by TC. This AAS also exerted deleterious effects on anxiety, aggressivity, nociception, exploratory and sexual behavior in female mice. Concurrently, TC attenuated ovarian follicle maturation, interrupted the estrous cycle, induced oviductal and uterine hypotrophy. Estrous cyclicity was reestablished 60 days after AAS treatment. However, TC-treated mice still exhibited impaired reproductive capacity, a disturbance potentially related to deficiency in folliculogenesis, sex hormones production, and endometrial receptivity mediate by ER-α, PR, HOXA-10 and LIF down-regulation. Taken together, our findings indicated that in addition to female behavior, reproductive organs microstructure and function are markedly impaired by TC in a dose-dependent manner, whose time-dependent reversibility remains to be clarified.

Creation of bioactive molecules for treatment of cardiovascular diseases based on natural peptides is the focus of intensive experimental research. In the recent years, it has been established that C-terminal fragments of apelin, an endogenous ligand of the APJ receptor, reduce metabolic and functional disorders in experimental heart damage. The review presents literature data and generalized results of our own experiments on the effect of apelin-13, [Pyr]apelin-13, apelin-12, and their chemically modified analogues on the heart under normal and pathophysiological conditions in vitro and in vivo. It has been shown that the spectrum of action of apelin peptides on the damaged myocardium includes decrease in the death of cardiomyocytes from necrosis, reduction of damage to cardiomyocyte membranes, improvement in myocardial metabolic state, and decrease in formation of reactive oxygen species and lipid peroxidation products. The mechanisms of protective action of these peptides associated with activation of the APJ receptor and manifestation of antioxidant properties are discussed. The data presented in the review show promise of the molecular design of APJ receptor peptide agonists, which can serve as the basis for the development of cardioprotectors that affect the processes of free radical oxidation and metabolic adaptation.

Schistosomiasis mansoni is a parasitic infection that causes enterohepatic morbidity associated with severe granulomatous inflammation triggered by parasite eggs. In this disease, granulomatous inflammation leads to intestinal erosion and environmental excretion of S. mansoni eggs from feces, an essential process for propagating the parasite and infecting host organisms. Metalloproteinases (MMP) are involved in S. mansoni-induced hepatic granulomatous inflammation and fibrosis. However, the relationship between MMP and collagen accumulation with the intestinal excretion of parasite eggs remains unclear. Thus, the present study investigated whether MMP inhibition is capable of modulating granulomatous inflammation, collagen accumulation and mechanical resistance to the point of influencing the dynamics between intestinal retention and excretion of S. mansoni eggs in infected mice. Our findings indicated that doxycycline (a potent MMP inhibitor) aggravates intestinal inflammation and subverts collagen dynamics in schistosomiasis. By attenuating MMP-2 and MMP-9 activity, this drug is capable of enhancing fibrosis and mechanical resistance of the intestinal wall, hindering S. mansoni eggs translocation. Although collagen content was not correlated with MMP activity, intestinal retention and fecal excretion of parasite eggs in untreated mice; these correlations were observed for doxycycline-treated animals. Thus, our study provides evidence that doxycycline is able to attenuate fecal elimination of S. mansoni eggs by inhibiting MMP-2 and MMP-9 activity, events potentially associated with excessive collagen accumulation, which increases intestinal mechanical resistance and hinders eggs translocation through the intestinal wall. Variations in intestinal collagen dynamics are relevant since they may represent changes in the environmental dispersion of S. mansoni eggs, bringing repercussions for schistosomiasis propagation.

In the winter of 2010-2011, Japan experienced a large outbreak of infections caused by clade 2.3.2.1 H5N1 high pathogenicity avian influenza viruses (HPAIVs) in wild birds. Interestingly, many tufted ducks (Aythya fuligula), which are migratory diving ducks, succumbed to the infection, whereas only one infection case was reported in migratory dabbling duck species, the major natural hosts of the influenza A virus, during the outbreak. To assess whether the susceptibility of each duck species to HPAIVs was correlated with the number of cases, tufted duck and dabbling duck species (Eurasian wigeon, Mareca penelope; mallard, Anas platyrhynchos; Northern pintail, Anas acuta) were intranasally inoculated with A/Mandarin duck/Miyazaki/22M807-1/2011 (H5N1), an index clade 2.3.2.1 virus previously used for experimental infection studies in various bird species. All ducks observed for 10 days post-inoculation (dpi) mostly shed the virus via the oral route and survived. The tufted ducks shed a higher titer of the virus than the other dabbling duck species, and one of them showed apparent neurological symptoms after 7 dpi, which were accompanied by eye lesions. No clinical symptoms were observed in the dabbling ducks, although systemic infection and viremia were observed in some of them sacrificed at 3 dpi. These results suggest that the susceptibility of clade 2.3.2.1 HPAIVs might differ by duck species.

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development.

OBJECTIVE: Plastic particles (PP) pollution is a global environmental concern. Although the reproductive toxicity of PP is primarily understood for invertebrates, the evidence for mammals is still fragmented. We used a systematic review framework to investigate the reproductive impact of microplastics and nanoplastics (MNP) on mammals.
METHODS: Research records were screened from Embase, Medline, Scopus and Web of Science. Twelve original papers were identified and reviewed. Immunological, oxidative and morphofunctional outcomes, and the risk of bias in all studies reviewed were analyzed.
RESULTS: These studies indicated that PP can accumulate in the gonads, triggering seminiferous degeneration, Sertoli cells death, blood-testis barrier disruption, sperm degeneration, malformation, reduced number and mobility, ovarian cysts, reduced follicular growth and granulosa cells death. Gonadal damage was associated with upregulation of prooxidant mediators (oxygen reactive species, lipid and DNA oxidation), cell death, proinflammatory molecular pathways and cytokines, as well as inhibition of enzymatic and non-enzymatic antioxidant defense mechanisms. Spermatogenesis, folliculogenesis, testosterone, progesterone and estrogen levels were also impaired in PP-treated animals, which were potentially associated with down-regulation of molecules involved in germ cells microstructural organization (occludin, N-cadherin, β-catenin and connexin 43) and steroidogenesis, such as hydroxysteroid dehydrogenases, steroidogenic acute regulatory proteins, follicle stimulating and luteinizing hormones. Selection, performance and detection bias were the main limitations identified.
CONCLUSIONS: Current evidence indicates that PP can induce dose-dependent microstructural and functional gonadal damage, which is orchestrated by pro-oxidant and pro-inflammatory mechanisms that disrupt genes, molecular effectors, and hormones that control spermatogenesis and folliculogenesis.

We investigated the relationship between inducible nitric oxide synthase (iNOS) and arginase pathways, cytokines, macrophages, oxidative damage and lung granulomatous inflammation in S. mansoni-infected and doxycycline-treated mice.
Swiss mice were randomized in four groups: (i) uninfected, (ii) infected with S. mansoni, (iii) infected + 200 mg/kg praziquantel (Pzt), (iv) and (v) infected + 5 and 50 mg/kg doxycycline. Pzt (reference drug) was administered in a single dose and doxycycline for 60 days.
S. mansoni-infection determined extensive lung inflammation, marked recruitment of M2 macrophages, cytokines (IL-4, IL-5, IFN-γ, TNF-α) upregulation, intense eosinophil peroxidase (EPO) levels, arginase expression and activity, reduced iNOS expression and nitric oxide (NO) production. The higher dose of doxycycline aggravated lung granulomatous inflammation, downregulating IL-4 levels and M2 macrophages recruitment, and upregulating iNOS expression, EPO, NO, IFN-γ, TNF-α, M1 macrophages, protein carbonyl and malondialdehyde tissue levels. The number and size of granulomas in doxycycline-treated animals was higher than untreated and Pzt-treated mice. Exudative/productive granulomas were predominant in untreated and doxycycline-treated animals, while fibrotic/involutive granulomas were more frequent in Pzt-treated mice. The reference treatment with Pzt attenuated all these parameters.
Our findings indicated that doxycycline aggravated lung granulomatous inflammation in a dose-dependent way. Although Th1 effectors are protective against several intracellular pathogens, effective schistosomicidal responses are dependent of the Th2 phenotype. Thus, doxycycline contributes to the worsening of lung granulomatous inflammation by potentiating eosinophils influx and downregulating Th2 effectors, reinforcing lipid and protein oxidative damage in chronic S. mansoni infection.

The design of new drugs for treatment of cardiovascular diseases based on endogenous peptide hormones is of undoubted interest and stimulates intensive experimental research. One of the approaches for development in this area is synthesis of the short bioactive peptides that mimic effects of the larger peptide molecules and have improved physicochemical characteristics. In recent years, it has been found that the N-terminal fragments of the neuropeptide galanin reduce metabolic and functional disorders in the experimental heart damage. The review presents literature data and generalized results of our own experiments on the effects of the full-size galanin and its chemically modified N-terminal fragments (2-11) and (2-15) on the heart in normal conditions and in modeling pathophysiological conditions in vitro and in vivo. It has been shown that the spectrum of the peptide actions on the damaged myocardium includes decrease in the necrotic death of cardiomyocytes, decrease in the damage of sarcolemma, improvement in the metabolic state of myocardium, decrease in the formation of reactive oxygen species (ROS) and lipid peroxidation (LPO) products. Mechanisms of the protective action of the modified galanin fragments associated with activation of the GalR2 receptor subtype and manifestation of antioxidant properties are discussed. The data summarized in the review indicate that the molecular design of pharmacological agonists of the GalR2 receptor is a promising approach, because they can serve as a basis for the development of cardioprotectors influencing processes of free radical oxidation and metabolic adaptation.

Correlation of in vivo imaging to histomorphological pathology in animal models requires comparative interdisciplinary expertise of different fields of medicine. From the morphological point of view, there is an urgent need to improve histopathological evaluation in animal model-based research to expedite translation into clinical applications. While different other fields of translational science were standardized over the last years, little was done to improve the pipeline of experimental pathology to ensure reproducibility based on pathological expertise in experimental animal models with respect to defined guidelines and classifications. Additionally, longitudinal analyses of preclinical models often use a variety of imaging methods and much more attention should be drawn to enable for proper co-registration of in vivo imaging methods with the ex vivo morphological read-outs. Here we present the development of the Comparative Experimental Pathology (CEP) unit embedded in the Institute of Pathology of the Technical University of Munich during the Collaborative Research Center 824 (CRC824) funding period together with selected approaches of histomorphological techniques for correlation of in vivo imaging to morphomolecular pathology.