A bivalent recombinant mycobacterium bovis BCG expressing the S1 subunit of the pertussis toxin induces a polyfunctional CD4 ⁺ T cell immune response

© 2019 Alex I. Kanno et al. Background. A recombinant BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (rBCG-S1PT), previously constructed by our research group, demonstrated the ability to develop high protection in mouse models of pertussis challenge which correlated with the induction of a Th1 immune response pattern. The Th1 immune response induced by rBCG-S1PT treatment was also confirmed in the murine orthotopic bladder cancer model, in which the intravesical instillation of rBCG-S1PT resulted in an improved antitumor effect. Based on these observations, we hypothesize that the reengineering of the S1PT expression in BCG could increase the efficiency of the protective Th1 immune response in order to develop a new alternative of immunotherapy in bladder cancer treatment. Objectives. To construct rBCG strains expressing S1PT from extrachromosomal (rBCG-S1PT) and integrative vectors (rBCG-Sli), or their combination, generating the bivalent strain (rBCG-S1+S1i), and to evaluate the respective immunogenicity of rBCG strains in mice. Methods. Mycobacterial plasmids were constructed by cloning the s1pt gene under integrative and extrachromosomal vectors and used to transform BCG, individually or in combination. Antigen expression and localization were confirmed by Western blot. Mice were immunized with wild-type BCG or the rBCG strains, and cytokines quantification and flow cytometry analysis were performed in splenocytes culture stimulated with mycobacterial-specific proteins. Findings. S1PT expression was confirmed in all rBCG strains. The extrachromosomal vector directs S1PT to the cell wall-associated fraction, while the integrative vector directs its expression mainly to the intracellular fraction. Higher levels of IFN-γ were observed in the splenocytes culture from the group immunized with rBCG-S1i in comparison to BCG or rBCG-S1PT. rBCG-S1+S1i showed higher levels of CD4 + IFN-γ + and double-positive CD4 + IFN-γ + TNF-α + T cells. Conclusions. rBCG-S1+S1i was able to express the two forms of S1PT and elicited higher induction of polyfunctional CD4 + T cells, indicating enhanced immunogenicity and suggesting its use as immunotherapy for bladder cancer

Comparison of the frequency of bacterial and viral infections among children with community-acquired pneumonia hospitalized across distinct severity categories: a prospective cross-sectional study

Background: The comparison of the frequencies of bacterial and viral infections among children with community-acquired pneumonia (CAP) admitted in distinct severity categories, in an original study, is lacking in literature to-date. We aimed to achieve this goal.Methods: Children aged 2-59-months-old hospitalized with CAP were included in this prospective study in Salvador, Brazil. Clinical data and biological samples were collected to investigate 11 viruses and 8 bacteria. Severity was assessed by using the World Health Organization criteria.Results: One hundred eighty-one patients were classified as "non-severe" (n = 53; 29.3 %), "severe" (n = 111; 61.3 %), or "very severe" (n = 17; 9.4 %) CAP. Overall, aetiology was detected among 156 (86.2 %) cases; viral (n = 84; 46.4 %), bacterial (n = 26; 14.4 %) and viral-bacterial (n = 46; 25.4 %) infections were identified. Viral infection frequency was similar in severe/very severe and non-severe cases (46.1 % vs. 47.2 %; p = 0.9). Pneumococcal infection increased across "non-severe" (13.2 %), "severe" (23.4 %), and "very severe" (35.3 %) cases (qui-squared test for trend p = 0.04). Among patients with detected aetiology, after excluding cases with co-infection, the frequency of sole bacterial infection was different (p = 0.04) among the categories; non-severe (12.5 %), severe (29.3 %) or very severe (55.6 %). Among these patients, sole bacterial infection was independently associated with severity (OR = 4.4 [95 % CI:1.1-17. 6]; p = 0.04) in a model controlled for age (OR = 0.7 [95 % CI:0.5-1.1]; p = 0.1).Conclusions: A substantial proportion of cases in distinct severity subgroups had respiratory viral infections, which did not differ between severity categories. Bacterial infection, particularly pneumococcal infection, was more likely among severe/very severe cases

Bearing My Heart: The Role of Extracellular Matrix on Cardiac Development, Homeostasis, and Injury Response

The extracellular matrix (ECM) is an essential component of the heart that imparts fundamental cellular processes during organ development and homeostasis. Most cardiovascular diseases involve severe remodeling of the ECM, culminating in the formation of fibrotic tissue that is deleterious to organ function. Treatment schemes effective at managing fibrosis and promoting physiological ECM repair are not yet in reach. Of note, the composition of the cardiac ECM changes significantly in a short period after birth, concurrent with the loss of the regenerative capacity of the heart. This highlights the importance of understanding ECM composition and function headed for the development of more efficient therapies. In this review, we explore the impact of ECM alterations, throughout heart ontogeny and disease, on cardiac cells and debate available approaches to deeper insights on cell–ECM interactions, toward the design of new regenerative therapies.This work was funded by the European Regional Development Fund (ERDF) through COMPETE 2020, Portugal 2020, and by the FCT (Fundação para a Ciência e Tecnologia) (POCI-01-0145-FEDER-030985) and by the FCT/Ministério da Ciência, Tecnologia e Inovação in the framework of individual funding (CEECINST/00091/2018) to DN

Bioenergetic dysfunction in Huntington's disease human cybrids

In this work we studied the mitochondrial-associated metabolic pathways in Huntington's disease (HD) versus control (CTR) cybrids, a cell model in which the contribution of mitochondrial defects from patients is isolated. HD cybrids exhibited an interesting increase in ATP levels, when compared to CTR cybrids. Concomitantly, we observed increased glycolytic rate in HD cybrids, as revealed by increased lactate/pyruvate ratio, which was reverted after inhibition of glycolysis. A decrease in glucose-6-phosphate dehydrogenase activity in HD cybrids further indicated decreased rate of the pentose-phosphate pathway. ATP levels of HD cybrids were significantly decreased under glycolysis inhibition, which was accompanied by a decrease in phosphocreatine. Nevertheless, pyruvate supplementation could not recover HD cybrids' ATP or phosphocreatine levels, suggesting a dysfunction in mitochondrial use of that substrate. Oligomycin also caused a decrease in ATP levels, suggesting a partial support of ATP generation by the mitochondria. Nevertheless, mitochondrial NADH/NAD(t) levels were decreased in HD cybrids, which was correlated with a decrease in pyruvate dehydrogenase activity and protein expression, suggesting decreased tricarboxylic acid cycle (TCA) input from glycolysis. Interestingly, the activity of alpha-ketoglutarate dehydrogenase, a critical enzyme complex that links the TCA to amino acid synthesis and degradation, was increased in HD cybrids. In accordance, mitochondrial levels of glutamate were increased and alanine was decreased, whereas aspartate and glutamine levels were unchanged in HD cybrids. Conversely, malate dehydrogenase activity from total cell extracts was unchanged in HD cybrids. Our results suggest that inherent dysfunction of mitochondria from HD patients affects cellular bioenergetics in an otherwise functional nuclear background

Aberrant p15, p16, p53, and DAPK Gene Methylation in Myelomagenesis: Clinical and Prognostic Implications

BACKGROUND: Aberrant DNA methylation is considered a crucial mechanism in the pathogenesis of monoclonal gammopathies. We aimed to investigate the contribution of hypermethylation of 4 tumor suppressor genes to the multistep process of myelomagenesis. METHODS: The methylation status of p15, p16, p53, and DAPK genes was evaluated in bone marrow samples from 94 patients at diagnosis: monoclonal gammopathy of uncertain significance (MGUS) (n = 48), smoldering multiple myeloma (SMM) (n = 8) and symptomatic multiple myeloma (MM) (n = 38), and from 8 healthy controls by methylation-specific polymerase chain reaction analysis. RESULTS: Overall, 63% of patients with MM and 39% of patients with MGUS presented at least 1 hypermethylated gene (P < .05). No aberrant methylation was detected in normal bone marrow. The frequency of methylation for individual genes in patients with MGUS, SMM, and MM was p15, 15%, 50%, 21%; p16, 15%, 13%, 32%; p53, 2%, 12,5%, 5%, and DAPK, 19%, 25%, 39%, respectively (P < .05). No correlation was found between aberrant methylation and immunophenotypic markers, cytogenetic features, progression-free survival, and overall survival in patients with MM. CONCLUSIONS: The current study supports a relevant role for p15, p16, and DAPK hypermethylation in the genesis of the plasma cell neoplasm. DAPK hypermethylation also might be an important step in the progression from MGUS to MM.info:eu-repo/semantics/publishedVersio

Mitochondrial-dependent apoptosis in Huntington's disease human cybrids

We investigated the involvement of mitochondrial-dependent apoptosis in Huntington's disease (HD) vs. control (CTR) cybrids, obtained from the fusion of human platelets with mitochondrial DNA-depleted NT2 cells, and further exposed to 3-nitropropionic acid (3-NP) or staurosporine (STS). Untreated HD cybrids did not exhibit significant modifications in the activity of mitochondrial respiratory chain complexes I-IV or in mtDNA sequence variations suggestive of a primary role in mitochondrial susceptibility in the subpopulation of HD carriers studied. However, a slight decrease in mitochondrial membrane potential and increased formation of intracellular hydroperoxides was observed in HD cybrids under basal conditions. Furthermore, apoptotic nuclei morphology and a moderate increase in caspase-3 activation, as well as increased levels of superoxide ions and hydroperoxides were observed in HD cybrids upon 3-NP or STS treatment. 3-NP-evoked apoptosis in HD cybrids involved cytochrome c and AIF release from mitochondria, which was associated with mitochondrial Bax translocation. CTR cybrids subjected to 3-NP showed increased mitochondrial Bax and Bim levels and the release of AIF, but not cytochrome c, suggesting a different mode of cell death, linked to the loss of membrane integrity. Additionally, increased mitochondrial Bim and Bak levels, and a slight release of cytochrome c in untreated HD cybrids may help to explain their moderate susceptibility to mitochondrial-dependent apoptosi