RESUMO

COVID-19 is marked by extensive damage to the respiratory system, often accompanied by systemic manifestations, due to both viral cytopathic effects and hyperinflammatory syndrome. Therefore, the development of new therapeutic strategies or drug repurposing aiming to control virus replication and inflammation are required to mitigate the impact of the disease. Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol-sequestering agent with antiviral activity that has been demonstrated against enveloped viruses in in vitro and in vivo experimental models. We also demonstrated that HP-BCD has an immunomodulatory effect, inhibiting the production of selected proinflammatory cytokines induced by microbial products. Importantly, this drug has been used in humans for decades as an excipient in drug delivery systems and as a therapeutic agent in the treatment of Niemann pick C disease. The safety profile for this compound is well established. Here, we investigated whether HP-BCD would affect SARS-CoV-2 replication and virus-induced inflammatory response, using established cell lines and primary human cells. Treating virus or cells with HP-BCD significantly inhibited SARS-CoV-2 replication with a high selective index. A broad activity against distinct SARS-CoV-2 variants was evidenced by a remarkable reduction in the release of infectious particles. The drug did not alter ACE2 surface expression, but affected cholesterol accumulation into intracellular replication complexes, lowering virus RNA and protein levels, and reducing virus-induced cytopathic effects. Virus replication was also impaired by HP-BCD in Calu-3 pulmonary cell line and human primary monocytes, in which not only the virus, but also the production of proinflammatory cytokines were significantly inhibited. Given the pathophysiology of COVID-19 disease, these data indicate that the use HP-BCD, which inhibits both SARS-CoV2 replication and production of proinflammatory cytokines, as a potential COVID-19 therapeutic warrants further investigation.

Assuntos

Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Colesterol/metabolismo , Citocinas/metabolismo , Humanos , RNA Viral , Replicação Viral

RESUMO

Monocytes from HIV-infected patients produce increased levels of inflammatory cytokines, which are associated with chronic immune activation and AIDS progression. Chronic immune activation is often not restored even in patients showing viral suppression under ART. Therefore, new therapeutic strategies to control inflammation and modulate immune activation are required. Hydroxypropyl-beta-cyclodextrin (HP-BCD) is a cholesterol-sequestering agent that has been reported to be safe for human use in numerous pharmaceutical applications and that has been shown to inactivate HIV in vitro and to control SIV infection in vivo Since cellular cholesterol content or metabolism has been related to altered cellular activation, we evaluated whether HP-BCD treatment could modulate monocyte response to inflammatory stimuli. Treatment of monocytes isolated from HIV-positive and HIV-negative donors with HP-BCD inhibited the expression of CD36 and TNF-α after LPS stimulation, independent of raft disruption. Accordingly, HP-BCD-treated cells showed significant reduction of TNF-α and IL-10 secretion, which was associated with lower mRNA expression. LPS-induced p38MAPK phosphorylation was dampened by HP-BCD treatment, indicating this pathway as a target for HP-BCD-mediated anti-inflammatory response. The expression of HLA-DR was also reduced in monocytes and dendritic cells treated with HP-BCD, which could hinder T cell activation by these cells. Our data suggest that, besides its well-known antiviral activity, HP-BCD could have an immunomodulatory effect, leading to decreased inflammatory responses mediated by antigen-presenting cells, which may impact HIV pathogenesis and AIDS progression.IMPORTANCE Chronic immune activation is a hallmark of HIV infection and is often not controlled even in patients under antiretroviral therapy. Indeed, chronic diseases with inflammatory pathogenesis are being reported as major causes of death for HIV-infected persons. Hydroxypropyl-beta cyclodextrin (HP-BCD) is a cholesterol-sequestering drug that inhibits HIV replication and infectivity in vitro and in vivo Recent studies have demonstrated the importance of cholesterol metabolism and content in different inflammatory conditions; therefore, we investigated the potential of HP-BCD as an immunomodulatory drug, regulating the activation of cells from HIV-infected patients. Treatment of monocytes with HP-BCD inhibited the expression and secretion of receptors and mediators that are usually enhanced in HIV patients. Furthermore, we investigated the molecular mechanisms associated with the immunomodulatory effect of HP-BCD. Our results indicate that, besides reducing viral replication, HP-BCD treatment may contribute to modulation of chronic immune activation associated with AIDS.

Assuntos

2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Anti-Inflamatórios/farmacologia , Imunossupressores/farmacologia , Monócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Antígenos CD36/análise , Células Cultivadas , Feminino , Infecções por HIV/patologia , Antígenos HLA-DR/análise , Humanos , Interleucina-10/análise , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/análise , Proteínas Quinases p38 Ativadas por Mitógeno/análise

RESUMO

Leaves from over 1000 Brazilian native plants growing in the cerrado and neighbouring regions were sampled for C and N content. Half of these were analysed for 15N and further samples for 13C and ash content. Nodulated legumes from all three sub-families were included, together with two types of reference plant, non-nodulated legumes and non-legumes. Particular emphasis was placed on the large caesalpinioid genus Chamaecrista which is here for the first time reported to fix nitrogen in its native habitats. Woody and herbaceous species of this and other nodulated genera, with the exception of the mimosoid tree Stryphnodendron, showed evidence of nitrogen fixation. Amounts fixed were site-specific as was the 15N signature of reference plants. There was no evidence that nodulated legumes had higher leaf N than non-nodulated legumes: both were higher than non-legumes. Several species of Chamaecrista from section absus and species of Stryphnodendron had carbon contents of 50-55%, higher than previously reported for leaves. This was coupled with low (1-3%) ash contents. The 13C values of plants with ≥49% C were significantly more negative than those with <49% C: most species in the former group were woody and most in the latter group herbaceous. Mimosa pudica was unusual in having a wide range of percent C, percent ash and 13C values; these parameters were significantly correlated. It is concluded that Brazilian native legumes can fix significant amounts of nitrogen in the nutrient-poor cerrado soils. Consideration of mineral and lipid nutrition will be necessary in order fully to understand relations between 13C, carbon content and other physiological parameters.

RESUMO

Aeschynomene fluminensis Veil., originally obtained from flooded areas of the Pantanal Matogrossense region of Brazil, was grown under stem-flooded or non-flooded conditions for 70 d after inoculation with isolates of photosynthetic stem nodule rhizobia obtained from native A. fluminensis. Stem nodules formed only on submerged stems of flooded plants (mean of 25 per plant), and did not form on aerial parts, although they were capable of growing and fixing N2 after drainage of the stems. Root nodules formed on both non-flooded and flooded plants but were usually decreased in number by flooding (from means of 124 to 51 per plant, respectively). Flooding (and stem-nodulation) resulted in an increase in shoot (and a decrease in root) dry weight, regardless of rhizobial isolate. Stem nodules were attached by a wide collar of aerenchymatous tissue at the base of the nodule. There were large air spaces in the stem where nodules were subtended and these were continuous with nodule aerenchyma/outer cortex. In addition, aerenchyma and spongy tissue at the base of the nodule connected both flooded and non-flooded root nodules to large intercellular spaces in the root cortex. The stem and root nodules were ovoid in shape, and essentially aeschynomenoid in type, i.e. the central infected tissue was without uninfected, interstitial cells. Root nodules had a similar structure to stem nodules (although stem nodules were generally larger), and flooded root nodules were approximately twice the size of non-flooded nodules. The infected tissue of root and stem nodules consisted of spherical, bacteroid-containing cells containing one or two rod-shaped bacteroids per peribacteroid unit and prominent organelles. Infection threads were observed in root but not in stem nodules. The cortex of stem and root nodules had an apparent oxygen diffusion barrier, consisting of concentric layers of small cells with interlocking cell walls and few intercellular spaces. Cell layers external to these consisted of larger cells and intercellular spaces, with some spaces being occluded with an electron-dense material that contained a glycoprotein recognized by the monoclonal antibodies MAC236 and MAC265. The amount of glycoprotein occlusions did not appear to differ between nodule types or treatments, although stem nodules contained intracellular glycoprotein vesicles adjacent to cell walls. The exterior of the nodules consisted of an epidermis of thin flattened cells with occasional lenticels. Amyloplasts were common in lower stem and hypocotyl nodules, but fewer in flooded or non-flooded root nodules. Upper stem nodules (i.e. those within 6 cm of the water surface) differed from more profoundly submerged stem nodules by having chloroplasts throughout the cortex. Root nodules did not contain chloroplasts, and undifferentiated plastids were found mainly in lower stem nodules.

RESUMO

We report on the structure of N2 -fixing nodules formed on the stem of Discolobium pulchellum Benth., an aquatic legume in the subfamily Pupilionoideae, tribe Aeschynomeneae, from the Hooded areas in the 'Pantanal Matogrossense' region of Brazil. The stern (and root) nodules were obligately aquatic, requiring permanent submergence in water or flooded soil, and receive oxygen via profuse aerenchyma covering The lower stem. Of the 69 isolates of rhizobia isolated from stem and root nodules, 70% were fast-growing acid producers and 38% were slow growers. The rhizobia were not photosynthetic. Nodules were connected to the stem, and the vascular system from the stem branched throughout the nodule, penetrating the infected, tissue within finger-like ingrowths of cortex. In both stem and root nodules, infected tissue was aeschynomenoid or desmodioid, that is, without uninfected (interstitial) cells. The infected cells in stem nodules were vacuolate, with visible infection threads. The inner cortex was rich in amyloplasts and contained the components of an oxygen diffusion barrier (a boundary cell layer without intercellular spaces and glycoprotein occlusions of intercellular spaces in other cell layers). The mid-cortex, external to the boundary layer, consisted of loosely-packed cells and these were continuous with stem aerenchyma. The outer part of the nodules was made up of phellogen-derived cells forming a periderm, or 'corky' layer of cells. The periderm formed large lenticels above cortical vascular bundles. These lenticels also connected with the stem aerenchyma. Root nodules differed only in that infected cells were not vacuolate, bacteroids were larger and contained more poly-ß-hydroxybutyrate (PHB) and there was less aerenchyma/lenticellular tissue. Stem and root nodule structure is discussed in terms of adaptations to O2 constraints in an aquatic environment.