Microglial NF-κB Signaling Deficiency Protects Against Metabolic Disruptions Caused by Volatile Organic Compound via Modulating the Hypothalamic Transcriptome.

Prolonged exposure to benzene, a prevalent volatile organic compound (VOC), at concentrations found in smoke, triggers hyperglycemia, and inflammation in mice. Corroborating this with existing epidemiological data, we show a strong correlation between environmental benzene exposure and metabolic impairments in humans. To uncover the underlying mechanisms, we employed a controlled exposure system and continuous glucose monitoring (CGM), revealing rapid blood glucose surges and disturbances in energy homeostasis in mice. These effects were attributed to alterations in the hypothalamic transcriptome, specifically impacting insulin and immune response genes, leading to hypothalamic insulin resistance and neuroinflammation. Moreover, benzene exposure activated microglial transcription characterized by heightened expression of IKKß/NF-κB-related genes. Remarkably, selective removal of IKKß in immune cells or adult microglia in mice alleviated benzene-induced hypothalamic gliosis, and protected against hyperglycemia. In summary, our study uncovers a crucial pathophysiological mechanism, establishing a clear link between airborne toxicant exposure and the onset of metabolic diseases.

Antibacterial activity and carbapenem re-sensitizing ability of 1,10-phenanthroline-5,6-dione and its metal complexes against KPC-producing Klebsiella pneumoniae clinical strains.

Infections caused by KPC-producing Klebsiella pneumoniae (Kp-KPC) are associated with high mortality rates due to the increased number of resistant isolates and the scarcity of therapeutic options. This scenario reinforces the urgent need for new chemotherapeutics. Herein, we investigated the effects of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based complexes, [Cu(phendione)3 ](ClO4 )2 .4H2 O (Cu-phendione) and [Ag(phendione)2 ]ClO4 (Ag-phendione), both alone and also combined with carbapenems (meropenem (MEM), and imipenem), against 46 clonally distinct clinical strains of Kp-KPC. All isolates were found to be multidrug resistant in accordance with their susceptibility patterns by disk diffusion method. Compounds geometric mean (GM)-MIC and GM-MBC values (µmol l-1 ), respectively, were: phendione, 42·06 and 71·27; Cu-phendione, 9·88 and 13·75; and Ag-phendione, 10·10 and 13·06. Higher synergism rates of MEM-containing combinations were observed by the checkerboard assay, particularly with the two metal complexes. Moreover, drug combinations were able to re-sensitize 87% of the phenotypically non-susceptible strains. Time-kill studies, with MEM plus Cu-phendione or Ag-phendione, indicated that combinations with 0·5× MIC of each agent produce synergistic effects after 9-12 h. The MEM plus Ag-phendione eradicated about 106  CFU per ml of bacteria. These findings support the effectiveness of the re-sensitizing combinatorial approach and provide evidence that phendione-based compounds offer real promise in the fight against Kp-KPC infections.

Preventive oral kefir supplementation protects mice from ovariectomy-induced exacerbated allergic airway inflammation.

Asthma is an inflammatory lung disease that affects more women than men in adulthood. Clinical evidence shows that hormonal fluctuation during the menstrual cycle and menopause are related to increased asthma severity in women. Considering that life expectancy has increased and that most women now undergo menopause, strategies to prevent the worsening of asthma symptoms are particularly important. A recent study from our group showed that re-exposure of ovariectomised allergic mice to antigen (ovalbumin) leads to an exacerbation of lung inflammation that is similar to clinical conditions. However, little is known about the role of probiotics in the prevention of asthma exacerbations during the menstrual cycle or menopause. Thus, our objective was to evaluate the effects of supplementation with kefir, a popular fermented dairy beverage, as a preventive strategy for modulating allergic disease. The results show that the preventive kefir administration decreases the influx of inflammatory cells in the airways and exacerbates the production of mucus and the interleukin 13 cytokine. Additionally, kefir changes macrophage polarisation by decreasing the number of M2 macrophages, as shown by RT-PCR assay. Thus, kefir is a functional food that potentially prevents allergic airway inflammation exacerbations in ovariectomised mice.

A rhamnose-binding lectin from Rhodnius prolixus and the impact of its silencing on gut bacterial microbiota and Trypanosoma cruzi.

Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.

Factors affecting the physical stability (aggregation) of peptide therapeutics.

The number of biological therapeutic agents in the clinic and development pipeline has increased dramatically over the last decade and the number will undoubtedly continue to increase in the coming years. Despite this fact, there are considerable challenges in the development, production and formulation of such biologics particularly with respect to their physical stabilities. There are many cases where self-association to form either amorphous aggregates or highly structured fibrillar species limits their use. Here, we review the numerous factors that influence the physical stability of peptides including both intrinsic and external factors, wherever possible illustrating these with examples that are of therapeutic interest. The effects of sequence, concentration, pH, net charge, excipients, chemical degradation and modification, surfaces and interfaces, and impurities are all discussed. In addition, the effects of physical parameters such as pressure, temperature, agitation and lyophilization are described. We provide an overview of the structures of aggregates formed, as well as our current knowledge of the mechanisms for their formation.

A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics.

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKa values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

Innate immunomodulation to trypanosomatid parasite infections.

The recognition of invading pathogens by the innate immune system is essential for host protection against human parasites and the initiation of an effective adaptive immune response. Innate immune cells such as macrophages and dendritic cells (DCs) are involved in the first line of defense against protozoan parasites via sensing the invaders through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). Activation of macrophages and dendritic cells starts with the interaction between microbial ligands (pathogen-associated molecular patterns - PAMPs) and PRRs, and these activated cells influence the overall immune response. Trypanosomatid PAMPs are sensed by TLRs; for example, TLR2 recognizes alkylacylglycerol and lipophosphoglycan in Trypanosoma cruzi and Leishmania, respectively; TLR2/TLR4 recognize glycoisnositolphospholipids and glycosylphosphatidyl inositol in Trypanosoma species; and TLR9 recognizes genomic DNA in Trypanosoma. TLR signaling includes the recruitment of different adaptor molecules that activate various transcription factors, such as NF-kB, IRF3/7, and MAP kinases, to induce the production of pro-inflammatory cytokines and type I interferons. Moreover, activated macrophages and dendritic cells produce ROS and NOS, which limit pathogen survival, and large amounts of cytokines; additionally, antigen presentation enhances the adaptive immune response. In this review, we highlight the recent findings on PAMP recognition in trypanosomatid infections and the signaling pathways activated by PRRs.

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K(+) cycling and to active Na(+) extrusion.

BACKGROUND: Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. METHODS: (32)Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na(+), H(+) and K(+) fluxes were also investigated. The transport capacities of different evolutive forms were compared. RESULTS: Epimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na(+). We found that the parasites express TcPho84, a H(+):Pi-symporter, and TcPho89, a Na(+):Pi-symporter. Both Pi influx mechanisms showed Michaelis-Menten kinetics, with a one-order of magnitude higher affinity for the Na(+)-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K(+) ionophore) or SCH28028 (inhibitor of (H(+)+K(+))ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H(+) gradient energizes uphill Pi entry and that K(+) recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na(+)-ATPase, decreased only the Na(+)-dependent Pi uptake, indicating that this Na(+) pump generates the Na(+) gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. CONCLUSIONS: Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na(+) or H(+)/K(+) fluxes. GENERAL SIGNIFICANCE: This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.

Na+-dependent and Na+-independent mechanisms for inorganic phosphate uptake in Trypanosoma rangeli.

BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.

In vivo study of the anti-inflammatory effect of Rhus toxicodendron.

BACKGROUND: Homeopathic Rhus toxicodendron (Rhus tox) is used in various inflammatory conditions. We screened its effect compared to succussed ethanol controls and appropriate active controls. METHOD: We initially experimented with Rhus tox 6, 12, 30 and 200 cH, using carrageenan-induced paw oedema in rats. The 6 cH dilution appeared most effective and was used in subsequent assays. We used pre-treatment and single treatment regimes in Wistar rats, and mice. RESULTS: We found significant reductions compared to control in carrageenan-induced paw oedema, vascular permeability, writhing induced by intraperitoneal acetic acid and stress induced gastric lesions. CONCLUSIONS: Rhus tox in homeopathic dilution appears to interfere with inflammatory processes involving histamine, prostaglandins and other inflammatory mediators.

Intraocular delivery of oligonucleotides.

Anti-mRNA and particularly antisense oligonucleotides are molecules able to inhibit gene expression after intracellular penetration being potentially very interesting for the treatment of ocular diseases where growth factors are involved such as ocular scarring diseases or for the inhibition of viral multiplication. In most cases, the site of action of oligonucleotides has shown to be the posterior segment of the eye and these molecules are injected mainly by the intravitreal route. However, oligonucleotides are poorly stable in biological fluids, have a low intracellular penetration and are quickly eliminated form the vitreous. These issues request repeated administration of oligonucleotides which are able to induce severe damages to the retina. This is the reason why drug delivery systems were developed to improve the stability and intracellular penetration of oligonucleotides and, by sustained release, to increase their long term activity in the treatment of ocular diseases.

A new species of Nasicola Yamaguti, 1968 (Monogenea: Capsalidae) from the nasal cavities of Thunnnus obesus and a redescription of N. klawei (Stunkard, 1962) from T. albacares off Brazil.

Two species of Nasicola Yamaguti, 1968 are described from the nasal cavities of tunas ( Thunnus spp.) from off the coast of Brazil: N. brasiliensis n. sp. from T. obesus (Lowe) and the type-species, N. klawei (Stunkard, 1962), from T. albacares (Bonn.). The new species is differentiated from N. klawei on the basis of the large number of testes and from N. hogansi Wheeler & Beverley-Burton, 1986 by its greater body-size, proportionately smaller haptor and smaller number of marginal spines. The host-specificity of Nasicola spp. is commented upon.

Cell-associated and extracellular proteinases in Blastocrithidia culicis: influence of growth conditions.

The proteinase profile of Blastocrithidia culicis was analyzed, as well as how different growth conditions influenced its expression by gelatin-SDS-PAGE and the use of specific proteinase inhibitors. Multiple cell-associated proteinases with molecular masses corresponding to 33, 55, 60 kDa (cysteine proteinases) and 77, 80, 90, and 108 kDa (metalloproteinases) were detected using these methods. All the metalloproteinases identified were partitioned into the detergent phase after Triton X-114 extract, suggesting that they are membrane-bound, while all cysteine proteinases were partitioned into the aqueous phase. The proteolytic zymograms were similar when three different media were used for variable times of incubation. However, few quantitative and qualitative changes were observed. The secreted proteinase profile showed an heterogeneous pattern of enzymatic activities whose expression was dependent on time of growth and medium composition. However, the extracellular proteinase activities were abolished by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinases.

Developmentally regulated protein expression mediated by dimethylsulfoxide in Herpetomonas samuelpessoai.

We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver-staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells.

Characterization of proteinases in Herpetomonas anglusteri and Herpetomonas roitmani.

We have analyzed the proteinase profile of two Herpetomonas species, H. anglusteri and H. roitmani (a symbiont-bearing trypanosomatid), by in situ detection of enzyme activities on SDS-PAGE gels containing copolymerized gelatin as substrate. Two major cell-associated proteolytic activities, a 60 kDa zinc-metalloproteinase and a 45 kDa cysteine proteinase could be detected based on the inhibition of their activities by 1, 10-phenathroline and E-64, respectively. The trypanosomatids released into the growth medium distinct proteinases. H. anglusteri expressed three digestion haloes in the gels of approximately 60, 50, and 40 kDa, whereas H. roitmani secreted only a 60 kDa enzyme. However, these activities were inhibited by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinase.

Extradural hematomas in children.

The authors studied 58 patients, under 12 years of age, admitted to the Emergency Room of the Sao Paulo University Hospital between September 1987 and November 1991 with the diagnosis of traumatic extradural hematoma. Emphasis was given to the etiology of injury, the time interval from head injury to emergency room evaluation, the clinical features and the outcome after surgical and nonsurgical management. The site of hematoma was defined and correlated with the presence of skull fractures. Computerized tomography is the main diagnostic method, although it is not definitive. Fifteen patients were treated nonsurgically according to rigid clinical and radiological parameters. The overall mortality rate was 3.4%.

[Conservative treatment of laminar extradural hematomas in children]. / Tratamento conservador dos hematomas extradurais laminares na infância.

We successfully treated by nonsurgical methods 15 children with laminar epidural hematomas (EH), with minimal neurological symptoms and no signs of brain herniation. These EH were discovered 30 minutes to 5 days after head injury. The majority were localized in the parietal region. All children recovered without surgery from 2 to 12 days after hospitalization and all had evidence on CT scan of spontaneous clot reabsorption. We discuss the criteria for patient selection for this kind of treatment on EH in children.

[Traumatic extradural hematoma in childhood and normal early computed tomography: report of 2 cases]. / Hematoma extradural traumático na infância e tomografia computadorizada precoce normal. Relato de dois casos.

The cases of two children with traumatic extradural hematoma with an early normal CT scan are reported. Consciousness impairment occurred in them respectively 20 and 60 hours after admission, and CT at this occasion evidenced extradural hematoma. Serial neurological examinations are recommended for children with large traumatic cephalohematoma associated or not to skull fracture and in whom an initial early CT was normal, since they can lately develop extradural hematoma.