Role of ErbB and IL-1 signaling pathways in the dermonecrotic lesion induced by Loxosceles sphingomyelinases D.

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

Role of ErbB and IL-1 signaling pathways in the dermonecrotic lesion induced by Loxosceles sphingomyelinases D

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

Human Chondrocyte Activation by Toxins From Premolis semirufa, an Amazon Rainforest Moth Caterpillar: Identifying an Osteoarthritis Signature.

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

Human chondrocyte activation by toxins from premolis semirufa, an Amazon rainforest moth caterpillar: identifying an osteoarthritis signature

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

A Cross-Sectional Study on the Prevalence and Risk Correlates of Mental Disorders: The GRANADΣP Study.

This is a cross-sectional study of participants from a population census living in the province of Granada (Spain). A total of 1176 persons were contacted, 367 (31%) refused and 54 (6.7%) needed substitution. A final sample of 809 participants (response rate, 69.3%) were screened for mental disorder (MD) using the MINI International Neuropsychiatric Interview, a comprehensive interview validated to generate diagnoses compatible with ICD-10/DSM-4 criteria. Current (1-month) prevalence for any MD was 11.3% (95% confidence interval [CI], 9.7%-13.4%; affective 8.2%, anxiety 9.6%, psychotic 2.1%, addiction 1.8%, personality disorder 3.6%). Lifetime MD prevalence was 24.6% (95% CI, 21.6-27.6; affective 14.9%, anxiety 15.5%, psychotic 3.4%, addiction 4.4%, personality disorder 3.6%). Female sex was associated with MD, but this appeared partially due to higher levels of neuroticism among women. MD also correlated significantly with cannabis use, family history of MD, higher social adversity, higher suicide risk, poorer physical health, poorer cognitive performance, and personality problems.

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase.

Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase

Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.

Leptospira interrogans outer membrane protein LipL21 is a potent inhibitor of neutrophil myeloperoxidase

Leptospirosis is a widespread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. After entrance in the host, pathogenic leptospires evade the host natural defense mechanisms in order to propagate and disseminate to multiple organs. Myeloperoxidase is an enzyme stored in neutrophils azurophilic granules, and is released upon neutrophil activation to produce mainly hypochlorous acid, a strong oxidant and potent antimicrobial agent. In the present investigation, we studied the modulation of myeloperoxidase activity by L. interrogans serovar Copenhageni. We show that leptospires and their culture supernatants are able to inhibit both peroxidase and chlorination activities of myeloperoxidase, without interfering with neutrophil degranulation. By leptospiral outer membrane protein extraction and fractionation, we identified the proteins LipL21 and LipL45 as myeloperoxidase inhibitors, constituting new Leptospira virulence factors. Accordingly, we propose a function for the protein LipL21, one of the most expressed leptospiral outer membrane proteins. Our results show a novel innate immune evasion mechanism by which leptospires interfere with the host response in order to cope with the host oxidative stress and efficiently achieve dissemination and colonization.

Colubrid Venom Composition: An -Omics Perspective.

Snake venoms have been subjected to increasingly sensitive analyses for well over 100 years, but most research has been restricted to front-fanged snakes, which actually represent a relatively small proportion of extant species of advanced snakes. Because rear-fanged snakes are a diverse and distinct radiation of the advanced snakes, understanding venom composition among "colubrids" is critical to understanding the evolution of venom among snakes. Here we review the state of knowledge concerning rear-fanged snake venom composition, emphasizing those toxins for which protein or transcript sequences are available. We have also added new transcriptome-based data on venoms of three species of rear-fanged snakes. Based on this compilation, it is apparent that several components, including cysteine-rich secretory proteins (CRiSPs), C-type lectins (CTLs), CTLs-like proteins and snake venom metalloproteinases (SVMPs), are broadly distributed among "colubrid" venoms, while others, notably three-finger toxins (3FTxs), appear nearly restricted to the Colubridae (sensu stricto). Some putative new toxins, such as snake venom matrix metalloproteinases, are in fact present in several colubrid venoms, while others are only transcribed, at lower levels. This work provides insights into the evolution of these toxin classes, but because only a small number of species have been explored, generalizations are still rather limited. It is likely that new venom protein families await discovery, particularly among those species with highly specialized diets.

Colubrid Venom Composition: An -Omics Perspective

Snake venoms have been subjected to increasingly sensitive analyses for well over 100 years, but most research has been restricted to front-fanged snakes, which actually represent a relatively small proportion of extant species of advanced snakes. Because rear-fanged snakes are a diverse and distinct radiation of the advanced snakes, understanding venom composition among colubrids is critical to understanding the evolution of venom among snakes. Here we review the state of knowledge concerning rear-fanged snake venom composition, emphasizing those toxins for which protein or transcript sequences are available. We have also added new transcriptome-based data on venoms of three species of rear-fanged snakes. Based on this compilation, it is apparent that several components, including cysteine-rich secretory proteins (CRiSPs), C-type lectins (CTLs), CTLs-like proteins and snake venom metalloproteinases (SVMPs), are broadly distributed among colubrid venoms, while others, notably three-finger toxins (3FTxs), appear nearly restricted to the Colubridae (sensu stricto). Some putative new toxins, such as snake venom matrix metalloproteinases, are in fact present in several colubrid venoms, while others are only transcribed, at lower levels. This work provides insights into the evolution of these toxin classes, but because only a small number of species have been explored, generalizations are still rather limited. It is likely that new venom protein families await discovery, particularly among those species with highly specialized diets

Adhesin activity of Leptospira interrogans lipoprotein identified by in vivo and in vitro shotgun phage display.

Leptospira interrogans causes leptospirosis, one of the most common zoonotic diseases in the world. This pathogenic spirochete is able to bind to extracellular matrix, to express virulent factors and to cause host death. Until now, there is no effective human vaccine for the disease. Shotgun phage display genomic libraries of L. interrogans were constructed and used for in vivo biopanning in hamsters and screened for ligands able to bind to LLC-PK1 epithelial cells. In both panning procedures, clones coding for the putative lipoprotein LIC12976 were identified and, in order to confirm its adhesin activity, a recombinant protein was produced in Escherichia coli and showed to interact with A31 fibroblasts, LLC-PK1 and Vero epithelial cells in vitro. Moreover, rLIC12976 was shown to bind to laminin, indicating an adhesin function. This protein was also detected in extracts of L. interrogans from different serovars and it was found to be conserved among pathogenic leptospires. Further, the protein was tested as vaccine candidate and immunization of hamsters with LIC12976 did not confer protection against a lethal challenge with the homologous L. interrogans serovar Copenhageni. Nevertheless, LIC12976 seems to act as an adhesin, and may be important for the host-pathogen interaction, so that its study can contribute to the understanding of the virulence mechanisms in pathogenic leptospires.

Leptospira interrogans shotgun phage display identified LigB as a heparin-binding protein.

LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue.

Breaking the bond: recent patents on bacterial adhesins.

Adhesins need to be exposed on the surface of pathogenic bacteria to properly interact with host tissues and allow establishment of the infection. This fact implies that, in theory, one could manage or avoid infection by controlling adhesins' function, and also by indirectly detecting bacteria through their surface-exposed adhesins. Besides, binding of anti-adhesin immunoglobulins on the bacterial surface tend to promote the opsonization of the pathogen. Therefore, bacterial adhesins represent a great target to develop new biopharmaceuticals, which may become commercially and medically important products. In this review, we will summarize the biological importance of bacterial adhesins, and also discuss some recent patents related to these molecules, as well as their use and possible new future developments in this area.

Induction of TNF-alfa and CXCL-2 mRNAs in different organs of mice infected with pathogenic Leptospira.

The role of innate immune response in protection against leptospirosis is poorly understood. We examined the expression of the chemokine CXCL2/MIP-2 and the cytokine TNF-α in experimental resistant and susceptible mice models, C3H/HeJ, C3H/HePas and BALB/c strains, using a virulent strain of Leptospira interrogans serovar Copenhageni. Animals were infected intraperitoneally with 10(7) cells and the development of the disease was followed. Mortality of C3H/HeJ mice was observed whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. The infection was confirmed by the presence of leptospiral DNA in the organs of the animals, demonstrated by PCR. Sections of the organs were analyzed, after H&E stain. The relative expression of mRNA of chemokine CXCL2/MIP-2 and cytokine TNF-α was measured in lung, kidney and liver of the mice by qPCR. The concentrations of these proteins were measured in extracts of tissues and in serum of the animals, by ELISA. Increasing levels of transcripts and protein CXCL2/MIP-2 were detected since the first day of infection. The highest expression was observed at third day of infection in kidney, liver and lung of BALB/c mice. In C3H/HeJ the expression of CXCL2/MIP-2 was delayed, showing highest protein concentration in lung and kidney at the 5th day. Increasing in TNF-α transcripts were detected after infection, in kidney and liver of animals from the three mice strains. The expression of TNF-α protein in C3H/HeJ was also delayed, being detected in kidney and lung. Our data demonstrated that Leptospira infection stimulates early expression of CXCL2/MIP-2 and TNF-α in the resistant strain of mice. Histological analysis suggests that the expression of those molecules may be related to the influx of distinct immune cells and plays a role in the naturally acquired protective immunity.

Venomics profiling of Thamnodynastes strigatus unveils matrix metalloproteinases and other novel proteins recruited to the toxin arsenal of rear-fanged snakes.

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus , in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (∼47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy's (venom) gland transcriptome.

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C-type lectins, Crisps, and a C-type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.