Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients.

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

Microbiology of chronic mesh infection.

PURPOSE: Mesh infection following hernia repair is one of the most dreaded complications of hernia surgery. Mesh sinus, infected seromas, mesh extrusion, and mesh-related enteric fistulas are common complications associated with synthetic mesh. This study aimed to review the microbiota of mesh infection in 100 patients submitted to mesh explantation. METHODS: We reviewed the charts of patients presenting with a history of mesh infection lasting or arising six months or more after mesh placement. All patients who submitted to abdominal wall repair with complete removal of an infected mesh and presenting a positive culture were included. The microbiology analysis was based on positive cultures obtained from the fluids and tissues surrounding the mesh or positive cultures of the mesh. Microorganisms were divided into gram-positive or gram-negative, aerobic or anaerobic, and fungi. RESULTS: Pure aerobic gram-positive cultures were encountered in 50% of the patients, followed by a combination of aerobic gram-positive/gram-negative (8%) and pure gram-negative cultures (6%). Anaerobes were recovered from 31% of patients. Fungi were recovered from 6%. Staphylococcus aureus was identified in 64% of cultures, with methicillin-resistant Staphylococcus aureus present in 42% and methicillin-sensitive Staphylococcus aureus in 22%. Among aerobic gram-negative infections, six (17%) were caused by multi-resistant bacteria, including Pseudomonas aeruginosa, Proteus mirabilis, Acinetobacter baumanii, Klebsiella pneumoniae complex, and Enterobacter cloacae complex. CONCLUSION: Staphylococcus aureus plays a significant role in the pathogenesis of synthetic mesh infection. Staphylococcus aureus, isolated in 64% of cultures, accounted for most single bacterial infections and was the prevalent germ in mesh sinus and infected seromas. Gram-negative infection occurred in 35%. Anaerobes occurred in 31%, commonly encountered in polymicrobial infections. Most fungi cultures happened in patients with enteric fistulas.

Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

Could polypropylene mesh impair male reproductive organs? Experimental study with different methods of implantation.

PURPOSE: To evaluate the vas deferens and testicles of rats submitted to bilateral inguinotomy and polypropylene (PP) mesh placement. METHOD: Sixty Wistar rats were randomized into three groups: Control (inguinotomy only), mesh placement over the vas deferens (Mesh-DD) or under the spermatic funiculus (Mesh-SF). The following analyses were performed: vas deferens morphometry (lumen area and wall thickness), quantification of collagen fibers, spermatogenesis, apoptosis (cleaved caspase-3 and TUNEL) and cellular proliferation (Ki67). Quantitative gene expression (qPCR) for apoptosis and inflammatory cytokines were evaluated by RT-PCR. RESULTS: In the apoptosis pathway, Mesh-DD showed one upregulated gene (Il10) and three downregulated genes (Fadd, Tnfrsf1b and Xiap). In Mesh-SF, 17 genes were downregulated. In the inflammation pathway (Mesh-DD), one gene was upregulated (Il1r1), and one gene was downregulated (Ccl12). In Mesh-SF, three genes were upregulated (Il1r1, Tnfsf13b and Csf1), and two were downregulated (Ccl12 and Csf2). PP mesh placement preserved spermatogenesis and did not alter the vas deferens or the testicle. In the ductus deferens, there was reduced luminal area (30 days), increased wall thickness (90 days), and increased type III collagen and cell proliferation (30 and 90 days) (p < 0.05). In the testicle, cell proliferation was greater in the Mesh-DD (p < 0.05). CONCLUSIONS: PP mesh, whether or not in direct contact with spermatic funicular structures, induces changes that were not sufficient to cause damage to the evaluated organs.

The use of synthetic mesh in contaminated and infected abdominal wall repairs: challenging the dogma-A long-term prospective clinical trial.

BACKGROUND: Abdominal wall reconstruction in patients presenting with enteric fistulas and mesh infection is challenging. There is a consensus that synthetic mesh must be avoided in infected operations, and the alternatives to using synthetic mesh, such as component separation techniques and biologic mesh, present disappointing results with expressive wound infection and hernia recurrence rates. METHODS: A prospective clinical trial designed to evaluate the short- and long-term outcomes of 40 patients submitted to elective abdominal wall repair with synthetic mesh in the dirty-infected setting, and compared to a cohort of 40 patients submitted to clean ventral hernia repairs. Patients in both groups were submitted to a single-staged repair using onlay polypropylene mesh reinforcement. RESULTS: Groups' characteristics were similar. There were 13 (32.5%) surgical site occurrences in the infected mesh (IM) group, compared to 11 (27.5%) in the clean-control (CC) group, p = 0.626. The 30-day surgical site infection rate was 15% for the IM group vs. 10% for the CC cases, p = 0.499. One patient required a complete mesh removal in each group. The mean overall follow-up was 50.2 ± 14.8 months, with 36 patients in the IM group and 38 clean-controls completing a follow-up of 36 months. There was one hernia recurrence (4.2%) in the IM group and no recurrences in the CC group. CONCLUSION: We demonstrated that using polypropylene mesh in the infected setting presented similar outcomes to clean repairs. The use of synthetic mesh in the onlay position resulted in a safe and durable abdominal wall reconstruction. TRIAL REGISTRATION: Study registered at Plataforma Brasil (plataformabrasil.saude.gov.br), CAAE 30836614.7.0000.0068. Study registered at Clinical Trials (clinicaltrials.gov), Identifier NCT03702153.

Maternal exposure to an environmentally relevant dose of triclocarban results in perinatal exposure and potential alterations in offspring development in the mouse model.

Triclocarban (TCC) is among the top 10 most commonly detected wastewater contaminants in both concentration and frequency. Its presence in water, as well as its propensity to bioaccumulate, has raised numerous questions about potential endocrine and developmental effects. Here, we investigated whether exposure to an environmentally relevant concentration of TCC could result in transfer from mother to offspring in CD-1 mice during gestation and lactation using accelerator mass spectrometry (AMS). 14C-TCC (100 nM) was administered to dams through drinking water up to gestation day 18, or from birth to post-natal day 10. AMS was used to quantify 14C-concentrations in offspring and dams after exposure. We demonstrated that TCC does effectively transfer from mother to offspring, both trans-placentally and via lactation. TCC-related compounds were detected in the tissues of offspring with significantly higher concentrations in the brain, heart and fat. In addition to transfer from mother to offspring, exposed offspring were heavier in weight than unexposed controls demonstrating an 11% and 8.5% increase in body weight for females and males, respectively. Quantitative real-time polymerase chain reaction (qPCR) was used to examine changes in gene expression in liver and adipose tissue in exposed offspring. qPCR suggested alterations in genes involved in lipid metabolism in exposed female offspring, which was consistent with the observed increased fat pad weights and hepatic triglycerides. This study represents the first report to quantify the transfer of an environmentally relevant concentration of TCC from mother to offspring in the mouse model and evaluate bio-distribution after exposure using AMS. Our findings suggest that early-life exposure to TCC may interfere with lipid metabolism and could have implications for human health.

A retrospective review and observations over a 16-year clinical experience on the surgical treatment of chronic mesh infection. What about replacing a synthetic mesh on the infected surgical field?

PURPOSE: To review the short- and long-term results in patients who underwent removal of infected or exposed mesh and reconstruction of the abdominal wall with simultaneous mesh replacement. METHODS: Patients undergoing removal of an infected or exposed mesh and single-staged reconstruction of the abdominal wall with synthetic mesh replacement over a 16-year period were retrospectively reviewed from a prospectively maintained database. Patients were operated and followed by a single surgeon. Outcome measures included wound complications and hernia recurrence. RESULTS: From 1996 until 2012, 41 patients (23 F, 18 M), with a mean age of 53.4 years and mean BMI of 31.2 ± 8 kg/m(2), were treated for chronic mesh infection (CMI). A suppurative infection was present in 27 patients, and 14 had an exposed mesh. The need for recurrent incisional hernia repair was observed in 25 patients; bowel resections or other potentially contaminated procedures were associated in 15 patients. The short-term results showed an uneventful post-operative course after mesh replacement in 27 patients; 6 (14.6%) patients developed a minor wound infection and were treated with dressings and antibiotics; 5 (12%) patients had wound infections requiring debridement and one required complete mesh removal. On the long-term follow-up, there were three hernia recurrences, one of which demanded a reoperation for enterocutaneous fistula; 95% of the patients submitted to mesh replacement were considered cured of CMI after a mean follow-up of 74 months. CONCLUSIONS: CMI can be treated by removal of infected mesh; simultaneous mesh replacement prevents hernia recurrence and has an acceptable incidence of post-operative acute infection. Standard polypropylene mesh is a suitable material to be used in the infected surgical field as an onlay graft.

The anti-promyelocytic leukemia mode of action of two endophytic secondary metabolites unveiled by a proteomic approach.

As a result of a program to find antitumor compounds of endophytes from medicinal Asteraceae, the steroid (22E,24R)-8,14-epoxyergosta-4,22-diene-3,6-dione (a) and the diterpene aphidicolin (b) were isolated from the filamentous fungi Papulaspora immersa and Nigrospora sphaerica, respectively, and exhibited strong cytotoxicity against HL-60 cells. A proteomic approach was used in an attempt to identify the drugs' molecular targets and their respective antiproliferative mode of action. Results suggested that the (a) growth inhibition effect occurs by G2/M cell cycle arrest via reduction of tubulin alpha and beta isomers and 14-3-3 protein gamma expression, followed by a decrease of apoptotic and inflammatory proteins, culminating in mitochondrial oxidative damage that triggered autophagy-associated cell death. Moreover, the decrease observed in the expression levels of several types of histones indicated that (a) might be disarming oncogenic pathways via direct modulation of the epigenetic machinery. Effects on cell cycle progression and induction of apoptosis caused by (b) were confirmed. In addition, protein expression profiles also revealed that aphidicolin is able to influence microtubule dynamics, modulate proteasome activator complex expression, and control the inflammatory cascade through overexpression of thymosin beta 4, RhoGDI2, and 14-3-3 proteins. Transmission electron micrographs of (b)-treated cells unveiled dose-dependent morphological characteristics of autophagy- or oncosis-like cell death.

Mutations in NPHS2 (podocin) in Mexican children with nephrotic syndrome who respond to standard steroid treatment.

Human nephrotic syndrome has been related to mutations in glomerular proteins. Mutations in the NPHS2 gene that encodes podocin have been described as responsible for steroid-resistant nephrotic syndrome. It has been advised to test for NPHS2 mutations in parallel or before giving steroid treatment in nephrotic syndrome patients in order to avoid unnecessary therapy. We identified NPHS2 mutations in Mexican children with nephrotic syndrome. The study included 13 children with nephrotic syndrome and 2 healthy control individuals; 8 patients were steroid-resistant and 5 were steroid-sensitive. We analyzed the 3rd exon of NPHS2 by DNA sequencing. Podocin heterozygous missense mutations L139R and L142P were found; the former was found in both steroid-sensitive and steroid-resistant children, while the latter was found in a steroid-resistant child. We conclude that NPHS2 mutations should be investigated to help decide the course of treatment in nephrotic syndrome patients.

Carbon turnover in the water-soluble protein of the adult human lens.

PURPOSE: Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse. METHODS: Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse. RESULTS: The water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction. CONCLUSIONS: As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.