Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides.

Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1-4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7-11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

Mucins MUC5AC and MUC5B Are Variably Packaged in the Same and in Separate Secretory Granules.

Rationale: MUC5AC (mucin 5AC, oligomeric gel-forming) and MUC5B (mucin 5B, oligomeric gel-forming) are the predominant secreted polymeric mucins in mammalian airways. They contribute differently to the pathogenesis of various muco-obstructive and interstitial lung diseases, and their genes are separately regulated, but whether they are packaged together or in separate secretory granules is not known. Objectives: To determine the packaging of MUC5AC and MUC5B within individual secretory granules in mouse and human airways under varying conditions of inflammation and along the proximal-distal axis. Methods: Lung tissue was obtained from mice stimulated to upregulate mucin production by the cytokines IL-1ß and IL-13 or by porcine pancreatic elastase. Human lung tissue was obtained from donated normal lungs, biopsy samples of transplanted lungs, and explanted lungs from subjects with chronic obstructive pulmonary disease. MUC5AC and MUC5B were labeled with antibodies from different animal species or, in mice only, by transgenic chimeric mucin-fluorescent proteins and imaged using widefield deconvolution or Airyscan fluorescence microscopy. Measurements and Main Results: In both mouse and human airways, most secretory granules contained both mucins interdigitating within the granules. Smaller numbers of granules contained MUC5B alone, and even fewer contained MUC5AC alone. Conclusions: MUC5AC and MUC5B are variably stored both in the same and in separate secretory granules of both mice and humans. The high fraction of granules containing both mucins under a variety of conditions makes it unlikely that their secretion can be differentially controlled as a therapeutic strategy. This work also advances knowledge of the packaging of mucins within secretory granules to understand mechanisms of epithelial stress in the pathogenesis of chronic lung diseases.

Intermediary Role of Lung Alveolar Type 1 Cells in Epithelial Repair upon Sendai Virus Infection.

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ (Sry-box 2+) clusters without differentiating into AT1 or AT2 cells. Large AT2 cell-depleted areas remain covered by AT1 cells, which we name "AT2-less regions", and are replaced by SOX2+ clusters spreading both basally and luminally. AT2 cell proliferation and differentiation are largely confined to topologically distal regions and form de novo alveolar surface, with limited contribution to in situ repairs of AT2-less regions. Time-course single-cell RNA sequencing profiling and RNAscope validation suggest enhanced immune responses and altered growth signals in AT1 cells. Our comprehensive spatiotemporal and genomewide study highlights the hitherto unappreciated role of AT1 cells in lung injury-repair.

Immune Modulation to Improve Survival of Viral Pneumonia in Mice.

Viral pneumonias remain global health threats, as exemplified in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, requiring novel treatment strategies both early and late in the disease process. We have reported that mice treated before or soon after infection with a combination of inhaled Toll-like receptor (TLR) 2/6 and 9 agonists (Pam2-ODN) are broadly protected against microbial pathogens including respiratory viruses, but the mechanisms remain incompletely understood. The objective of this study was to validate strategies for immune modulation in a preclinical model of viral pneumonia and determine their mechanisms. Mice were challenged with the Sendai paramyxovirus in the presence or absence of Pam2-ODN treatment. Virus burden and host immune responses were assessed to elucidate Pam2-ODN mechanisms of action and to identify additional opportunities for therapeutic intervention. Enhanced survival of Sendai virus pneumonia with Pam2-ODN treatment was associated with reductions in lung virus burden and with virus inactivation before internalization. We noted that mortality in sham-treated mice corresponded with CD8+ T-cell lung inflammation on days 11-12 after virus challenge, after the viral burden had declined. Pam2-ODN blocked this injurious inflammation by minimizing virus burden. As an alternative intervention, depleting CD8+ T cells 8 days after viral challenge also decreased mortality. Stimulation of local innate immunity within the lungs by TLR agonists early in disease or suppression of adaptive immunity by systemic CD8+ T-cell depletion late in disease improves outcomes of viral pneumonia in mice. These data reveal opportunities for targeted immunomodulation to protect susceptible human subjects.

Platelet Munc13-4 regulates hemostasis, thrombosis and airway inflammation.

Platelet degranulation is crucial for hemostasis and may participate in inflammation. Exocytosis in platelets is mediated by SNARE proteins and should be controlled by Munc13 proteins. We found that platelets express Munc13-2 and -4. We assessed platelet granule exocytosis in Munc13-2 and -4 global and conditional knockout (KO) mice, and observed that deletion of Munc13-4 ablates dense granule release and indirectly impairs alpha granule exocytosis. We found no exocytic role for Munc13-2 in platelets, not even in the absence of Munc13-4. In vitro, Munc13-4-deficient platelets exhibited defective aggregation at low doses of collagen. In a flow chamber assay, we observed that Munc13-4 acted as a rate-limiting factor in the formation of thrombi. In vivo, we observed a dose-dependency between Munc13-4 expression in platelets and both venous bleeding time and time to arterial thrombosis. Finally, in a model of allergic airway inflammation, we found that platelet-specific Munc13-4 KO mice had a reduction in airway hyper-responsiveness and eosinophilic inflammation. Taken together, our results indicate that Munc13-4-dependent platelet dense granule release plays essential roles in hemostasis, thrombosis and allergic inflammation.

Diffusion-weighted magnetic resonance imaging evaluation of intra-abdominal sites of implants to predict likelihood of suboptimal cytoreductive surgery in patients with ovarian carcinoma.

OBJECTIVES: To analyse the diagnostic accuracy and to establish a predictive score based on diffusion-weighted magnetic resonance imaging (DWMRI) compared to exploratory laparotomy (EL) for predicting suboptimal cytoreductive surgery for different intra-abdominal sites of implants in patients with ovarian cancer. METHODS: Thirty-four patients with advanced ovarian carcinoma were studied. Preoperative DWMRI of the abdomen and pelvis was performed. DWMRI findings were compared with EL. Ten anatomical sites were selected for inclusion in the score. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for suboptimal cytoreduction were calculated for both DWMRI and EL. Receiver operating characteristic (ROC) curve analysis was used to assess the ability to predict suboptimal cytoreduction. RESULTS: Using predictive score, ROC curves were generated with an area under the curve of 0.938 for DWMRI and 0.947 for EL (P < 0.0001). For DWMRI, a score ≥6 had the highest overall accuracy at 91.1 % and identified patients with unnecessary EL with a sensitivity of 75 %. For EL, a score ≥4 had the highest overall accuracy at 88.2 % and was able to identify patients with unnecessary EL with a sensitivity of 87.5 %. CONCLUSIONS: DWMRI is an emerging technique that may be useful to predict suboptimal cytoreduction in ovarian cancer. KEY POINTS: • DWMRI is increasingly used in ovarian cancer. • DWMRI is an accurate technique for depicting intra-abdominal sites of implants • DWMRI is useful for predicting optimal cytoreductive surgical outcome. • We report a high predictive value similar to exploratory laparotomy.

Repair of ovine peripheral nerve injuries with xenogeneic human acellular sciatic nerves prerecellularized with allogeneic Schwann-like cells-an innovative and promising approach.

INTRODUCTION: The iatrogenic effects of repairing peripheral nerve injuries (PNIs) with autografts (AGTs) encouraged the present study to involve a new approach consisting of grafting xenogeneic prerecellularized allogeneic cells instead of AGTs. METHODS: We compared sheep's AGT regenerative and functional capacity with decellularized human nerves prerecellularized with allogeneic Schwann-like cell xenografts (onwards called xenografts). Mesenchymal stem cells were isolated from ovine adipose tissue and induced in vitro to differentiate into Schwann-like cells (SLCs). Xenografts were grafted in ovine sciatic nerves. Left sciatic nerves (20 mm) were excised from 10 sheep. Then, five sheep were grafted with 20 mm xenografts, and five were reimplanted with their nerve segment rotated 180° (AGT). RESULTS: All sheep treated with xenografts or AGT progressively recovered the strength, movement, and coordination of their intervened limb, which was still partial when the study was finished at sixth month postsurgery. At this time, numerous intrafascicular axons were observed in the distal and proximal graft extremes of both xenografts or AGTs, and submaximal nerve electrical conduction was observed. The xenografts and AGT-affected muscles appeared partially stunted. CONCLUSIONS: Xenografts and AGT were equally efficacious in starting PNI repair and justified further studies using longer observation times. The hallmarks from this study are that human xenogeneic acellular scaffolds were recellularized with allogenic SCL and were not rejected by the nonhuman receptors but were also as functional as AGT within a relatively short time postsurgery. Thus, this innovative approach promises to be more practical and accessible than AGT or allogenic allografts and safer than AGT for PNI repair.

Improved HUMARA for the Detection of X-Linked Agammaglobulinemia Carriers.

Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.

Preneoplastic and neoplastic changes in the Leydig cells population in mice exposed to low doses of cadmium.

The morphological consequences of chronic exposition to low doses of cadmium (Cd) in the Leydig cells population were investigated in 40 sexually mature male mice at morphological and ultrastructural levels. Animals were orally exposed to cadmium (0.015 g/L of CdCl(2) in drinking water) for 3, 6, 12 and 18 months and then sacrificed, samples were collected for toxicological, light and electron microscope studies. Vascular lesions were evident from 6 months of Cd exposure, the severity of the morphological changes observed in the testicular vases were highly and clearly correlated to the time of exposure to Cd. The severity of the Leydig cells morphological changes were increasing along the time of exposure. Presence of cytoplasm vacuolization and degenerative images of the cells were frequent after 12 months of Cd exposure. Also two Leydig cells tumours after 12 and 18 months Cd exposure were presented. These results indicate that prolonged exposures to low doses of Cd are able to induce morphological damage on the Leydig cells.

Inducible epithelial resistance against acute Sendai virus infection prevents chronic asthma-like lung disease in mice.

BACKGROUND AND PURPOSE: Respiratory viral infections play central roles in the initiation, exacerbation and progression of asthma in humans. An acute paramyxoviral infection in mice can cause a chronic lung disease that resembles human asthma. We sought to determine whether reduction of Sendai virus lung burden in mice by stimulating innate immunity with aerosolized Toll-like receptor (TLR) agonists could attenuate the severity of chronic asthma-like lung disease. EXPERIMENTAL APPROACH: Mice were treated by aerosol with 1-µM oligodeoxynucleotide (ODN) M362, an agonist of the TLR9 homodimer, and 4-µM Pam2CSK4 (Pam2), an agonist of the TLR2/6 heterodimer, within a few days before or after Sendai virus challenge. KEY RESULTS: Treatment with ODN/Pam2 caused ~75% reduction in lung Sendai virus burden 5 days after challenge. The reduction in acute lung virus burden was associated with marked reductions 49 days after viral challenge in eosinophilic and lymphocytic lung inflammation, airway mucous metaplasia, lumenal mucus occlusion and hyperresponsiveness to methacholine. Mechanistically, ODN/Pam2 treatment attenuated the chronic asthma phenotype by suppressing IL-33 production by type 2 pneumocytes, both by reducing the severity of acute infection and by down-regulating Type 2 (allergic) inflammation. CONCLUSION AND IMPLICATIONS: These data suggest that treatment of susceptible human hosts with aerosolized ODN and Pam2 at the time of a respiratory viral infection might attenuate the severity of the acute infection and reduce initiation, exacerbation and progression of asthma.

Relationship Between Osteoporosis and Marginal Bone Loss in Osseointegrated Implants: A 2-Year Retrospective Study.

BACKGROUND: Fitting implants in osteoporotic patients has traditionally been controversial, and there is little scientific evidence relating osteoporosis to marginal bone loss (MBL). The aims of this study are as follows: 1) to evaluate the possibility of a correlation between osteoporosis, as measured by the mandibular cortical index (MCI), and MBL and 2) to assess how various systemic diseases, periodontitis, and placement of implants in regenerated bone are correlated with MBL and MCI. METHODS: This retrospective study examines 212 implants inserted in 67 patients. To take a possible cluster failure into account, an implant for each patient was selected (n = 67 implants). MBL was assessed. Osteoporosis was evaluated using the MCI. Both MBL and MCI were assessed from panoramic radiographs. χ(2) test was performed (Haberman post hoc test). Significance was P <0.05. RESULTS: When the total sample implant (N = 212) was evaluated, a significant association was found between the presence of osteoporosis and MCI (P <0.001) and between the presence of diabetes mellitus and MCI (P <0.01). Significant associations were also found between MBL and placement of implants in regenerated sites (P <0.001) and between MBL and a previous history of periodontitis (P <0.05). When the sample is evaluated only in selected implants (one per patient, n = 67), significant differences appear to relate only to the MBL with the placement of implants in regenerated bone sites (P <0.001). CONCLUSIONS: Osteoporosis (as evaluated by MCI) does not pose a risk for the development of greater MBL. Parameters adversely affecting the development of increased MBL are a previous history of periodontitis and especially the placement of implants at sites of bone regeneration.

SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion.

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated.

Use of anesthetics associated to vasoconstrictors for dentistry in patients with cardiopathies. Review of the literature published in the last decade.

OBJECTIVE: The use of local anesthetics associated to vasoconstrictor agents in dentistry is thoroughly justified and is widely extended, but we cannot ignore the fact that anesthetic infiltration poses risk of complications throughout the dental treatment period. The objective of the present review is to document the reported effects the use of the local anesthetics most widely employed in dentistry, with or without association to vasoconstrictor agents may have in patients with any sort of cardiopathy. STUDY DESIGN: We have searched for randomized clinical trials on the assessment of the cardiovascular effects of local anesthetics used in dentistry, without limits as regards age or sex, conducted in patients with any type of cardiopathy which were published during the last decade and were index-linked in Cochrane, Embase and Medline. RESULTS: We have found six randomized clinical trials index-linked in Medline and Cochrane in the past ten years. These trials compare different types of anesthetics: lidocaine 2%, mepivacaine 2%, prilocaine 2% , associated or not to different vasoconstrictor concentrations such as adrenaline or felypressin. The cardiopathies affecting the patients included in the different trials range from hypertension, ischemic heart disease, arrythmias, chronic coronary disease to heart transplantation. CONCLUSIONS: The use of anesthetics associated to vasoconstrictor agents is justified in the case of patients with cardiopathies (once we get over the period in which any type of dental manipulation is contraindicated) and in controlled hypertensive patients. In any case, we must be very careful with the choice and execution of the anesthetic technique, being it possible to use a dose between 1.8 and 3.6 ml, on a general basis. Further studies are necessary to establish the effects of these drugs on severe hypertensive patients or in patients with other more advanced cardiopathies. Key words:Vasoconstrictor agents, epinephrine/adverse effects, local anesthetics, dental restoration, oral surgery, cardiovascular diseases, coronary arteriosclerosis, heart disease, hypertension, arrhythmias, coronariopathy.