A novel method for efficient generation of antigen-specific effector T-cells using dendritic cells transduced with recombinant adeno-associated virus and p38 kinase blockade.

BACKGROUND: The inefficacy of standard therapeutic strategies for ovarian cancer is reflected by the enduring poor prognosis of this malignancy. Due to the potential for exquisite specificity, sensitivity and long-term memory, immunotherapy offers an alternative modality for durable control of the disease, provided appropriate antigens can be identified and presented in the right context. METHODS: We tested a novel dendritic cell vaccine formulation to reprogram autologous antigen-specific T-cells in vitro, in vivo in a murine model of ovarian cancer, and ex vivo using human cells from patients. RESULTS: We show that dendritic cells (DCs) treated with a p38 MAPK inhibitor and transduced with a recombinant adenovirus associated vector (AAV) expressing Sperm protein (Sp) 17 are highly effective in generating antigen-specific T-cell cytotoxic response against ovarian cancer cells. Additionally, these DCs enhanced the differentiation of effector T-cells while reducing the frequency of Foxp3+ T-reg cells in vitro. CONCLUSIONS: This work provides a rationale for translation of pharmacologically reprogrammed DCs into clinical trials for prevention of tumor recurrence and progression in high-risk ovarian cancer patients.

Neonatal Opioid Withdrawal Syndrome: A Developmental Care Approach.

Tens of thousands of infants are impacted yearly by prenatal opioid exposure. The term neonatal opioid withdrawal syndrome (NOWS) is now replacing the more familiar term neonatal abstinence syndrome (NAS). Ongoing debate continues related to standard regimens for treatment of this oftentimes perplexing condition. Historically, treatment has focused on pharmacologic interventions. However, there is limited research that points to nonpharmacologic methods of treatment as viable options, whether alone or in addition to pharmacologic interventions. This article, utilizing a review of pertinent literature, outlines the physical aspects of NOWS, including its pathophysiology and the resulting physical clinical signs. In addition, we present an overview of how age-appropriate, nonpharmacologic interventions, centered on developmental care, may be a valuable approach to organize and prioritize routine care for these infants, their families, and the health care team facing the challenges of NOWS. Finally, the need for further research to better define evidence-based standards of care for these infants and their families is discussed.

Different Munc18 proteins mediate baseline and stimulated airway mucin secretion.

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell-deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.

Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells.

Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.

Assessment of intracellular mucin content in vivo.

Airway mucus presents a first line of defense against inhaled materials. It also, however, is a significant pathological contributor to chronic lung diseases, such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Thus, gaining a better understanding of the mechanisms of mucus production and secretion is an important goal for improving respiratory health. Mucins, the chief glycoprotein components of airway mucus, are very large polymeric glycoproteins, and measuring their production and secretion in experimental animals presents significant technical challenges. Over the past several years, we have developed assays for accurately quantifying mucin production and secretion using histological and biochemical assays. These methods are described here.

Selective cytochrome c displacement by phosphate and Ca(2+) in brain mitochondria.

In brain mitochondria, phosphate- and Ca(2+)-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca(2+). Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2-[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC(12)-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC(12)-HPC liposomes by either phosphate or Ca(2+), but only Ca(2+) produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Deltapsi(m)) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca(2+) caused inhibition of complex IV activity and impairment of Deltapsi(m). In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.

Exogenous phospholipids specifically affect transmembrane potential of brain mitochondria and cytochrome C release.

Release of cytochrome c, a decrease of membrane potential (Deltapsi(m)), and a reduction of cardiolipin (CL) of rat brain mitochondria occurred upon incubation in the absence of respiratory substrates. Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes. Fusion was triggered by potassium phosphate at concentrations producing mitochondrial permeability transition pore opening but not cytochrome c release, which was observed only at >10 mm. Cyclosporin A inhibited phosphate-induced CL fusion, whereas Pronase pretreatment of mitochondria abolished it, suggesting that mitochondrial permeability transition pore and protein(s) are involved in the fusion process. Phosphate-dependent fusion was enhanced in respiratory state 3 and influenced by phospholipid classes in the order CL > phosphatidylglycerol (PG) > phosphatidylserine. The probe 10-nonylacridine orange indicated that fused CL had migrated to the inner mitochondrial membrane. In state 3, CL enrichment of mitochondria resulted in a pH decrease in the intermembrane space. Cytofluorimetric analysis of mitochondria stained with 3,3'-diexyloxacarbocyanine iodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzymidazolylcarbocyanine iodide showed Deltapsi(m) increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required Deltapsi(m) consumption, suggesting that Deltapsi(m) is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high Deltapsi(m) values and prevented phosphate-dependent cytochrome c release.