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Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) stimulate immune cells via Toll-like receptor 9 (TLR9). Because oligodeoxynucleotides (ODNs) are susceptible to gastric degradation, clinical trials designed to evaluate their therapeutic utility have relied solely on parenteral routes of administration. A strategy to improve the activity of orally delivered ODNs by reducing their susceptibility to gastrointestinal (GI) digestion via encapsulation in calcium carbonate nanoparticles (ODNcaps) was recently described. This study compares the in vitro and in vivo activity of encapsulated (ODNcaps) versus free CpG ODNs delivered orally or parenterally. ODNcaps mirrored the ability of free ODNs to stimulate splenic B cells and macrophages in vitro. ODNcaps activated immune cells in the Peyer’s patches and mesenteric lymph nodes after oral delivery. Their effect on GI immunity was evaluated in studies of dextran sulfate sodium (DSS)-induced colitis and enteric infection, whereas systemic immunity was examined by monitoring their effect on lipopolysaccharide (LPS)-induced cytokine production and systemic pathogen challenge. Results indicate that orally delivered CpG ODNs predominantly induce GI rather than systemic immunity, and that calcium carbonate encapsulation does not significantly alter this behavior. Keywords: TLR9 agonist, delivery, activation, oral, encapsulated

Background: Mechanical ventilation (MV) is commonly used to improve blood oxygenation in critically ill patients and for general anesthesia. Yet the cyclic mechanical stress induced at even moderate ventilation volume settings [tidal volume (Vt) Methods: Balb/cJ mice were mechanically ventilated for 4 h using clinically relevant Vt and a positive end-expiratory pressure of 3 cmH 2 O under 2–3% isoflurane anesthesia. Lung tissue and bronchoalveolar lavage fluid were collected to assess lung inflammation and lung function was monitored using a FlexiVent ® . Results: MV induced significant pulmonary inflammation characterized by the influx and activation of CD11c + /F4/80 + macrophages and CD11b + /Ly6G + polymorphonuclear cells into the lung and bronchoalveolar lavage fluid. The concurrent administration of Sup ODN attenuated pulmonary inflammation as evidenced by reduced cellular influx and production of inflammatory cytokines. Oligonucleotide treatment did not worsen lung function as measured by static compliance or resistance. Conclusions: Treatment with Sup ODN reduces the lung injury induced by MV in mice.

Background 18:1/18:1-Dioleoyl-phosphatidylgycerol (DOPG) is a surfactant phospholipid that is nearly non-detectable in neonatal surfactant films. When alveolar macrophages are exposed to DOPG in vitro , secretory phospholipase A2 (sPLA2) production is blocked, resulting in suppressed macrophage activity and improved surfactant function. We investigated whether the addition of DOPG to a commercially available surfactant preparation would improve lung function in a neonatal piglet model of acute respiratory distress syndrome. Materials and methods Respiratory failure was achieved by triple-hit lung injury (repeated broncho-alveolar lavage, injurious ventilation, tracheal lipopolysaccharide instillation, each intervention 24 h apart) in twenty-four domestic piglets aged 2–6 days and subject to mechanical ventilation. Following each lung injury protocol the piglets were treated with surfactant alone or with surfactant + DOPG. Results Within 72 h of mechanical ventilation, we observed significantly improved gas exchange (oxygenation and ventilation), lung mechanics (compliance and resistance of the respiratory system), and pulmonary oedema (extra-vascular lung water index) in the surfactant + DOPG group. This favourable clinical effect could be attributed to improved surfactant function, reduced sPLA2 secretion, inhibition of macrophage migration, reduced alveolar epithelial apoptosis, and suppression of amphiregulin and TGF-β1 expression in pulmonary tissues as a prerequisite for fibrous lung repair. Conclusions We conclude that surfactant fortified by DOPG preserves lung function, and prevents alveolar epithelial injury and fibrous stimulus by reduction of sPLA2 in a neonatal model of acute respiratory distress syndrome without any relevant discernable side effects. Hence, DOPG supplementation in a neonatal lung exerts important function protecting effects and seems to be justified in cases of overwhelming pulmonary inflammation.

Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.

Sporadic or familial germline mutations in GATA2 frequently result in a bone marrow failure syndrome characterized by recurrent life-threatening infections, cytopenias, and progression to myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), or chronic myelomonocytic leukemia (CMML). Hematopoietic stem cell transplantation (HSCT) represents the only definitive therapy for GATA2 deficiency. However, the highly variable disease penetrance in different families, and in mutation positive individuals within a single family, make the timing of HSCT challenging. We assessed the utility of scoring patient characteristics according to the Revised International Prognostic Scoring System (IPSS-R) to predict survival after HSCT in a cohort of 51 patients with GATA2 deficiency who had progressed to MDS. Higher IPSS-R scores significantly correlated with reduced overall survival after transplant. In evaluating the components of the IPSS-R score for patients with GATA2 deficiency, cytopenias, including anemia, neutropenia, and thrombocytopenia, were important variables in placing these patients in higher risk IPSS-R categories. Since GATA2 deficiency patients have very low peripheral blood monocyte, B cell, and NK cell counts, we evaluated these cytopenias in conjunction with IPSS-R scoring. These cytopenias also had prognostic value for outcome after HSCT in patients with GATA2 deficiency. Cytogenetics, another component of the IPSS-R score, also placed patients with GATA2 deficiency into higher risk categories. In particular, patients harboring monosomy 7 had poorer survival outcomes than patients with normal cytogenetics or trisomy 8. Of note, all five patients with GATA2 deficiency and trisomy 1q successfully underwent HSCT. Clonal cytogenetic progression with the development of additional cytogenetic aberrations was a harbinger of myeloid progression and led to HSCT. In a cohort of four patients who underwent HSCT following progression to AML/CMML, there was only one survivor. Thus, HSCT is recommended in patients with GATA2 deficiency and intermediate or high-risk IPSS-R scores before the development of AML or CMML. Disclosures No relevant conflicts of interest to declare.

Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-β1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant “fortified” by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN) stimulate immune cells via TLR9. CpG ODN are being evaluated clinically for use as vaccine adjuvants and in cancer immunotherapy. CpG ODNs are generally delivered parenterally as they are susceptible to degradation by acids and nucleases in the GI tract if delivered orally. Wang et al. developed a method to protect ODNs from digestion by encapsulating them in calcium (ODNcaps). They reported that orally delivered ODNcaps could alter susceptibility to atopic dermatitis in mice. To understand the effect of orally delivered CpG ODNcap on local and systemic immune responses, their in vivo and in vitro activity was evaluated in comparison to free (unencapsulated) ODN. TLR9-expressing spleen cells stimulated with CpG ODNcaps in vitro responded by producing cytokines. Oral delivery of CpG ODNcaps increased cytokine production in the Peyer’s patches and mesenteric lymph nodes. Oral administration of CpG ODNcaps exacerbated disease severity in a murine model of dextran sulfate induced inflammatory colitis similar to oral CpG ODN. Oral CpG ODNcap had no detectable systemic effects: they induced no change in serum cytokine levels or spleen cell activation. However, oral CpG ODNcap did enhance host responsiveness to subsequent LPS challenge while reducing susceptibility to L. monocytogenes challenge. Of note, the protection conferred by CpG ODNcap did not exceed that provided when free CpG ODN were delivered orally. These findings indicate that ODNcaps retain biological activity but that calcium encapsulation does not improve their bioavailability or activity in vivo.

Synthetic oligonucleotides (ODN) that express unmethylated “CpG motifs” trigger cells that express Toll-like receptor 9. In humans this includes plasmacytoid dendritic cells and B cells. CpG ODN induce an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. Their utility as vaccine adjuvants was evaluated in a number of clinical trials. Results indicate that CpG ODN improve antigen presentation and the generation of vaccine-specific cellular and humoral responses. This work provides an up-to-date overview of the utility of CpG ODN as adjuvants for vaccines targeting infectious agents and cancer.