Case report: HLA-haploidentical HSCT rescued with donor lymphocytes infusions in a patient with X-linked chronic granulomatous disease.

Chronic granulomatous disease is an inborn error of immunity due to disrupted function of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. This results in impaired respiratory burst of phagocytes and insufficient killing of bacteria and fungi. Patients with chronic granulomatous disease are at increased risk for infections, autoinflammation and autoimmunity. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only widely available curative therapy. While HSCT from human leukocyte antigen (HLA) matched siblings or unrelated donors are standard of care, transplantation from HLA-haploidentical donors or gene therapy are considered alternative options. We describe a 14-month-old male with X-linked chronic granulomatous disease who underwent a paternal HLA-haploidentical HSCT using T-cell receptor (TCR) alpha/beta+/CD19+ depleted peripheral blood stem cells followed by mycophenolate graft versus host disease prophylaxis. Decreasing donor fraction of CD3+ T cells was overcome by repeated infusions of donor lymphocytes from the paternal HLA-haploidentical donor. The patient achieved normalized respiratory burst and full donor chimerism. He remained disease-free off any antibiotic prophylaxis for more than three years after HLA-haploidentical HSCT. In patients with x-linked chronic granulomatous disease without a matched donor paternal HLA-haploidentical HSCT is a treatment option worth to consider. Administration of donor lymphocytes can prevent imminent graft failure.

Effect of Calcium Carbonate Encapsulation on the Activity of Orally Administered CpG Oligonucleotides.

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.

Three distinct pneumotypes characterize the microbiome of the lung in BALB/cJ mice.

Bacteria can rarely be isolated from normal healthy lungs using conventional culture techniques, supporting the traditional belief that the lungs are sterile. Yet recent studies using next generation sequencing report that bacterial DNA commonly found in the upper respiratory tract (URT) is present at lower levels in the lungs. Interpretation of that finding is complicated by the technical limitations and potential for contamination introduced when dealing with low biomass samples. The current work sought to overcome those limitations to clarify the number, type and source of bacteria present in the lungs of normal mice. Results showed that the oral microbiome is diverse and highly conserved whereas murine lung samples fall into three distinct patterns. 33% of the samples were sterile, as they lacked culturable bacteria and their bacterial DNA content did not differ from background. 9% of samples contained comparatively higher amounts of bacterial DNA whose composition mimicked that detected in the URT. A final group (58%) contained smaller amounts of microbial DNA whose composition was correlating to that of rodent chow and cage bedding, likely acquired by inspiration of food and bedding fragments. By analyzing each sample independently rather than working with group averages, this work eliminated the bias introduced by aspiration-contaminated samples to establish that three distinct microbiome pneumotypes are present in normal murine lungs.

Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature.

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.

Suppressive oligonucleotides inhibit inflammation in a murine model of mechanical ventilator induced lung injury.

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) <10 mL/kg] can injure the lungs and induce an inflammatory response. This work explores the effect of treatment with suppressive oligonucleotides (Sup ODN) in a mouse model of ventilator-induced lung injury (VILI). METHODS: Balb/cJ mice were mechanically ventilated for 4 h using clinically relevant Vt and a positive end-expiratory pressure of 3 cmH2O 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.

Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer.

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.

18:1/18:1-Dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome.

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-ß1 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.

Topical application of phosphatidyl-inositol-3,5-bisphosphate for acute lung injury in neonatal swine.

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-ß1) 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.

Inositol-trisphosphate reduces alveolar apoptosis and pulmonary edema in neonatal lung injury.

D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-ß1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.