Riva Eydelman

Azithromycin Suppresses Activation of Nuclear Factor-kappa B and Synthesis of Pro-inflammatory Cytokines in Tracheal Aspirate Cells From Premature Infants

Nuclear factor-kappaB (NF-κB) plays a central role in regulating key proinflammatory mediators. The activation of NF-κB is increased in tracheal aspirate (TA) cells from premature infants developing bronchopulmonary dysplasia (BPD). We studied the effect of azithromycin (AZM) on the suppression of NF-κB activation and the synthesis of pro-inflammatory cytokines IL-6 and IL-8 by TA cells obtained from premature infants. Tracheal aspirate cells were stimulated with tumor necrosis factor-alpha (TNF-α) and incubated with AZM. The nuclear NF-κB-DNA binding activity, the levels of inhibitory kappaB-alpha (IκB-α) in the cytoplasmic fraction and IL-6 and IL-8 release in the cell culture media were measured. Stimulation of TA cells by TNF-α increased the activation of NF-κB, which was suppressed by the addition of AZM. Increased activation of NF-κB was also associated with increased levels of pro-inflammatory cytokines (IL-6 and IL-8). AZM significantly reduced the IL-6 and IL-8 production to the levels similar to control. TNF-α stimulation also increased the degradation of IκB-α, which was restored with the addition of AZM. Our data suggest that AZM therapy may be an effective alternative to steroids in reducing lung inflammation and prevention of BPD in ventilated premature infants.

Angiopoietin 2 concentrations in infants developing bronchopulmonary dysplasia: attenuation by dexamethasone

Objectives:To study the association between angiopoietin 2 (Ang2) concentrations in tracheal aspirates (TAs) and adverse outcome (bronchopulmonary dysplasia (BPD)/death) in ventilated premature infants (VPIs) and modulation of Ang2 concentrations with dexamethasone (Dex) use.Study Design:Serial TA samples were collected on days 1, 3, 5 and 7, and Ang2 concentrations were measured. Ang2 TA concentrations were compared prior to and after 48 to 72 h of using Dex.Result:A total of 151 TA samples were collected from 60 VPIs. BPD was defined as the oxygen requirement at 36 weeks postmenstrual age (PMA). Twelve infants (mean±s.d.) (gestational age (GA) 26.5±2.1 weeks, birth weight (BW) 913±230 g) had no BPD, 32 infants (GA 25.8±1.4 weeks, BW 768±157 g) developed BPD and 16 infants (GA 24.5±1.1 weeks, BW 710±143 g) died before 36 weeks PMA. Ang2 concentrations were significantly lower in infants with no BPD (median, 25th and 75th percentile) (157, 16 and 218 pg mg−1) compared with those who developed BPD (234, 138 and 338 pg mg−1, P=0.03) or BPD and/or death (234, 157 and 347 pg mg−1, P=0.017), in the first week of life. Twenty-six VPIs (BW 719±136 g, GA 25.1±1.3 weeks) received 27 courses of Dex. Ang2 concentrations before starting Dex were 202, 137 and 278 pg mg−1 and significantly decreased to 144, 0 and 224 pg mg−1 after therapy (P=0.007).Conclusions:Higher Ang2 concentrations in TAs are associated with the development of BPD or death in VPIs. Dex use suppressed Ang2 concentrations.

An extraperitoneal isolated vascularized bone marrow transplant model in the rat.

An isolated vascularized bone marrow transplant (iVBMT) model was previously developed in the rat to specifically study the role of bone marrow and its environment in a composite tissue allotransplant. An extraperitoneal model was successfully created to avoid laparotomy and cross-clamping of the great vessels. The extraperitoneal iVBMT model consisted of a left donor femur that was harvested with its nutrient vessels, anastomosed to the right femoral vessels in a syngeneic host, and then placed subcutaneously in the abdominal wall. At explant, the graft vessels were grossly patent, and histology of the graft bones showed a viable marrow compartment. Polymerase chain reaction demonstrated peripheral chimerism in the recipients. This model is technically simple with minimal morbidity in the recipient animals. By using the iVBMT, future studies across semiallogeneic and allogeneic barriers will help define the role of the bone marrow compartment in composite tissue allotransplants to potentially induce immune tolerance.

Impact of histological chorioamnionitis on tracheal aspirate cytokines in premature infants.

BACKGROUND Histological chorioamnionitis (CHORIO) may increase inflammatory mediators in the lungs of preterm infants. OBJECTIVE To study the impact of CHORIO on tracheal aspirate (TA) cytokines in ventilated infants. DESIGN/METHODS TA samples collected within 48 hours after birth from 40 ventilated neonates (gestational age [GA] <30 weeks, body weight [BW] <1250 g) were analyzed. Levels of 12 cytokines (interleukin [IL]-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, epidermal growth factor [EGF], interferon-γ [IFN-γ], monocyte chemotactic protein-1 [MCP-1], tumor necrosis factor-α [TNF-α], vascular endothelial growth factor [VEGF]) were measured using a biochip multianalyte immunoassay (Randox Laboratories, Antrim, UK). Total protein was measured by the Bradford assay. CHORIO assessment was done by a blinded pathologist. RESULTS Twenty-six infants (GA 26.6 ± 1.4 weeks, BW 852 ± 162 g) had no CHORIO and 14 (GA 25.1 ± 1.0 weeks, BW 776 ± 164 g) had CHORIO. IL-1α, IL-1β, IL-8, and VEGF were significantly higher in TA of infants with CHORIO. After correction for dilution, IL-1α, IL-1β, and IL-8 were significantly elevated. Increased TA total cell count correlated with CHORIO, VEGF, EGF, MCP-1, IL-8, and IL-6 TA levels (all p ≤ 0.02). Ventilator, oxygen supplementation, and hospital days correlated with TA IFN-γ levels (all p ≤ 0.01). CONCLUSION CHORIO is associated with increased specific proinflammatory mediators in TA samples of preterm infants.

High-mobility group box-1 protein in tracheal aspirates from premature infants: relationship with bronchopulmonary dysplasia and steroid therapy

Objective:High-mobility group box-1 (HMGB1) is a potent inflammatory mediator and contributes to acute lung injury in adults. The role of HMGB1 in neonatal lung injury and the development of bronchopulmonary dysplasia (BPD) is unknown. We studied the association between HMGB1 levels in tracheal aspirates (TAs) and adverse outcomes (BPD/death) in ventilated premature infants (VPIs) and modulation of HMGB1 levels with dexamethasone (Dex) use.Study Design:Infants born before 32 weeks gestation and requiring mechanical ventilation were enrolled. Serial TA samples were collected on days 1, 3, 5 and 7 and HMGB1 levels were measured. HMGB1 levels in TA samples were compared between infants with no BPD and infants who developed BPD or died. HMGB1 TA levels were also compared before and after using Dex.Result:In all, 24 infants (gestational age 26.4±1.9 weeks, birth weight 859±200 g) had no BPD, 60 infants (gestational age 25.4±1.8 weeks, birth weight 749±156 g) developed BPD or died before 36 weeks postmenstrual age. Mean HMGB1 level in first week of life was significantly lower in infants with no BPD (27.3±16.5 ng mg–1) compared with those who developed BPD or died (45.1±30.9 ng mg–1, P=0.004). In total, 29 VPIs received Dex. There was no significant change in HMGB1 levels with steroid therapy (before 47.0±43.9, after 60.1.5±58.8, P=0.3).Conclusion:Our data suggest that higher HMGB1 levels in TA are associated with the development of BPD or death in VPI. Dex use had no effect on HMGB1 levels.

Dexamethasone Suppresses Expression of Nuclear Factor-kappaB in the Cells of Tracheobronchial Lavage Fluid in Premature Neonates with Respiratory Distress

Nuclear Factor-kappaB (NF-κB) plays a central role in regulating the key mediators of inflammation involved in acute lung injury. The anti-inflammatory effect of steroids by suppressing pro-inflammatory cytokines may be mediated by inhibition of transcription factor NF-κB. The objective of this study was to determine the effect of glucocorticoid therapy on the expression of NF-κB in the cells of tracheobronchial lavage fluid (TBLF) in premature neonates with respiratory distress. Nineteen premature neonates requiring mechanical ventilation and receiving glucocorticoids were enrolled. Their gestational age (mean ± SD) was 25.0 ± 1.2 wk, birth weight 714 ± 105 g and age of starting dexamethasone was 33 ± 15 d. Tracheobronchial lavage fluid was collected before and 48–72 h after starting dexamethasone. NF-κB expression was measured by immunocytochemistry using mouse MAb against the p65 subunit of NF-κB on cytospin slides. The percent of cells stained and the intensity staining index were significantly higher before starting dexamethasone compared with after steroid therapy. Localization of NF-κB was significantly decreased in the cytoplasm and nuclei of mononuclear cells after initiation of dexamethasone therapy. The concentration of IL-8 was also significantly lower after starting dexamethasone. In conclusion, dexamethasone suppressed the expression of NF-κB in the cytoplasm and nuclei of mononuclear cells and decreased levels of IL-8 in TBLF from premature neonates with respiratory distress. The anti-inflammatory effects of corticosteroids may be mediated through NF-κB.

Absence of graft-versus-host disease in the isolated vascularized bone marrow transplant.

An isolated vascularized bone marrow transplant (iVBMT) model was developed to study the contribution of the bone marrow component in a composite tissue allograft. We hypothesized that the iVBMT would be functional and cause graft-versus-host disease (GVHD) in a fraction of the recipients. Lewis iVBMT grafts were transplanted to Lewis-Brown Norway recipients. Animals were sacrificed at various times from 1 to 14 weeks. Polymerase chain reaction for microchimerism was performed on the hosts marrow. No animals exhibited signs of GVHD at death. Histologic examination of the grafts showed a normal mix of hematopoietic and fatty elements and appeared to be functional. Tissues usually affected-tongue, ear, liver, and gut-also showed no evidence of disease. Polymerase chain reaction demonstrated microchimerism in both groups. These findings suggest that the vascularized bone marrow within a composite tissue allograft is not the component that causes GVHD; rather, it may serve an immunomodulatory function for tolerance induction.

IFN-γ and IP-10 in tracheal aspirates from premature infants: Relationship with bronchopulmonary dysplasia†‡

Interferon‐gamma (IFN‐γ) and interferon‐inducible protein of 10 kDa (IP‐10) are potent inflammatory mediators and contribute to acute lung injury in adults. Recently, a potential role for IFN‐γ and IP‐10 in the pathogenesis of bronchopulmonary dysplasia (BPD) has been reported in animal models.

Sirtuin1 in tracheal aspirate leukocytes: possible role in the development of bronchopulmonary dysplasia in premature infants.

Objective: To study the association between Sirtuin1 (Sirt1), a class III histone deacetylator, in tracheal aspirate (TA) leukocytes and the development of bronchopulmonary dysplasia (BPD) in premature infants and modulation of Sirt1 with dexamethasone (Dex) use. Design/methods: Serial TA samples were collected on days 1, 3, 5 and 7 from ventilated premature neonates. Sirt1 was localized by immunocytochemistry and quantified on a scale of 0–4 by blinded observers. BPD was defined as the need of supplemental oxygen at 36 weeks postmenstrual age (PMA). Results: A total of 130 TA samples were collected from 51 infants (mean ± SD: GA 25.5 ± 1.4 w, BW 762 ± 174 g). Eleven infants survived without BPD and 40 infants died before 36 weeks PMA or developed BPD. Sirt1 was localized in the cytoplasm and nuclei of mononuclear (MONO) as well as polymorphonuclear cells. Sirt1 was significantly more localized in the nuclei of MONO cells in infants without BPD compared to infants who developed BPD or died before 36 weeks PMA. Twenty six infants received Dex. There was no significant change in Sirt1 localization with steroid therapy. Conclusions: Lower Sirt1 in TA leukocytes is associated with the development of BPD or death in premature infants. Dex use had no effect on Sirt1.