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Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

AIM Neonatal haemolysis increases bilirubin production rates, which can then lead to severe hyperbilirubinaemia and bilirubin neurotoxicity. During haemolysis, haem is degraded by haem oxygenase (HO), which can be induced under stress conditions. It is known that neonatal sepsis is a risk factor for haemolysis and severe hyperbilirubinaemia. Here, we evaluated whether an exposure to lipopolysaccharide (LPS) to induce sepsis can upregulate HO-1, further increasing in vivo bilirubin production rates in mouse pups under haem loads. METHODS Three-day-old pups were given LPS (1250 μg/kg) or saline (controls). Liver HO enzyme activity, HO-1 mRNA and HO-1 protein were measured post-LPS exposure. We then assessed the effects of LPS treatment on in vivo bilirubin production rates after haem loading. RESULTS Liver HO activity significantly increased (142%) over controls 24 hours after treatment with LPS (1250 μg/kg) without mortality. Liver HO-1 mRNA was significantly upregulated 2.47-fold at 24 hours post-LPS administration, while liver HO-1 protein increased 1.29-fold 24 hours post-LPS treatment. After haem loading, pups exposed to LPS had significantly higher bilirubin production rates (1.30-fold) compared with age-matched, saline-treated controls. CONCLUSION Low-dose LPS treatment can upregulate liver HO-1 expression and may underlie the severe hyperbilirubinaemia seen in septic infants, particularly when undergoing haemolysis.

Background: Endogenous carbon monoxide (CO) production is primarily due to heme degradation, which also results in the equimolar production of bilirubin. Thus, estimates of total body CO production can serve as indices of total body bilirubin formation. The elimination rate of CO from a person’s body (CO washout rate) after exposure to an elevated ambient CO concentration is determined by a variety of factors, and is very different between babies and adults. Objective: We determined CO washout rates for babies using a simplified technique to measure total body CO excretion rates (VeCO). Methods: Using a simplified technique, we measured the times to reach an approximate steady state after a change in ambient CO concentration (decay time constant) and CO washout rates in normal newborn infants using non-linear least squares curve fitting. Results: We found a mean CO washout time of 18.7 ± 4.2 min and a CO equilibration (decay time) constant of 0.12 ± 0.04/min (0.08–0.21) for newborn infants. Conclusions: We confirm that CO washout rates for babies are much faster than those for adults. Therefore, measurements of carboxyhemoglobin (COHb) or end-tidal CO (ETCO), corrected for ambient CO, (COHbc and ETCOc, respectively) can be used as surrogates for VeCO and can provide accurate estimates of endogenous CO (VCO) and bilirubin production rates under normal environmental conditions. Such measurements can be used to identify infants with severe hyperbilirubinemia due to hemolysis and thus at high risk for bilirubin neurotoxicity.

Oxygen levels in the placental microenvironment throughout gestation are not constant, with severe hypoxic conditions present during the first trimester. This hypoxic phase overlaps with the most critical stages of placental development, i.e., blastocyst implantation, cytotrophoblast invasion, and spiral artery remodeling initiation. Dysregulation of any of these steps in early gestation can result in pregnancy loss and/or adverse pregnancy outcomes. Hypoxia has been shown to regulate not only the self-renewal, proliferation, and differentiation of trophoblast stem cells and progenitor cells, but also the recruitment, phenotype, and function of maternal immune cells. In this review, we will summarize how oxygen levels in early placental development determine the survival, fate, and function of several important cell types, e.g., trophoblast stem cells, extravillous trophoblasts, syncytiotrophoblasts, uterine natural killer cells, Hofbauer cells, and decidual macrophages. We will also discuss the cellular mechanisms used to cope with low oxygen tensions, such as the induction of hypoxia-inducible factor (HIF) or mammalian target of rapamycin (mTOR) signals, regulation of the metabolic pathway, and adaptation to autophagy. Understanding the beneficial roles of hypoxia in early placental development will provide insights into the root cause(s) of some pregnancy disorders, such as spontaneous abortion, preeclampsia, and intrauterine growth restriction.

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell–cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

Extreme hyperbilirubinemia can cause bilirubin neurotoxicity. Infants with glucose-6-phosphate dehydrogenase (G6PD) deficiency can develop hemolysis and thus are at high risk. We evaluated a device that quantitatively measures G6PD activity kinetically using digital microfluidics (DMF). Intra- and inter-instrument and -day imprecision (CVs) were first assessed. G6PD activity in 86 samples was then measured and compared between DMF and 2 reference methods. Overall DMF reproducibility was 3.8% over 5 days by 2 operators on 2 instruments. Mean intra- and inter-instrument variabilities were 3.6% and 3.9%, respectively (n = 28), with a user variability of 4.3%. Mean G6PD activity was 6.40±4.62 and 6.37±4.62 U/g hemoglobin for DMF and Reference Methods 1 (n = 46) and 12.15±3.86 and 11.48±1.55 for DMF and 2 (n = 40), respectively, and strongly correlated (r = 0.95 and 0.95) with mean biases of +0.04±2.90 and +0.67±1.55 for methods 1 and 2, respectively. The novel device could be used for early newborn G6PD screening.

Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 mins by using stable isotope-labeled ceramides as internal standards (ISs). This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring (MRM) for mass spectrometric detection. To qualify for clinical applications, our assay was validated against the FDA guidelines: the Lower Limit of Quantitation (LLOQ as low as 1 nM), linearity (R2>0.99), precision (Coefficient of Variation90%), stability (>85%), and carryover (

Bilirubin is the end product of heme catabolism formed during a process that involves oxidation-reduction reactions and conserves iron body stores. Unconjugated hyperbilirubinemia is common in newborn infants, but rare later in life. The basic physiology of bilirubin metabolism, such as production, transport, and excretion, has been well described. However, in the neonate, numerous variables related to nutrition, ethnicity, and genetic variants at several metabolic steps may be superimposed on the normal physiological hyperbilirubinemia that occurs in the first week of life and results in bilirubin levels that may be toxic to the brain. Bilirubin exists in several isomeric forms that differ in their polarities and is considered a physiologically important antioxidant. Here we review the chemistry of the bilirubin molecule and its metabolism in the body with a particular focus on the processes that impact the newborn infant, and how differences relative to older children and adults contribute to the risk of developing both acute and long-term neurological sequelae in the newborn infant. The final section deals with the interplay between the brain and bilirubin and its entry, clearance, and accumulation. We conclude with a discussion of the current state of knowledge regarding the mechanism(s) of bilirubin neurotoxicity.

Purpose:PI3K signaling is a common feature of B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL), and PI3K inhibitors have been introduced into the clinic. However, there remains a clear need to develop new strategies to target PI3K signaling. PI3K activity is countered by Src homology domain 2-containing inositol-5′-phosphatase 1 (SHIP1) and, here, we have characterized the activity of a novel SHIP1 activator, AQX-435, in preclinical models of B-cell malignancies.Experimental Design:In vitro activity of AQX-435 was evaluated using primary CLL cells and DLBCL-derived cell lines. In vivo activity of AQX-435, alone or in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, was assessed using DLBCL cell line and patient-derived xenograft models.Results:Pharmacologic activation of SHIP1 using AQX-435 was sufficient to inhibit anti–IgM-induced PI3K-mediated signaling, including induction of AKT phosphorylation and MYC expression, without effects on upstream SYK phosphorylation. AQX-435 also cooperated with the BTK inhibitor ibrutinib to enhance inhibition of anti–IgM-induced AKT phosphorylation. AQX-435 induced caspase-dependent apoptosis of CLL cells preferentially as compared with normal B cells, and overcame in vitro survival-promoting effects of microenvironmental stimuli. Finally, AQX-435 reduced AKT phosphorylation and growth of DLBCL in vivo and cooperated with ibrutinib for tumor growth inhibition.Conclusions:Our results using AQX-435 demonstrate that SHIP1 activation may be an effective novel therapeutic strategy for treatment of B-cell neoplasms, alone or in combination with ibrutinib.

Secondary phosphorylation develops in myocytes expressing phospho-mimetic cardiac troponin I (cTnI) but it is not known whether multiple substitutions (e.g. cTnISDTD and cTnIS4D) cause preferential phosphorylation of the remaining endogenous or the phospho-mimetic cTnI in intact myocytes. Western analysis was performed to determine whether the FLAG/total cTnI ratios are similar for phosphorylated versus total cTnI in myocytes expressing phospho-mimetic cTnI with Asp(D) substitutions at S43/45 plus S23/24 (cTnIS4D) or T144 (cTnISDTD). Representative Western analysis of phosphorylated S23/24 (p-S23/24) and S150 (p-S150) are presented along with re-probes using an antibody which detects all cTnI (MAB1691 Ab). The level of p-S150 also is compared to results obtained using single S43D and/or S45D phospho-mimetic substitutions. These results are discussed in more detail in Lang et al. [1].

Purpose: In chronic lymphocytic leukemia (CLL), disease progression associates with surface IgM (sIgM) levels and signaling capacity. These are variably downmodulated in vivo and recover in vitro, suggesting a reversible influence of tissue-located antigen. Therapeutic targeting of sIgM function via ibrutinib, an inhibitor of Bruton tyrosine kinase (BTK), causes inhibition and tumor cell redistribution into the blood, with significant clinical benefit. Circulating CLL cells persist in an inhibited state, offering a tool to investigate the effects of drug on BTK-inhibited sIgM. Experimental Design: We investigated the consequences of ibrutinib therapy on levels and function of sIgM in circulating leukemic cells of patients with CLL. Results: At week 1, there was a significant increase of sIgM expression (64% increase from pretherapy) on CLL cells either recently released from tissue or persisting in blood. In contrast, surface IgD (sIgD) and a range of other receptors did not change. SIgM levels remained higher than pretherapy in the following 3 months despite gradual cell size reduction and ongoing autophagy and apoptotic activity. Conversely, IgD and other receptors did not increase and gradually declined. Recovered sIgM was fully N-glycosylated, another feature of escape from antigen, and expression did not increase further during culture in vitro. The sIgM was fully capable of mediating phosphorylation of SYK, which lies upstream of BTK in the B-cell receptor pathway. Conclusions: This specific IgM increase in patients underpins the key role of tissue-based engagement with antigen in CLL, confirms the inhibitory action of ibrutinib, and reveals dynamic adaptability of CLL cells to precision monotherapy. See related commentary by Burger, p. 2372

The action spectrum for bilirubin photodegradation has been intensively studied. However, questions still remain regarding which light wavelength most efficiently photodegrades bilirubin. In this study, we determined the in vitro effects of different irradiation wavelength ranges on bilirubin photodegradation. In our in vitro method, normalized absolute irradiance levels of 4.2 × 1015 photons/cm2/s from light-emitting diodes (ranging from 390–530 nm) and 10-nm band-pass filters were used to irradiate bilirubin solutions (25 mg/dL in 4% human serum albumin). Bilirubin and its major photoisomer concentrations were determined; the half-life time of bilirubin (t1/2) was calculated for each wavelength range, and the spectral characteristics for bilirubin photodegradation products were obtained for key wavelengths. The in vitro photodegradation of bilirubin at 37 °C decreased linearly as the wavelength was increased from 390 to 500 nm with t1/2 decreasing from 63 to 17 min, respectively. At 460 ± 10 nm, a significantly lower rate of photodegradation and thus higher t1/2 (31 min) than that at 500 nm (17 min) was demonstrated. In our system, the optimum bilirubin photodegradation and lumirubin production rates occurred between 490 and 500 nm. Spectra shapes were remarkably similar, suggesting that lumirubin production was the major process of bilirubin photodegradation.

Population-based biorepositories are important resources, but sample handling can affect data quality. Identify metabolites of value for clinical investigations despite extended postcollection freezing delays, using protocols representing a California mid-term pregnancy biobank. Blood collected from non-pregnant healthy female volunteers (n = 20) underwent three handling protocols after 30 min clotting at room temperature: (1) ideal—samples frozen (− 80 °C) within 2 h of collection; (2) delayed freezing—samples held at room temperature for 3 days, then 4 °C for 9 days, the median times for biobank samples, and then frozen; (3) delayed freezing with freeze–thaw—the delayed freezing protocol with a freeze–thaw cycle simulating retrieved sample sub-aliquoting. Mass spectrometry-based untargeted metabolomic analyses of primary metabolism and complex lipids and targeted profiling of oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were performed. Metabolite concentrations and intraclass correlation coefficients (ICC) were compared, with the ideal protocol as the reference. Sixty-two percent of 428 identified compounds had good to excellent ICCs, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze–thaw effects were assay-specific with lipids being most stable. Despite extended post-collection freezing delays characteristic of some biobanks of opportunistically collected clinical samples, numerous metabolomic compounds had both stable levels and good concordance.

Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 min. by using stable isotope-labeled ceramides as internal standards. This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring for mass spectrometric detection. To qualify for clinical applications, our assay has been validated against the FDA guidelines for Lower Limit of Quantitation (1 nM), linearity (R2>0.99), precision (imprecision 90 %), stability (>85 %), and carryover (

A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca2+ transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.

Phototherapy has been used to treat newborns with jaundice for more than 50 years with the presumption that it is safe and effective for all infants. In fact, this presumption may not be true for all infants, especially the smallest and most immature. The safety and efficacy of phototherapy have never really been questioned or adequately tested in the latter, yet clinical applications of phototherapy have been further refined as its mechanisms of action have been better understood and alternative light sources have become available. This article addresses what is known about the possible risks of photo-oxidative injury in extremely low birth weight infants.

Aim Relative contributions of increased production [by end-tidal carbon monoxide concentrations (ETCOc)] and decreased elimination of bilirubin to predischarge hour-specific total bilirubin (TB) levels were assessed in healthy late-preterm and term newborns. Secondly, we report predischarge ETCOc ranges to guide clinical management of hyperbilirubinemia. Methods TB and ETCOc (≤3 timepoints) determinations of newborns aged between six hours and 1.5 (p < 0.00003). Conclusion Near-simultaneous ETCOc and TB measurements in infants with TB >75th percentile accurately identify haemolytic hyperbilirubinemia.

The circulating tumor cells of chronic lymphocytic leukemia (CLL) are characterized by variably low surface IgM (sIgM) levels and signaling capacity, consequent to stimulation by tissue-based antigen. The variability is evident in both CLL subsets with unmutated and mutated immunoglobulin genes and affects tumor cell survival and proliferation. This appears clinically important because CLL with relatively higher sIgM levels/signaling capacity progress more rapidly than those with lower sIgM levels/signaling capacity (D'Avola, Blood 2016), likely due to a larger proliferative component at tissue sites. B-cell receptor (BCR)-signaling inhibitor ibrutinib exerts its therapeutic activity in CLL by irreversibly binding the Bruton's tyrosine kinase (BTK) at Cys481 in the active site. This leads to redistribution of CLL cells from tissue sites into peripheral blood and to a selective recovery of sIgM levels and function, consequent to release from antigen drive at tissue sites (Drennan, Clin Cancer Res 2019). We have now investigated the hypothesis that sIgM levels on the CLL cells affect duration of response to ibrutinib therapy possibly consequent to circumvention of the pharmacological BTK inhibition. Seventy CLL patients participating in the "real-world" observational study at the University of Southampton (NIHR/UKCRN ID: 31076) were investigated for phenotypic, molecular and functional characteristics associated with response to single-agent ibrutinib. Time to progression requiring a new treatment from ibrutinib start (TTNT) was used as primary endpoint to measure duration of response. The levels of sIgM were measured by flow cytometry. Anti-IgM induced signaling was determined by immunoblotting or iCa2+ mobilization. BTK and PLCγ2 mutations were determined using the Illumina NexteraXT kit and a MiSeq at the University of Southampton and validated independently at Karolinska Institutet. Our previous cut-offs of 50 (MFI) or 5 (iCa2+ % mobilization) were used to distinguish patients with high/low sIgM levels or signaling capacity, respectively. Median follow-up from ibrutinib start was 42 months (range 12-70). Pre-ibrutinib sIgM levels (range 7-618, median 65; high sIgM MFI 46/70, 66%) and sIgM signaling capacity (range 1-98; median 45; high sIgM signalers 56/70, 80%) were broadly heterogeneous. However, median values were significantly higher than the general CLL population at diagnosis, as expected in progressive CLL. By univariate analyses of clinical, phenotypic, FISH and immunogenetic characteristics for TTNT, high sIgM was the only parameter predicting shorter duration of response to ibrutinib (p=0.02). Only 2/24 CLL with low sIgM levels (CLLIgM_lo) had progressed (median TTNT not reached) compared to 15/46 CLL with high sIgM (CLLIgM_hi, 14% at 12 months, 18% at 24 months, 34% at 36 months, 44% at 42 months) during therapy. The levels of sIgM correlated significantly with sIgM signaling capacity prior to treatment (r= 0.68; p From in vitro analysis of 13 therapy-naive CLL samples, the degree of inhibition by ibrutinib of anti-IgM induced signals downstream to BTK correlated negatively with sIgM levels (r=-0.68,p=0.01) and signaling (r=-0.71,p=0.009). Anti-IgM induced signals were then measured in 12 patients (7 CLLIgM_hi, 5 CLLIgM_lo) after 1, 4 and 12 weeks of ibrutinib therapy. These patients had not acquired BTK or PLCγ2 mutations and BTK phosphorylation appeared completely inhibited at all time-points. Mean anti-IgM induced iCa2+ mobilization and ERK1/2 phosphorylation were significantly reduced but not abolished during therapy and degree of inhibition was variable between individuals. Remarkably, degree of inhibition of anti-IgM-induced iCa2+ mobilization and pERK1/2 was significantly lower in CLLIgM_hi than in CLLIgM_lo (p These results confirm that sIgM signaling is dependent on sIgM levels and that it can circumvent BTK blockade when sIgM levels are high. They suggest that high sIgM signaling can drive ibrutinib resistance despite ability of ibrutinib to fully occupy the BTK phosphorylation pocket. A possibility is that those CLL cells with high sIgM represent a potentially dangerous subpopulation equipped to migrate to tissue and to receive proliferative stimuli. These cells might be targeted by a combinatorial therapeutic approach with ibrutinib. Disclosures Johnson: Takeda: Honoraria; Kite: Honoraria; Bristol-Myers Squibb: Honoraria; Genmab: Honoraria; Incyte: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria; Epizyme: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria. Duncombe:Abbvie: Other: Advisory Board membership;Educational support; Gilead: Other: Advisory Board membership; Janssen: Other: Advisory Board membership;Educational support; Novartis: Other: Advisory Board membership. Scarfo:Janssen: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria. Sutton:Gilead: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Ghia:Pharmacyclics LLC, an AbbVie Company: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; ArQule: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy, Honoraria; Novartis: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding. Forconi:Roche: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Gilead Sciences: Research Funding.

Background: Increased bilirubin production due to hemolysis can lead to severe neonatal hyperbilirubinemia and, if left untreated, to bilirubin neurotoxicity. Post-cardiac surgery newborns have been shown to be at an increased risk for developing hyperbilirubinemia and also hemolysis. Isoflurane (ISO), a volatile anesthetic agent routinely used in newborn surgery, has been reported to upregulate heme oxygenase 1 (HO-1) expression. HO is the rate-limiting enzyme in the bilirubin production pathway. Objective: Here, we evaluated whether ISO exposure induces HO-1 and further increases bilirubin production in a hemolytic newborn mouse model. Methods: Three-day-old newborn mice were exposed to 2% ISO for 18 min or air. Liver HO activity and HO-1 protein were measured after exposure to ISO. Next, we evaluated the effect of ISO exposure on bilirubin production as indexed by the total body excretion rate of carbon monoxide following heme loading. Results: ISO significantly increased liver HO activity 120% and 116% at 24 and 48 h, respectively, after exposure. HO-1 protein levels also similarly increased after ISO exposure, but the increases were not statistically significant compared with controls. After heme loading, ISO-exposed pups had significantly higher bilirubin production rates (1.24-fold), and also peaked earlier, than age-matched nonexposed pups. Conclusions: ISO exposure can induce HO-1 expression in the liver and may explain the development of severe hyperbilirubinemia in postsurgical infants, especially in those undergoing hemolysis.

The risk of developing treatment-warranted Type 1 retinopathy of prematurity (ROP) might be reduced in preterm infants by modifying certain systemic factors. There are steps that can be taken both early and late in the course of retinal vascular maturation that may potentially reduce an infant's risk of developing Type 1 ROP. In prethreshold stage 2-3 ROP without plus disease, a combination of supplemental oxygen, correction of severe anemia, and light adaptation to reduce rod photoreceptor oxygen consumption helped us to reduce ROP severity, and encouraged a return to a more physiologic retinal vascular maturation pattern. Thus, it may be possible to reduce the risk of developing Type 1 ROP by making adjustments in certain systemic parameters aimed at reducing retinal hypoxia, thereby gently lowering pathologically elevated levels of vascular endothelial growth factor (VEGF) within the eye.

Phototherapy was introduced in the early 1950’s, and is the primary treatment of severe neonatal jaundice or Crigler-Najjar syndrome. Nevertheless, the potential biological effects of the products generated from the photodegradation of bilirubin during phototherapy remain unknown. This is very relevant in light of recent clinical observations demonstrating that the use of aggressive phototherapy can increase morbidity or even mortality, in extremely low birthweight (ELBW) infants. The aim of our study was to investigate the effects of bilirubin, lumirubin (LR, its major photo-oxidative product), and BOX A and B (its monopyrrolic oxidative products) on the central nervous system (CNS) using in vitro and ex vivo experimental models. The effects of bilirubin photoproducts on cell viability and expression of selected genes were tested in human fibroblasts, three human CNS cell lines (neuroblastoma SH-SY5Y, microglial HMC3, and glioblastoma U-87 cell lines), and organotypic rat hippocampal slices. Neither bilirubin nor its photo-oxidative products affected cell viability in any of our models. In contrast, LR in biologically-relevant concentrations (25 μM) significantly increased gene expression of several pro-inflammatory genes as well as production of TNF-α in organotypic rat hippocampal slices. These findings might underlie the adverse outcomes observed in ELBW infants undergoing aggressive phototherapy.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzyme deficiency affecting more than 300 million individuals worldwide. Extreme neonatal hyperbilirubinemia, with its severe sequelae of bilirubin neurotoxicity and the potential of death, is the most devastating manifestation of G6PD deficiency. In a recent review of Favism, Luzzatto and Arese state that the pathophysiology of jaundice in G6PD-deficient neonates is different from that of favism, as there is little evidence of hemolysis in these infants. Objectives: To explore the role of hemolysis in neonatal hyperbilirubinemia associated with G6PD deficiency. Methods: Previously published works including studies of endogenous production of carbon monoxide (CO), an index of heme catabolism, in hyperbilirubinemic G6PD-deficient neonates were reviewed to determine the role of hemolysis in this condition. Results: Three studies demonstrated that endogenous CO production is elevated in G6PD-deficient neonates with extreme hyperbilirubinemia. Conclusions: Hemolysis is an important pathogenetic factor in G6PD deficiency-associated neonatal hyperbilirubinemia.

We performed an in vitro evaluation of the effect of maraviroc or dolutegravir on bilirubin to albumin binding. At typical treatment and low albumin concentrations, maraviroc had no impact, while dolutegravir affected bilirubin to albumin binding to an equivalent extent as sulfisoxazole. However in vivo, neither is likely to significantly impact bilirubin to albumin binding because of their low concentrations relative to albumin.

Problem Uterine and placental macrophages play critical roles in maintaining a normal pregnancy. The majority of these macrophages are believed to be alternatively activated macrophages (M2). Method of study Mouse bone marrow cells were differentiated into macrophages and polarized to M2 in vitro by treatment with IL-4 [M2a] or IL-10 [M2c] and M1 with LPS/IFN-γ as controls. Macrophage subtypes were confirmed by surface markers and characterized by gene expression profiles. Results Compared to M1, M2 showed strong pro-angiogenic activity by expressing higher mRNA for angiogenic-associated factors (Cxcl12, Vegfa, PlGF, Mmp2). M2c produced the highest levels of PlGF, Mmp2, and Cxcr4. To mimic the normal placental microenvironment, M2 were exposed to hypoxia and sex hormones (progesterone, estrogen). In both M2c and M2a, severe hypoxic (1%-3% O2 ) exposure significantly suppressed PlGF, Cxcl12, and Mmp2 mRNA, but not Vegfa, compared to normoxia (21% O2 ) or physiological hypoxia (5% O2 ). mRNA expression returned to normal when hypoxic cells were returned to normoxia. Hypoxia (1%) reduced antioxidant levels in M2 and re-exposure to normoxia significantly increased superoxide dismutase (Sod1, Sod2) and heme oxygenase-1 (HO-1) levels in M2a, and only glutathione peroxidase (Gpx1, Gpx3, Gpx4) in M2c. However, progesterone and estrogen treatment had minimal effects on angiogenic factor expression in M2. Conclusion M2, particularly M2c, displayed strong pro-angiogenic potential, which decreased under severe hypoxia such as in early pregnancy. Thus, conditions that alter the placental oxygen microenvironment or macrophage response in early pregnancy might cause aberrant angiogenesis and vascular remodeling, and lead to abnormal placental vascular development.

Sepsis in preterm infants is associated with systemic inflammatory responses. The stress-response protein heme oxygenase-1 (HO-1) has protective anti-inflammatory properties. Recently, we reported a protective role of HO-1 using our non-surgical cecal slurry (CS) model in wild-type (WT) mouse pups. Here, we extend these findings to investigate the association of HO-1 deficiency with sepsis severity. Adapting the Wynn model, we induced sepsis in 4-day-old HO-1-deficient (HO-1+/−, Het) pups to determine if HO-1 deficiency affected survival rates at the LD40 (2.0 mg/g) of WT pups. To see if HO-1 induction affected sepsis severity, we gave 30-μmol heme/kg subcutaneously to 3-day-old mice 24 h prior to sepsis induction. Post-sepsis induction, Het pups had a mortality of 85.0% (n = 20) and increased expression of the pro-inflammatory gene in the livers and affected hematologic profiles. Heme treatment 24 h prior to sepsis induction significantly increased liver HO activity, reduced mortality to 24.5% (n = 17), attenuated inflammatory responses, reduced spleen bacterial counts, and significantly increased peripheral neutrophils. A partial deficiency in HO-1 increased the progression and mortality in sepsis. Furthermore, induction of HO-1 significantly reduced the mortality even in Het pups. Thus, we conclude that HO-1 plays an important role in the protection against preterm sepsis.

AIM To identify neonates at risk of haemolytic hyperbilirubinaemia through near-concurrent measurements of total serum/plasma bilirubin (TB) or transcutaneous bilirubin (TcB) and end-tidal breath carbon monoxide (CO), corrected for ambient CO (ETCOc), an index of bilirubin production and haemolysis. METHODS Paired TB/TcB (mg/dL) and ETCOc (ppm) measurements were obtained in newborns (n = 283) at 20 to

Background: Bilirubin-induced neurologic dysfunction (BIND) is a spectrum of preventable neurological sequelae in jaundiced newborns. Current total plasma bilirubin (BT) concentration thresholds for phototherapy and/or exchange transfusion poorly predict BIND. Methods: The unbound (free) bilirubin (Bf) measured at these BT thresholds provides additional information about the risk for BIND. Bf can be readily adapted to clinical use by determining Bf population parameters at current BT thresholds. These parameters can be established using a plasma bilirubin binding panel (BBP) consisting of BT, Bf, and two empiric constants, the maximum BT (BTmax) and the corresponding equilibrium association bilirubin constant (K). Results: BTmax and K provide the variables needed to accurately estimate Bf at BT

Background Heme oxygenase (HO) is the rate-limiting enzyme in the heme degradation pathway that produces bilirubin. The promoter region of human heme oxygenase-1 (HMOX1) contains a polymorphic (GT)n repeat that can regulate gene expression. Here, we investigated the association of (GT)n repeat length in the HMOX1 promoter region with neonatal hyperbilirubinemia in a population of Japanese term neonates. Methods Using polymerase chain reaction and fragment analysis, we determined the number of (GT)n repeats in 149 Japanese neonates. To omit the effects of the G71R mutation in uridine diphosphoglucuronosyltransferase on hyperbilirubinemia, we excluded 41 neonates with the G71R mutation. As a result, 25 neonates with hyperbilirubinemia and 83 non-hyperbilirubinemic controls were included in this prospective case–control study. Allele and genotype frequencies of (GT)n repeats in the HMOX1 gene were compared between hyperbilirubinemic and non-hyperbilirubinemic control neonates. Results The prevalence of short alleles (

We assessed the relative contributions of increased bilirubin production (indexed by end-tidal carbon monoxide (CO) concentrations, corrected for ambient CO (ETCOc)) to hour-specific total bilirubin (TB) levels in healthy late preterm and term newborns. Post hoc analyses of concurrent ETCOc and TB (at 30±6 h of age) and follow-up TB levels at age 96±12 h and up to 168 h after birth were performed in a cohort of 641 term and late preterm infants. Increased bilirubin production (hour-specific ETCOc ⩾1.7 p.p.m. at age 30±6 h) was noted in ~80%, 42% and 32% of infants in the high-, intermediate- and low-risk TB zones, respectively. One infant with TB

Bilirubin binding capacity (BBC) defines the dynamic relationship between an infant’s level of unbound or “free” bilirubin and his/her ability to “tolerate” increasing bilirubin loads. BBC is not synonymous with albumin (Alb) levels because Alb binding of bilirubin is confounded by a variety of molecular, biologic, and metabolic factors. We utilized a novel modification of a previously developed hematofluorometric method to directly assay BBC in whole blood from preterm and term neonates and then combined these data with an archived database. Total bilirubin (TB) was also measured, and multiple regression modeling was used to determine whether BBC in combination with TB measurements can assess an infant’s risk for developing bilirubin-induced neurotoxicity. TB and BBC levels ranged from 0.7–22.8 to 6.3–47.5 mg/dl, respectively. Gestational age (GA) correlated with BBC (r = 0.54; P < 0.0002) with a slope of 0.93 mg/dl/wk by logistic regression. Our calculations demonstrate that recently recommended GA-modulated TB thresholds for phototherapy and exchange transfusion correspond to 45 and 67% saturation of our observed regression line, respectively. We speculate that the spread of BBC levels around the regression line (±5.8 mg/dl) suggests that individualized BBC assays would provide a robust approach to gauge risk of bilirubin neurotoxicity compared with TB and GA.

BACKGROUND AND OBJECTIVES: We recently demonstrated that direct antiglobulin titer (DAT) positive, blood group A or B newborns born to group O mothers had a high incidence of hyperbilirubinemia, attributable to increased hemolysis. We reanalyzed our data asking whether increasing DAT strength plays a modulating role in the pathophysiology of the hemolysis and hyperbilirubinemia. METHODS: Data from previously published DAT-positive, ABO-heterospecific neonates were analyzed for hyperbilirubinemia and hemolysis according to strength of DAT. DAT was measured by using a gel agglutination technique and reported as values ranging from DAT ± to DAT ++++. Hemolysis was evaluated by blood carboxyhemoglobin corrected for inspired, ambient CO (COHbc), and expressed as percent total hemoglobin (tHb). Hyperbilirubinemia was defined as any plasma total bilirubin value >95th percentile on the hour-specific nomogram. RESULTS: Hyperbilirubinemia was more prevalent in those with DAT ++ readings (16 of 20, 80%) than those both DAT ± (37 of 87 [42.5%], relative risk: 1.88, 95% confidence interval: 1.35–2.61) and DAT + (32 of 56 [57.1%], relative risk: 1.40, 95% confidence interval: 1.02–1.92). COHbc values were higher for those with DAT ++ (1.45 ± 0.49% tHb [mean ± SD]) than those DAT ± (1.20 ± 0.37% tHb, P = .01) or DAT + (1.22 ± 0.37% tHb, P = .02). CONCLUSIONS: DAT ++ readings were associated with a higher incidence of hyperbilirubinemia and higher COHbc values than DAT ± or DAT + counterparts. Increasing DAT strength may be a modulator of hemolysis and hyperbilirubinemia in ABO-heterospecific neonates. DAT strength, and not merely DAT presence or absence, should be taken into consideration in the management of ABO-heterospecific newborns.

Hyperbilirubinemia is a benign transitional phenomenon that occurs in 60% to 80% of all term infants. The degree of hyperbilirubinemia and hence risk for developing bilirubin-induced neurologic dysfunction or BIND is dependent upon two major processes: (i) bilirubin production and its elimination.The aim of this review is to address the importance of hemolysis and its clinical detection in neonates with hyperbilirubinemia.In newborns, an increased bilirubin production rate due to hemolysis is often the primary cause of hyperbilirubinemia during the first week of life. If undiagnosed or untreated, it may lead to an increased risk for BIND. Therefore, the ability to identify infants with hemolytic disease is important in assessing those at risk for developing BIND. In addition, an infant's genetic profile and bilirubin binding status can also affect their overall capacity to cope with the resultant tissue bilirubin load and affect risk and guide appropriate management strategies.Therefore, the determination of a newborn's bilirubin production rate is critical to the assessment of a newborn's risk for developing unpredictable extreme hyperbilirubinemia and preventing BIND.

Increased protein kinase C (PKC) activity is associated with heart failure, and can target multiple cardiac troponin I (cTnI) residues in myocytes, including S23/24, S43/45 and T144. In earlier studies, cTnI-S43D and/or -S45D augmented S23/24 and T144 phosphorylation, which suggested there is communication between clusters. This communication is now explored by evaluating the impact of phospho-mimetic cTnI S43/45D combined with S23/24D (cTnIS4D) or T144D (cTnISDTD). Gene transfer of epitope-tagged cTnIS4D and cTnISDTD into adult cardiac myocytes progressively replaced endogenous cTnI. Partial replacement with cTnISDTD or cTnIS4D accelerated the time to peak (TTP) shortening and time to 50% re-lengthening (TTR50%) on day 2, but peak shortening was only diminished by cTnIS4D. Extensive cTnIS4D replacement continued to accelerate TTP, and decrease shortening amplitude, while TTR50% returned to baseline levels on day 4. In contrast, cTnISDTD modestly reduced shortening amplitude and continued to accelerate myocyte TTP and TTR50%. These results indicate cTnIS43/45 communicates with S23/24 and T144, with S23/24 exacerbating and T144 attenuating the S43/45D-dependent functional deficit. In addition, more severe functional alterations in cTnIS4D myocytes were accompanied by higher levels of secondary phosphorylation compared to cTnISDTD. These results suggest that secondary phosphorylation helps to maintain steady-state contractile function during chronic cTnI phosphorylation at PKC sites.

Background: Severe hemolytic disease of the newborn leads to the release of pro-oxidative free heme (FH). Heme oxygenase (HO) is primarily responsible for detoxifying FH. Objective: To investigate the protective effects of HO in a model of heme overload. Methods: For in vitro studies, NIH3T3 HO-1-luc cells were incubated with 10, 30, or 60 µM FH or methemalbumin (MHA). HO-1 promoter activity was assessed 3, 6, and 24 h after treatment. Cell survival was indexed by viability assays. For in vivo studies, 1- and 5-week-old wild-type (Wt) or HO-1-heterozygous (Het, HO-1+/-) mice were given 60 µmol FH or MHA/kg intraperitoneally. After 24 h, plasma aspartate aminotransferease (AST)/alanine transaminase (ALT) and hemopexin, liver HO activity, and lipid peroxidation (LP) were determined. Results: In HO-1-luc cells, HO-1 promoter activity peaked 6 h after incubation with 30 µM FH (1.6-fold) or 60 µM MHA (2.1-fold) over baseline. Twenty-four hours after exposure to 60 µM FH, a decrease in viability of 80% was found, compared with no decrease after exposure to 60 µM MHA. In 1-week-old Wt and HO-1 Het pups given 60 µmol FH/kg, HO activity significantly increased 3.5- and 3.1-fold, respectively. No changes in LP or AST/ALT levels were observed. In adult Wt and HO-1 Het mice, HO activity increased (3.0- and 2.6-fold, respectively). LP and AST levels significantly increased 28.4- and 2.7-fold, respectively, in adult HO-1 Het mice. Hemopexin levels at baseline were higher in adults compared with newborns for both Wt and Het mice. In addition, FH induced hemopexin levels in both adults and newborns, but to a lesser degree in newborns. Conclusions: FH is highly toxic in vitro, but its toxicity is abolished when bound to albumin. Newborns appear to be protected from the pro-oxidative effects of FH, which may be mediated by heme binding and a higher absolute HO activity at baseline and after FH-mediated induction.

Purpose: B-cell receptor (BCR)–associated kinase inhibitors, such as ibrutinib, have revolutionized the treatment of chronic lymphocytic leukemia (CLL). However, these agents are not curative, and resistance is already emerging in a proportion of patients. IL4, expressed in CLL lymph nodes, can augment BCR signaling and reduce the effectiveness of BCR kinase inhibitors. Therefore, simultaneous targeting of the IL4- and BCR signaling pathways by cerdulatinib, a novel dual Syk/JAK inhibitor currently in clinical trials (NCT01994382), may improve treatment responses in patients. Experimental Design: PBMCs from patients with CLL were treated in vitro with cerdulatinib alone or in combination with venetoclax. Cell death, chemokine, and cell signaling assay were performed and analyzed by flow cytometry, immunoblotting, q-PCR, and ELISA as indicated. Results: At concentrations achievable in patients, cerdulatinib inhibited BCR- and IL4-induced downstream signaling in CLL cells using multiple readouts and prevented anti-IgM- and nurse-like cell (NLC)–mediated CCL3/CCL4 production. Cerdulatinib induced apoptosis of CLL cells, in a time- and concentration-dependent manner, and particularly in IGHV-unmutated samples with greater BCR signaling capacity and response to IL4, or samples expressing higher levels of sIgM, CD49d+, or ZAP70+. Cerdulatinib overcame anti-IgM, IL4/CD40L, or NLC-mediated protection by preventing upregulation of MCL-1 and BCL-XL; however, BCL-2 expression was unaffected. Furthermore, in samples treated with IL4/CD40L, cerdulatinib synergized with venetoclax in vitro to induce greater apoptosis than either drug alone. Conclusions: Cerdulatinib is a promising therapeutic for the treatment of CLL either alone or in combination with venetoclax, with the potential to target critical survival pathways in this currently incurable disease. Clin Cancer Res; 23(9); 2313–24. ©2016 AACR.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours. Electronic supplementary material The online version of this article (10.1007/s00262-017-2111-y) contains supplementary material, which is available to authorized users.

Blood circulates throughout the entire body and contains molecules drawn from virtually every tissue, including the microbes and viruses which colonize the body. Through massive shotgun sequencing of circulating cell-free DNA from the blood, we identified hundreds of new bacteria and viruses which represent previously unidentified members of the human microbiome. Analysing cumulative sequence data from 1,351 blood samples collected from 188 patients enabled us to assemble 7,190 contiguous regions (contigs) larger than 1 kbp, of which 3,761 are novel with little or no sequence homology in any existing databases. The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification. When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.

Nature Reviews Disease Primers 3, 16096 (2017) In the version of the article originally published, a typographical error has now been corrected. The statement now reads: Approval of ibrutinib as initial therapy was based on the results of a randomized trial that showed a significant improvement in median PFS and overall survival in patients.

Background: Heme oxygenase (HO) is the initial, rate-limiting enzyme in the conversion of heme to bilirubin. Dinucleotide (GT)n repeat length in the promoter region of the encoding gene modulates transcription: shorter alleles, in contrast with longer allele counterparts, are associated with greater gene expression and should result in increased heme catabolism. Objective: We compared the rates of heme catabolism and plasma total bilirubin (TB) between HO-1 promoter genotypes of varying (GT)n repeat lengths in glucose-6-phosphate dehydrogenase (G6PD)-normal and -deficient neonates. Methods:HO-1 promoter length was determined from genomic DNA from previous studies by size discrimination of fluorescently-labeled PCR products with capillary electrophoresis. Sizing was confirmed by sequencing homozygote samples. Alleles were categorized as: short (≤24 GT repeats), medium (25-33 GT repeats), and long (≥34 GT repeats). Previously determined values for blood carboxyhemoglobin, corrected for inspired carbon monoxide (COHbc), and TB were used to determine the rate of heme catabolism and 3rd day TB values for each HO-1 promoter length genotype, respectively. G6PD Mediterranean was determined by PCR analysis. Results: Neither COHbc nor TB values were significantly different between various HO-1 promoter genotypes for either G6PD-normal or -deficient neonates. Conclusions: In the steady state, HO-1 promoter genotypes, based on the length of (GT)n repeats, do not modulate heme catabolism or 3rd day TB values in either G6PD-normal or -deficient neonates.

The role of genetic factors in the modulation of serum bilirubin levels and the pathophysiology of neonatal hyperbilirubinemia is being increasingly recognized. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme by which heme is catabolized to biliverdin and thence to bilirubin, with the simultaneous release of equimolar quantities of ferrous iron (Fe3+) and carbon monoxide. Polymorphisms of the HO-1 gene promoter may modulate transcriptional activity, thereby augmenting or attenuating HO-1 expression with resultant modulation of the production of bilirubin. Few studies have related these polymorphisms to neonatal bilirubin metabolism and have reported conflicting results. In this clinical review, we surveyed the role of HO-1 gene promoter polymorphisms in the control of bilirubin production and further considered their role, if any, in the pathophysiology of neonatal hyperbilirubinemia.

Acquisition of mannosylated glycans in the surface immunoglobulin (sIg) variable region (sIgV) antigen-binding site is a unique tumor-specific structural change of certain lymphomas, including all follicular lymphomas (FL) and ~40% diffuse large B-cell lymphomas (DLBCL). Mannosylation of the sIgV allows binding to environmental lectins including DC-SIGN (Coelho V et al, PNAS 2010). SIgV engagement is generally required for survival of DLBCL cells (Young RM et al, PNAS 2015), but how sIgV mannosylation distributes and affects behavior in the two germinal center B-cell-like (GCB-like) or activated B-cell-like (ABC-like) DLBCL subsets is unknown. While the mannosylation of the sIgV is tumor specific and irreversible, there are other natural N-glycosylation sites in the sIg constant region (sIgC). In secreted IgM these are mainly fully glycosylated and that is seen in sIgM of normal B cells (Krysov S et al, Blood 2010). However, engagement of sIgM by anti-IgM leads to expression ofan immature (mannosylated) form in both tumor and normal B cells. This conversion is dynamic, and tumor B cells restore expression of sIgC with mature glycans following BCR disengagement in vitro(Krysov S et al, Blood 2010). In this study, the glycosylation patterns of sIgV and sIgC were analyzed in GCB-like (n=6) vs ABC-like DLBCL lines (n=2) and primary samples (n=8) by IGHV-D-J sequencing, DC-SIGN binding and immunoblot of the biotinylated sIg following digestion by EndoH (specific for the mannosylated sugars) or by PNGase (removes all sugars). We found acquisition of N-mannosylation sequence motifs in the IGHV-D-J transcripts of all GCB-DLBCL lines with t(14;18), indicating a likely relationship with FL. In contrast, neither of the ABC-DLBCL lines had acquired sites, confirming a separate origin. DC-SIGN binding, which is specific for mannosylated IgV structures on the tumor cells, was observed in all GCB-DLBCL and not in the ABC-DLBCL, confirming that the acquired sites were glycosylated. These results allowed us to discriminate DLBCL cases into "DC-SIGN binders" (DB-DLBCL) vs "DC-SIGN non-binders" DLBCL (NB-DLBCL). Analysis of the carbohydrate structures on the sIgC revealed that the immature form was confined to the NB-DLBCL lines (2/2), while the DB-DLBCL expressed a mature fully glycosylated form (6/6). Consistent with the nature of ABC-DLBCL, these results revealed an activated BCR status of the NB-DLBCL. This was confirmed in the 8 primary samples (5/8 DB, 3/8 NB), which expressed an immature (activated) sIgC in 3/3 NB-DLBCL and a mature sIgC in 5/5 DB-DLBCL. However, engagement of anti-IgM F(ab')2 polyclonal antibody converted the inactive sIg form of DB-DLBCL into an activated sIg with relative increase of the immature sugars. It was evident that the mannosylated sites on the sIgC were not available for DC-SIGN binding, which is confined to the sIgV sites. We verified BCR activation status by investigating constitutive phosphorylation of SYK, BTK and PLCγ2, which are recruited to the membrane upon BCR activation, prior to endosome formation (Phelan JD et al, Nature 2018), in 2 DB-DLBCL lines (NU-DHL1 and SU-DHL6) and 2 NB-DLBCL (HBL-1 and TMD8). Basal phosphorylation of SYK, BTK and PLCγ2 was higher in the NB-DLBCL, consistent with the activated status associated with an immature sIgC. Our results reveal a functional dichotomy in DLBCL, which indicates: first, the cell of origin dictates whether sIgV carries mannoses in the antigen-binding site; second, reversible sIgC mannosylation associates with activation via sIg. Interestingly, this feature of activation is in ABC-DLBCL, which lacks IgV mannosylation. It is consistent with the suggestion that occupation of the antigen-binding sites with mannoses blocks further engagement of the receptor by 'antigen'. However, acquisition of mannoses in the sIgV sites appears to confer an ability to interact with environmental lectins such as DC-SIGN, whereas the sIgC sites fail to do this, suggesting an alternative function. Clearly, the post-translational modification targets several sites in sIg. Sites in the sIgC have a similar, possibly maturational, function in normal B cells, but in tumor cells the irreversible addition of mannoses to the sIgV adds a tumor-specific function. Disclosures Packham: Aquinox: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

Introduction: Follicular lymphoma (FL) cells retain expression of a functional B cell receptor (BCR) despite the loss of one Ig allele due to the hallmark t14:18 translocation and ongoing somatic hypermutation (SHM) of the variable genes (V genes) which increases the likelihood of crippling mutations. SHM introduces N-glycosylation (N-gly) motifs within the V genes, a feature exclusively restricted to germinal centre (GC)-derived lymphomas. Oligosaccharides of the high mannose type are added to motifs and interact with calcium-dependent lectins associated with cells of the microenvironment. This activates the BCR signalling pathway and likely contributes to the survival, retention and proliferation of tumor cells in the GC. Determining at what stage of disease evolution N-gly motifs are acquired and their behaviour during progression can ascertain their importance in pathogenesis and their potential as an effective therapeutic target. To achieve this, we analysed motifs within the Ig heavy chain variable gene (IGHV) of tumor-related subclones across temporal FL samples. Method: Genomic DNA from three FL patients taken at different time points of disease progression were analysed. In total, 8 samples were selected, all carrying an IGHV3 rearranged tumor clone. IGHV DNA amplicons were sent for 2x250bp paired-end sequencing using the Miseq Illumina platform (Genewiz, NJ). Tumor-related reads with counts greater than ten were selected following analysis on IMGT/HIGH-V-QUEST. Reads were aligned and unique sequences were assigned as subclones. Additional tumor related reads sequenced on the Roche 454 Life Sciences Genome Sequencer FLX were available (Patients 4 & 5). Subclones were analysed for N-gly motifs and evolutionary pathways were generated using the IgTree program, based on intraclonal SHM profiles and homology of tumor clones to the germline IGHV sequence. Results: The earliest time point samples for Patient's 1, 3, 4 & 5 contained one N-gly site within the IGHV of the MC defined by the largest count number. These sites were conserved in >97% of unique subclones (p97% of subclones in and across disease events, whereas the FR3 site was conserved in 95.5% of the diagnostic subclones and in ~80% of the relapsed and transformed populations. No subclones with loss of all four sites were detected for Patient 2. Patient 5 samples were taken from different anatomical sites with tumor populations acquiring distinct N-gly motifs, suggesting an early divergence in tumor evolution. Despite this, a minor population of motif positive clones are shared, suggesting a trafficking ability of subclones. Subclones with motifs made up ≥99% of the total tumor count, highlighting the motif as a feature of the tumor bulk, with motif negative clones representing a minor population. These negative clones are presumably lost during disease progression as they not shared between events. Evolutionary analysis revealed no additional sites are gained as motif positive clones expand while rare negative subclones cannot reacquire sites and do not undergo further diversification. Conclusion: We report for the first time that acquired N-gly motif sites are a clonal feature in FL disease as seen through their conservation both in the heterogeneous subclonal population and the overall tumor mass. The sites are also retained in progression-associated subclones while rare motif-negative subclones disappear. This suggests that although acquisition of additional driver mutations may dampen the tumor's microenvironment dependency, the motifs and added mannoses may retain functional significance at later stages of disease. The data indicates motifs as being a universal event of the reservoir cell pool responsible for propagating disease episodes. Targeting N-gly sites and their interacting partners may lead to the disruption of an early and vital FL-microenvironment interaction, presumably mediated through the mannose-lectin interaction, reducing relapse rates and progression of disease. Disclosures Forconi: Abbvie: Consultancy; Janssen-Cilag: Consultancy. Gribben:Abbvie: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Cancer Research UK: Research Funding; Wellcome Trust: Research Funding; Acerta Pharma: Honoraria, Research Funding; TG Therapeutics: Honoraria; NIH: Research Funding; Kite: Honoraria; Janssen: Honoraria, Research Funding; Unum: Equity Ownership; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Necrotizing enterocolitis (NEC) is typified by mucosal destruction, which subsequently can lead to intestinal necrosis. Prematurity, enteral feeding, and bacterial colonization are the main risk factors and, combined with other stressors, can cause increased intestinal permeability, injury, and an exaggerated inflammatory response. Heme oxygenase-1 (HO-1) mediates intestinal protection due to anti-inflammatory, antioxidative, and antiapoptotic effects of its products carbon monoxide, biliverdin, and bilirubin. This study investigates a possible role of HO-1 in the pathogenesis of NEC using a newborn mouse model. We induced NEC-like intestinal injury in 7-day-old HO-1 heterozygous (HO-1 Het, Hmox1+/-) and wild-type (Wt, Hmox1+/+) mice by gavage feeding and hypoxic exposures. Control (Con) pups of both genotypes were dam-fed. Intestines of HO-1 Het Con pups appeared predisposed to injury, with higher histological damage scores, more TUNEL-positive cells, and a significant reduction in muscularis externa thickness compared with Wt Con pups. The increase in HO activity after HO-1 induction by the substrate heme or by hypoxic stress was significantly impaired in HO-1 Het pups. After induction of intestinal injury, HO-1 Het pups displayed significantly higher NEC incidence (78 vs. 43%), mortality (83 vs. 54%), and median scores (2.5 vs. 1.5) than Wt NEC pups. PCR array analyses revealed increased expressions of IL-1β, P-selectin, matrix metallopeptidase 2, collagen type XVIII-α1, serpine 1, and others in NEC-induced HO-1 Het ileal and jejunal tissues. We conclude that a partial HO-1 deficiency promotes experimental NEC-like intestinal injury, possibly mediated by exaggerated inflammation and disruption in tissue repair.

Preterm neonates with increased bilirubin production loads are more likely to sustain adverse outcomes due to either neurotoxicity or overtreatment with phototherapy and/or exchange transfusion. Clinicians should rely on expert consensus opinions to guide timely and effective interventions until there is better evidence to refine bilirubin-induced neurologic dysfunction or benefits of bilirubin. In this article, we review the evolving evidence for bilirubin-induced brain injury in preterm infants and highlight the clinical approaches that minimize the risk of bilirubin neurotoxicity.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

Because heme catabolism leads to the formation of equimolar amounts of carbon monoxide (CO) and bilirubin, a variety of techniques have been developed to correlate CO production rates as indices of bilirubin production. The use of end-tidal breath CO measurements for estimating rates of bilirubin production in infants has been well documented and validated in a number of clinical studies for its use and predictive value in identifying infants who are high producers of bilirubin and hence at risk for developing pathologic neonatal hyperbilirubinemia. Recently, end-tidal breath CO has been suggested as a marker for chronic lung disease and developmental problems. Trace gas analysis remains an area for interesting investigation in the future.

Neonatal hyperbilirubinemia arises from increased bilirubin production and decreased bilirubin elimination. Although phototherapy safely and effectively reduces bilirubin levels, recent evidence shows that it has adverse effects. Therefore, alternative treatments are warranted. Metalloporphyrins, competitive inhibitors of heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, effectively reduce bilirubin formation; however, many are photoreactive. Here, we investigated possible photosensitizing effects of chromium mesoporphyrin (CrMP) and zinc deuteroporphyrin bis-glycol (ZnBG). Administration of CrMP or ZnBG to 3-d-old mouse pups (3.75–30.0 µmol/kg intraperitoneally) and exposure to cool white (F20T12CW) and blue (TL20W/52) fluorescent lights (+L) for 3 h, resulted in a dose-dependent mortality (50% lethal dose (LD50) = 21.5 and 19.5 µmol/kg, respectively). In contrast to ZnBG, there was no significant difference in survival between the CrMP+L and CrMP groups. Following 30 µmol/kg ZnBG+L, we found significant weight loss, decreased liver antioxidant capacities, and increased aspartate aminotransaminase levels. At 6-d post-light exposure, ZnBG+L-treated pups showed gross and histologic skin changes at doses >7.5 µmol/kg. No lethality was observed following treatment with 30 µmol ZnBG/kg plus exposure to blue light-emitting diodes. Phototoxicity of ZnBG was dependent on light source, emission spectrum, and irradiance. Low doses of ZnBG (