Separate Donors Research Articles

Polarization charge densities provide a predictive quantification of hydrogen bond energies

A systematic density functional theory based study of hydrogen bond energies of 2465 single hydrogen bonds has been performed. In order to be closer to liquid phase conditions, different from the usual reference state of individual donor and acceptor molecules in vacuum, the reference state of donors and acceptors embedded in a perfect conductor as simulated by the COSMO solvation model has been used for the calculation of the hydrogen bond energies. The relationship between vacuum and conductor reference hydrogen bond energies is investigated and interpreted in the light of different physical contributions, such as electrostatic energy and dispersion. A very good correlation of the DFT/COSMO hydrogen bond energies with conductor polarization charge densities of separated donor and acceptor atoms was found. This provides a method to predict hydrogen bond strength in solution with a root mean square error of 0.36 kcal mol(-1) relative to the quantum chemical dimer calculations. The observed correlation is broadly applicable and allows for a predictive quantification of hydrogen bonding, which can be of great value in many areas of computational, medicinal and physical chemistry.

Hydrogen Bonding in the Electronic Excited State

Because of its fundamental importance in many branches of science, hydrogen bonding is a subject of intense contemporary research interest. The physical and chemical properties of hydrogen bonds in the ground state have been widely studied both experimentally and theoretically by chemists, physicists, and biologists. However, hydrogen bonding in the electronic excited state, which plays an important role in many photophysical processes and photochemical reactions, has scarcely been investigated. Upon electronic excitation of hydrogen-bonded systems by light, the hydrogen donor and acceptor molecules must reorganize in the electronic excited state because of the significant charge distribution difference between the different electronic states. The electronic excited-state hydrogen-bonding dynamics, which are predominantly determined by the vibrational motions of the hydrogen donor and acceptor groups, generally occur on ultrafast time scales of hundreds of femtoseconds. As a result, state-of-the-art femtosecond time-resolved vibrational spectroscopy is used to directly monitor the ultrafast dynamical behavior of hydrogen bonds in the electronic excited state. It is important to note that the excited-state hydrogen-bonding dynamics are coupled to the electronic excitation. Fortunately, the combination of femtosecond time-resolved spectroscopy and accurate quantum chemistry calculations of excited states resolves this issue in laser experiments. Through a comparison of the hydrogen-bonded complex to the separated hydrogen donor or acceptor in ground and electronic excited states, the excited-state hydrogen-bonding structure and dynamics have been obtained. Moreover, we have also demonstrated the importance of hydrogen bonding in many photophysical processes and photochemical reactions. In this Account, we review our recent advances in electronic excited-state hydrogen-bonding dynamics and the significant role of electronic excited-state hydrogen bonding on internal conversion (IC), electronic spectral shifts (ESS), photoinduced electron transfer (PET), fluorescence quenching (FQ), intramolecular charge transfer (ICT), and metal-to-ligand charge transfer (MLCT). The combination of various spectroscopic experiments with theoretical calculations has led to tremendous progress in excited-state hydrogen-bonding research. We first demonstrated that the intermolecular hydrogen bond in the electronic excited state is greatly strengthened for coumarin chromophores and weakened for thiocarbonyl chromophores. We have also clarified that the intermolecular hydrogen-bond strengthening and weakening correspond to red-shifts and blue-shifts, respectively, in the electronic spectra. Moreover, radiationless deactivations (via IC, PET, ICT, MLCT, and so on) can be dramatically influenced through the regulation of electronic states by hydrogen-bonding interactions. Consequently, the fluorescence of chromophores in hydrogen-bonded surroundings is quenched or enhanced by hydrogen bonds. Our research expands our understanding of the nature of hydrogen bonding by delineating the interaction between hydrogen bonds and photons, thereby providing a basis for excited-state hydrogen bonding studies in photophysics, photochemistry, and photobiology.

The Effect of Graded Cyclic Stretching on Extracellular Matrix–Related Gene Expression Profiles in Cultured Primary Human Lamina Cribrosa Cells

Cyclic stretching of the glial fibrillary acidic protein (GFAP)-negative lamina cribrosa (LC) cell in vitro is associated with transcriptomic changes in genes involved in extracellular matrix (ECM) dynamics in vivo, thereby implicating this cell type in the pathophysiologic changes of the optic nerve head (ONH) in glaucoma. The purpose of the study was to determine whether exposure to different grades of mechanical stretch progressively alters the expression of ECM genes in cultured LC cells. Primary cultures of human LC cells from three separate donors were maintained in static culture or exposed to low-level strain (3% ± 0.5% elongation, 1 Hz) for 24 hours. A baseline comparison of the expression of 62 genes involved in ECM dynamics was performed with low-density gene arrays (LDAs). The 3% protocol was used in a 24-hour period of baseline dynamic low-level stretch, and gene expression was compared with that occurring in a further 24-hour exposure to a 12.5% or a 20% stretch. Gene expression levels were determined by qRT-PCR. LC cells displayed a nonlinear, transcriptional response to the mechanical stretch. Ten ECM-related and growth factor genes were altered by 3% strain versus static culture (nine downregulated and one upregulated). Increasing strain from 3% to 20% resulted in a significant increase in expression of 15 ECM-elated genes. Only one gene (epidermal growth factor) was increased between the 3% and 12.5% strains. Low-level, pulsatile, cyclic strain resets a lower baseline expression of several glaucoma-associated ECM genes. The LC ECM gene response occurs above a fourfold increase in baseline strain (12.5% strain) in vitro. The study supports the use of a nonstatic baseline when studying the effect of stretch (or strain) on the activation of ONH-derived, ECM-producing cells.

Alloreactive Nk Cells to Augment Antileukemia Cytotoxicity with Allogeneic Stem Cell Transplantation for Aml/Mds: A Phase I Study

AbstractAbstract 489Relapse remains the major cause of treatment failure after allogeneic hematopoietic transplantation for AML and MDS. Alloreactive NK cells mediate a potent antileukemic effect and may also enhance engraftment and reduce GVHD. We performed a phase I study infusing “third party” alloreactive NK cells from a haploidentical related donor as a component of the preparative regimen for allotransplantation from a separate HLA identical donor. The goal was to augment the antileukemia cytotoxicity of the preparative regimen by infusion of alloreactive NK cells and improve the overall outcome of hematopoietic transplantation.Patients with advanced AML or high risk MDS in relapse or beyond first remission were eligible. They received the busulfan-fludarabine preparative regimen, followed by infusion of NK cells predicted to be alloreactive by KIR:KIR ligand incompatibility. The NK cell enriched product was produced from a steady state apheresis product by depleting CD3+ cells using the CliniMACS device (Miltenyi Corp). A second step positively selecting CD56+ cells was performed for the first dose level, but discontinued thereafter in order to increase the cell yield. The NK cell product was then cultured in complete media containing rIL-2 for 16 hours and infused intravenously following completion of the busulfan-fludarabine chemotherapy. After 5 days, ATG was administered followed by PBSC infusion from the HLA identical sibling or unrelated donor. Patients received tacrolimus and methotrexate for GVHD prophylaxis. Patients were treated in 4 dose levels of the NK cell enriched product; 1) 106 cells/kg, 2) 5 × 106/kg, 3) 3 × 107/kg, and 4) 3 × 107/kg followed by systemic interleukin-2 0.5 million units/m2 SQ daily for 5 days. CD3+ cells in the NK cell product were required to be <105/kg (median infused 1.1 x104/kg). Median CD56+ cells infused (x106/kg) were from 0.9 (level 1), 1.5 (level 2), and 4.9 (levels 3 and 4).13 patients were entered. Median age was 51 years (range 2–60). 11 had active disease and 2 were in a second remission. Only mild infusion toxicity occurred with the NK cell infusion. Other toxicities were similar to that experienced with the preparative regimen without NK cells. The 2 patients at dose level 4 tolerated interleukin-2 systemic treatment without fever or increased toxicity. The haploidentical NK cells were transiently detected in the blood by chimerism studies in one patient. Rapid engraftment and hematologic recovery uniformly occurred from the HLA matched PBPC donor in all patients. None had graft failure. Grade 2 acute GVHD developed in 3 patients; all responded to corticosteroid treatment. None developed grade 3 or 4 acute GVHD. 9 of 11 patients with active disease achieved a complete remission. Only one patient died of nonrelapse mortality; she died in remission from infection 2 months post transplant.This trial confirms the feasibility of producing the haploidentical NK cell product, and the lack of major toxicity attributable to the NK cell infusion in combination with an HLA compatible allogeneic transplantation. Infusion of haploidentical alloreactive NK cells was well tolerated and did not interfere with engraftment or increase the rate of GVHD after allogeneic hematopoietic transplantation. This approach merits further phase II and III study designed to reduce relapse and improve the outcome of allogeneic hematopoietic transplantation for AML/MDS.NNK cell doseCells/kggrade 2 GVHDRelapseNRMAlive in CR4106140045 × 1060301 (27+ m)33 × 1072111 (11+ m)23 × 107 + IL-20002 (9+ m, 4+ m) Disclosures:No relevant conflicts of interest to declare.

Bis(propane-1,3-diaminium) hexafluoridoferrate(III) fluoride trihydrate

The asymmetric unit of the title iron hybrid fluoride, (C3H12N2)2[FeF6]F·3H2O, contains two propane-1,3-diamin­ium [(H2dap)2+] cations, an octa­hedral [FeF6]3− anion, an isolated F− anion and three water mol­ecules of solvation. Each [FeF6]3− anion is surrounded by four separate hydrogen-bonded water mol­ecules in the equatorial sites and by five separate aminium cation donor groups. The axial F atoms are only involved in N—H⋯F hydrogen bonds, resulting in a three-dimensional structure.

IL-4 stimulates production of TGF-β-induced (TGFBI) mRNA and protein in T cells but not in monocytes, fibroblasts, or pulmonary epithelial cells (44.16)

Abstract TGFBI is an extracellular matrix and an adhesion protein produced by a variety of cell types. It is not known whether cytokines other than TGF-β may regulate TGFBI production. RT-Q-PCR and Western blotting assays revealed production of TGFBI mRNA and protein, respectively, in cultured purified primary monocytes, primary pulmonary fibroblasts, and a pulmonary epithelial cell line A549. Stimulation of these cultures with 300 ng/ml of rhIL-4 did not cause a significant change in TGFBI mRNA after 3, 6, 12, or 24 h, or in TGFBI protein after 24 or 48h, (p &amp;gt; 0.05 in all cases, two separate cell donors tested each on two independent occasions). Primary T cell (&amp;gt; 95% purity) cultured from 4 separate donors and tested each on at least three occasions produced basal TGFBI mRNA and protein, although at the levels 10-15 fold below those observed in other tested cell types. Stimulation of T cells or PBMC with IL-4 revealed time dependent increases in the steady-state level of TGFBI mRNA and protein, but not in TGF-β mRNA. The maximal effect of IL-4 on TGFBI mRNA (2-6 fold increase, p &amp;lt; 0.05) was observed at 3 h and 6 h; and on TGFBI protein at 24 and 48 h of stimulation. The effect of IL-4 on TGFBI production was not attenuated by a TGF-β-blocking antibody 1D11. Thus, IL-4 induces TGFBI production in T cells in a TGF-β-independent fashion. T cells may use TGFBI as an adhesion molecule for binding to known TGFBI receptors integrins αVβ3, αVβ5, and α3β1, collagens, and fibronectin.

Phospholipid-esterified Eicosanoids Are Generated in Agonist-activated Human Platelets and Enhance Tissue Factor-dependent Thrombin Generation

Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 ± 1.42 (PE) or 18.35 ± 4.61 (PC), whereas free was 65.5 ± 17.6 ng/4 × 107 cells (n = 5 separate donors, mean ± S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca2+ mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

In vitroanalysis of cryopreserved alginate-poly-l-lysine-alginate-microencapsulated human hepatocytes

The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications. Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 degrees C for 12-24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed. Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious. The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 degrees C for 12-24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.

Recovering Absolute Fret Efficiency from Single Molecules: Comparing Methods of Gamma Correction

Fluorescence resonance energy transfer is widely thought of as a “spectroscopic ruler.” Because biological processes and cellular assemblies occur on the nanometer scale, FRET is a popular tool for structural biology. In contrast to ensemble solution FRET measurements which record the entire emission spectrum, microscopy- based FRET experiments separate donor and acceptor intensity by passing the emission through a series of optical elements. Observed FRET efficiency, determined from the uncorrected donor and acceptor intensities, has been called a relative proximity ratio, which is internally consistent only if the photophysical properties and instrument remain unchanged. However, it is desirable to measure absolute distances using FRET, which requires that FRET efficiency be corrected for both instrument response and fluorophore properties. Thus, “gamma” correction adjusts for differences between the donor and acceptor dyes in their probability of photon emission upon excitation and the probability that emitted photons will be detected. Methods of gamma correction vary depending on the single molecule methodology. To test different methods for correcting FRET efficiency, we recorded smFRET distributions for protein and DNA on different instruments and with different filter sets which altered the observed FRET efficiency. Knowledge of filter set transmission allows for comparison of results between groups using different instruments. Applying empirically-derived corrections for instrument response and quantum yield was only slightly better than corrections based solely on filter set transmission data. We found that gamma correction based on single molecule photobleaching was the most effective particularly when gamma was determined for each sample or even each molecule. Variations in focus of the two colors and sub-pixel errors in image mapping affect both FRET and gamma. As such, per molecule correction affects distribution width because FRET outliers may also have anomalous gamma values.

Ineffective Erythropoiesis Caused by Phenylhydrazine Activates the Expression of GDF15 in Maturing Erythroblasts.

Abstract 4032Poster Board III-968Several types of ineffective erythropoiesis (thalassemia, congenital dyserythropoietic anemia type I, and refractory anemia with ringed sideroblasts) develop anemia and eventually manifest secondary hemochromatosis even in the absence of transfusion therapy. The highly elevated levels of GDF15 in those patients may contribute to iron loading due to suppression of hepcidin. However, the underlying mechanism for high-level expression of GDF15 in association with ineffective erythropoiesis remains vague. Phenylhydrazine (PHZ), which causes hemoglobin denaturation and alpha-globin precipitation, was utilized in CD34+ cultures as an ex vivo model of ineffective erythropoiesis. All experiments were performed in triplicate with cells from three separate human donors. Initial studies were performed to determine the effects of phenylhydrazine upon reactive oxygen species (ROS) production and apoptosis in the erythroblasts. In dosed titrations (0-10 uM), PHZ was added to the culture medium before and after erythroblast hemoglobin accumulation on culture days 3-6 and 11-14, respectively. PHZ addition prior to the accumulation of hemoglobin had little effect on ROS production. In contrast, PHZ added to hemoglobinized erythroblasts resulted in significant and dosed increases in ROS production. In addition, apoptosis cascades that are downstream of ROS production were studied including activation of caspase-3, EGR-1 transcription, p53 stabilization, and phosphatidylserine on the cell surface (detected by Annexin V). There was a dose-dependent increase in caspase-3, p53 stabilization, and Annexin V positive cells that mirrored the ROS production after PHZ treatment only among the more mature erythroblasts. However, the PHZ addition did not cause a significant increase in the nuclear localization of EGR-1. Since GDF15 is a known target of p53, PHZ effects upon GDF15 expression were also measured. Production of GDF15 was not observed on culture days 3-6 in the absence or presence of PHZ. In contrast, dosed increases in GDF15 expression were detected among the hemoglobinized erythroblasts (culture days 11-14) in response to phenylhydrazine (PHZ = 0 uM, GDF15 = 16 ± 3 pg/1×104cells; PHZ = 1 uM, GDF15 = 23 ± 5 pg/1×104cells; PHZ = 5 uM, GDF15 = 48 ± 9 pg/1×104cells [p<0.01]; PHZ = 10 uM, GDF15 = 75 ± 22 pg/1×104cells [p<0.01]). Of note, GDF15 expression was not detected after enucleation in peripheral blood erythrocytes exposed to PHZ despite increased ROS. According to these data, it is proposed that ineffective erythropoiesis may be caused by increased ROS production during the later stages of erythroblast maturation that activates caspase-3, apoptosis and p53-mediated expression of GDF15. Disclosures:No relevant conflicts of interest to declare.

A Simple Isolation and Cryopreservation Method for Adult Human Hepatocytes

The objective of this study was to establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for the treatment of acute and chronic liver diseases by hepatocyte transplantation and bioartificial liver support systems, and for the use of hepatocytes as an in vitro model of the liver. Adult hepatocytes of 20 separate donors were isolated with a two-step extracoporeal collagenase perfusion technique. Seven preincubation time points (2h, 6h, 12h, 24h, 36h, 48h and 72h) were selected, then the hepatocytes were transferred to HepatoZYME-SFM medium containing 10% FBS and 10% DMSO, and were immediately put into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the postthawing culture period, viability, plating efficiency, albumin secretion and urea synthesis were analyzed. The viability and plating efficiency of hepatocytes after partial hepatectomy using two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0% respectively. Preincubation at 4? for 12 hours or 24 hours proved to be the optimal time at which the albumin secretion was higher than at other time points (p<0.05). Compared to the immediate cryopreservation groups (IC), we also found significant improvement in viability (61.4+/-4.8%/62.0+/-5.6% vs. 53.4+/-4.2%, p<0.05), plating efficiency (63.2+/-5.8%/62.6+/-3.6% vs. 55.2+/-4.6%, p<0.05), albumin secretion and urea synthesis (p<0.05) at these time points. The two-step extracorporeal collagenase perfusion technique after partial hepatectomy provides a novel, simple, and reliable method for hepatocyte isolation. The results of the present study suggest that recovery of human hepatocytes after isolation preincubation at 4 degrees C for 12 hours to 24 hours prior to cryopreservation can obtain hepatocytes ideal for use in pharmacotoxicology, bioartificial liver and cell therapy research purposes.

Preliminary Investigation on the Action Modes of an Oligosaccharide-Producing Multifunctional Amylase

The action modes of an oligosaccharide-producing multifunctional amylase (OPMA) were investigated using glucose and some oligosaccharides as its substrates. OPMA did not cause the hydrolysis of maltose or isomaltose, but it catalyzed the alpha-1,6-transglycosylation of maltose to produce isomaltose or did the self-condensation of isomaltose to form isomaltotetraose and 4-O-alpha-isomaltosyl isomaltose. OPMA exhibited strong alpha-1,6-transglycosylation activity in addition to its alpha-1,4-hydrolytic activity on higher oligosaccharides substrates rather than bisaccharides. OPMA displayed high acceptor specificity in its transglycosylation or condensation reaction. OPMA seemed to only take glucose or isomaltose as the acceptor molecule in its transglycosylation or condensation reaction, which made glucose or isomaltose form higher products, and as a result, glucose or isomaltose were absent in the final products. In view of the simultaneously formation of several transglycosylation or condensation products, it was predicted that there might be separate donor and acceptor sites in OPMA's active center and the fact that the catalytically active form of this enzyme included its homodimer or homotrimer supported this prediction. Accordingly, a special pathway, isomaltose pathway, for OPMA catalysis was proposed to emphasize the central or important signification of isomaltose in OPMA catalysis.

The use of Urgotul™ in the treatment of partial thickness burns and split‐thickness skin graft donor sites: a prospective control study

The use of paraffin-impregnated gauze for burns and skin graft donor sites is commonly associated with wound adherence with consequent pain and trauma upon removal. This prospective clinical study was performed to evaluate a new class of lipido-colloid dressings (Urgotul) in promoting healing and in reducing tissue adherence. In a 6-month period, 25 consecutive patients were recruited. Two separate burn or donor sites on each patient were dressed with tulle-gras (TG) or Urgotul and covered with standard secondary dressings. Objective assessment of wounds by two reviewers, and patients' subjective assessments were recorded. Twenty-three (92%) patients were followed up for a mean of 3 months. Mean time to complete epithelialisation was 9.6 and 11.9 days for the Urgotul and TG sites respectively (P < 0.05). Bleeding was seen in 52% of Urgotul sites compared with 100% of the TG sites at first dressing change (P < 0.05). Patients reported 'moderate pain' during dressing change in 22% and 57% in the Urgotul and TG groups respectively (P < 0.05), with 35% of TG sites being 'very painful' requiring extra analgesia. We found that compared with TG, Urgotul was associated with faster epithelialisation, less pain and trauma (bleeding) during dressing changes.

Determination of first-order molecular hyperpolarizability of EAADCy and EDMAADCy using steady-state spectroscopic data and quantum-chemical calculations

Ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate (EAADCy) and ethyl 5-(4-dimethylaminophenyl)-3-amino-2,4-dicyanobenzoate (EDMAADCy) organic molecules containing separate electron donor and electron acceptor groups belong to biphenyl derivatives in which a large dipole moment change between ground (S 0) and the first intramolecular charge transfer excited (S 1) states, as well as a large transition moment have been noted. The existence of electronically excited states with a strong intermolecular charge transfer (ICT) character is an essential prerequisite for large non-linear optical properties. Therefore, in this paper, we present a scrupulous analysis of the first-order hyperpolarizabilities of the studied molecules using an equivalent internal field model of an organic molecule. The calculated (using semiempirical calculations, CAChe WS 5.04) additive part of the first-order hyperpolarizability, β add , values are discussed in relationship to the experimental data of the charge transfer hyperpolarizability, β CT , obtained from steady-state spectroscopic measurements.

Two small flaps from one anterolateral thigh donor site for bilateral buccal mucosa reconstruction after release of submucous fibrosis and/or contracture

Oral submucous fibrosis causes health-related and social problems for affected patients. Free flap reconstruction has proved effective for maintaining mouth opening after release of fibrosis. Two independent free flaps from separate donor sites, such as bilateral forearm flaps or bilateral anterolateral thigh (ALT) flaps, were traditionally required for reconstruction. The former option sacrifices one of the two major arteries in the forearm. Both options are time consuming and required two donor sites. To eliminate these disadvantages, we developed a technical modification that allows harvesting of two independent flaps from one ALT thigh based on one descending branch of the lateral circumflex femoral artery (d-LCFA). Eighteen flaps from nine donor sites were harvested for post-release reconstruction of oral submucous fibrosis. Mean flap size was 4.1 x 7.5 cm, mean pedicle length was 7.6 cm, mean ischaemia time was 104 min and mean total operation time was 13 h and 19 min. All donor sites were closed primarily, with one exception. One flap failed and was replaced with a contralateral ALT flap. One patient developed a wound infection and another developed a seroma at the recipient site. Four flaps required secondary de-bulking in three patients. The improvement in mouth opening was evaluated by inter-incisor distance (IID): mean preoperative IID was 9.6mm (range: 0-20mm), mean follow-up time was 16.2 months (range: 10-33 months); mean postoperative IID was 23.8mm and mean improvement in IID was 15.3mm (range: 10-27 mm). In conclusion, two independent flaps can be harvested from d-LCFA of the same thigh, instead of from both thighs, to reconstruct bilateral buccal defects after release of submucous fibrosis and/or contracture.

Proteomic Characteristics of ex vivo-Enriched Adult Human Bone Marrow Mononuclear Cells in Continuous Perfusion Cultures

A major challenge in developing cell therapies is reliable characterization of the cell product at the molecular level. Fresh autologous and passaged human bone marrow enriched for stem and mesenchymal stromal stem cells have been used to regenerate bone. We report the proteome of an innovative autologous human bone marrow-derived mixed cell product (BMMCP), cultured ex vivo for 12 days, in automated continuous media perfusion system to avoid passaging, and discuss reproducibility of protein composition. Each BMMCP is compared to its originating human adult bone marrow mononuclear cells (BMMNC). With the use of 2-D LC-MS/MS approach, 638 (BMMNC) and 867 (BMMCP) distinct proteins were identified including cell adhesion molecules, extracellular matrix and growth factors. Overlap of protein identifications revealed that 67% of the BMMNC proteome was retained in the BMMCP, and protein expression of selected cell lineages was enhanced. Isotope-coded affinity tags (ICAT) and MS/MS were used to identify and quantify relative changes in the proteome of BMMNC and their related BMMCP, obtained from 3 separate donors. In 3 separate ICAT experiments, 57% of proteome identified was shared between donors. Measurable and definable proteomic characterization of BMMCP will facilitate their use in clinical trials and provide insight into cell functionality needed to support multiple therapeutic indications.

Modified Technique for Frontal Sinus Obliteration Using Calvarial Bone and Tisseel Glue

Frontal sinus obliteration is indicated in trauma and frontal sinus disease. We present our experience with a modified technique of frontal sinus obliteration. We reviewed the records of 17 patients who underwent frontal sinus obliteration with a combination of Tisseel fibrin glue (Baxter Healthcare Corporation, Deerfield, IL) and calvarial bone and describe our technique. One female and 16 male patients (mean age, 36.6) underwent frontal sinus obliteration from 2001 to 2005. Traumatic fracture was the most common indication. The patients were followed up for a median of 15 months. There were no major complications. Postoperatively, one patient developed a localized frontal wound infection at 3 weeks; this resolved with debridement. At latest follow-up, all were free from local sinus complications, disease recurrence, headaches, or intracranial events. The combined use of autogenous calvarial bone and Tisseel is suitable for frontal sinus obliteration. Our technique is associated with low donor site morbidity, avoids a separate donor surgical site, and has a low complication rate.

Iron Depleted Erythropoiesis: Slow but Effective

Iron deficiency affects billions worldwide, and anemia develops when iron stores become insufficient to maintain normal erythropoiesis. However, iron depleted erythropoiesis is incompletely understood. In this study, an ex vivo model of iron depleted erythropoiesis was developed using dosed titrations of the iron chelator deferoxamine (DFO) in CD34+ cell cultures. All experiments were performed in triplicate with cells from three separate donors. Hemoglobinization and expression patterns of erythroid markers (GPA/CD71) demonstrated minimal changes in DFO supplemented medium. DFO caused dose-related suppression of cell counts after 14 days (20uM DFO: 42% reduction, 30uM DFO: 86% reduction, 40uM DFO: 96% reduction of cell counts compared to 0uM DFO controls). Cultures supplemented with 30uM DFO were used for subsequent studies, and the DFO-mediated effects were completely reversed by addition of 30uM ferric chloride. To investigate whether apoptosis caused the reduction in cell counts, surface Annexin V was measured by flow cytometry. No significant increases in levels of apoptosis among the proliferating erythroblasts were detected (Annexin V positive cells; 0uM DFO 6.2 ± 0.95%, 30uM DFO 9.0 ± 1.55%, p = 0.058). Analyses of p53 protein quantitation and p53 DNA-binding activity further suggested that the suppression of cell counts by iron chelation was not due to p53 related apoptosis. In the absence of apoptosis or maturation arrest, we hypothesized that the growth suppressing effects of DFO were not due to ineffective erythropoiesis. To test this hypothesis, a recently-discovered marker of ineffective erythropoiesis named GDF15 (Tanno et al. Nat. Med. 2007) was measured in the culture supernatants, and no increase was detected (GDF15 concentration per 1×104 cells: 0uM DFO; 79.7 ± 4.4 pg, 30uM; 43.9 ± 4.3 pg). In the clinic, serum GDF15 levels from 17 blood donors with significantly reduced ferritin and iron saturation (279 ± 98 pg/ml) were not increased when compared to serum GDF15 levels in 17 healthy volunteers with normal iron parameters (410 ± 119 pg/ml). To determine if cell cycling, instead of apoptosis, caused the reduction in cell counts, carboxyfluorescein diacetate succinimidyl diester (CFSE) staining studies were performed during the first and second weeks of culture. CFSE is an intracellular fluorescent stain, the intensity of which halves with each cell division. Three or four fewer erythroblast cell divisions were detected in 30uM DFO during the 14-day culture period when compared to controls. Based upon the reduction in cell divisions, it was estimated that the average cell cycle time was prolonged by 40–70% in DFO. Propidium iodide analyses additionally demonstrated changes in the cell cycle kinetics including a significant reduction in the percentage of S-phase erythroblasts at the peak phase of proliferation (30uM DFO: 37.0 ± 1.8% vs. control: 48.7 ± 3.4%, p = 0.0063). These results suggest that iron depletion inhibits the overall growth of erythroblasts by prolonging the cell cycle rather than causing apoptosis or maturation arrest. Due to the large amount of iron required for hemoglobin production, slower cell cycles may facilitate effective erythropoiesis by allowing extra time for uptake when extracellular iron is scarce.

Gamma-Globin Gene Expression in Adult Human Erythroblasts Is Associated with Concurrent Changes in the Nuclear Protein Levels of at Least Seven Transcription Factors.

Alterations of globin-gene cluster expression mediated through cytokine signal transduction have been previously established. In adult erythroblasts, ex vivo combinatorial signaling from cytokines such as erythropoietin (EPO), stem cell factor (SCF), and transforming growth factor-beta (TGF-B) causes a robust reactivation of fetal hemoglobin. To determine if cytokine-activated expression of fetal hemoglobin significantly alters the transcript and protein abundance of nuclear transcription factors, profiling studies of transcription factor expression were performed. Primary CD34+ cells were cultured as donor-matched pairs using cytokine combinations that produced low vs. high levels of gamma-globin mRNA and fetal hemoglobin (Bhanu et al. Blood, 2005). To identify candidate genes, total RNA from 15 separate healthy volunteer donors was collected and combined into 5 pools. The pooled RNA was labeled and hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips. Informatics strategies were aimed toward identifying differential expression of those transcription factors with binding motif on any of the 10 human globin gene promoter regions (553 transcription factors; 115 binding motif families), versus all transcription factors demonstrating robust expression with changes of at least 3-fold. Combining both strategies, 11 candidate transcription factors were identified (BCL11A, CBFB, EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, SOX-6, TCF3, and THRB). RNA-based descriptions of gene activity do not necessarily reflect changes in protein levels; therefore, Western analyses were performed. GATA-1 was utilized as a control, since no significant change in GATA-1 mRNA was detected by expression array profiling. Nuclear protein extracts were obtained from 3 additional donors' CD34+ cells cultured under conditions of low vs. high gamma-globin mRNA synthesis (1.6E+06 ± 3.8E+05 and 1.5E+07 ± 6.3E+06 copies/ng total RNA, respectively). Western blot analysis revealed reproducible and robust differences in protein expression levels for 7 of the 11 candidate transcription factors (EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, and SOX-6). Two other candidates were expressed below the protein detection limit. GATA-1 and the remaining 2 candidates demonstrated no change in the nuclear protein levels. Among the group with confirmed changes in gene expression, ELK-1 and EGR-1 are downstream targets of the MAP kinase signal transduction cascade. MAFF, ID2, and SOX-6 are known regulators of globin gene expression. The differential expression of HHEX and MNDA warrants further investigation. These data demonstrate that cytokine signal transduction causes changes in the intranuclear levels of at least 7 transcription factors concurrently with activation of gamma-globin gene expression.

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.