IL-36γ is a crucial proximal component of protective type-1-mediated lung mucosal immunity in Gram-positive and -negative bacterial pneumonia.

Interleukin-36γ (IL-36γ) is a member of novel IL-1-like proinflammatory cytokine family that are highly expressed in epithelial tissues and several myeloid-derived cell types. Little is known about the role of the IL-36 family in mucosal immunity, including lung anti-bacterial responses. We used murine models of IL-36γ deficiency to assess the contribution of IL-36γ in the lung during experimental pneumonia. Induction of IL-36γ was observed in the lung in response to Streptococcus pneumoniae (Sp) infection, and mature IL-36γ protein was secreted primarily in microparticles. IL-36γ-deficient mice challenged with Sp demonstrated increased mortality, decreased lung bacterial clearance and increased bacterial dissemination, in association with reduced local expression of type-1 cytokines, and impaired lung macrophage M1 polarization. IL-36γ directly stimulated type-1 cytokine induction from dendritic cells in vitro in a MyD88-dependent manner. Similar protective effects of IL-36γ were observed in a Gram-negative pneumonia model (Klebsiella pneumoniae). Intrapulmonary delivery of IL-36γ-containing microparticles reconstituted immunity in IL-36γ-/- mice. Enhanced expression of IL-36γ was also observed in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome because of pneumonia. These studies indicate that IL-36γ assumes a vital proximal role in the lung innate mucosal immunity during bacterial pneumonia by driving protective type-1 responses and classical macrophage activation.

Assessment of resuscitation as measured by markers of metabolic acidosis and features of injury.

AIMS: The best time for definitive orthopaedic care is often unclear in patients with multiple injuries. The objective of this study was make a prospective assessment of the safety of our early appropriate care (EAC) strategy and to evaluate the potential benefit of additional laboratory data to determine readiness for surgery. PATIENTS AND METHODS: A cohort of 335 patients with fractures of the pelvis, acetabulum, femur, or spine were included. Patients underwent definitive fixation within 36 hours if one of the following three parameters were met: lactate < 4.0 mmol/L; pH ≥ 7.25; or base excess (BE) ≥ -5.5 mmol/L. If all three parameters were met, resuscitation was designated full protocol resuscitation (FPR). If less than all three parameters were met, it was designated an incomplete protocol resuscitation (IPR). Complications were assessed by an independent adjudication committee and included infection; sepsis; PE/DVT; organ failure; pneumonia, and acute respiratory distress syndrome (ARDS). RESULTS: In total, 66 patients (19.7%) developed 90 complications. An historical cohort of 1441 patients had a complication rate of 22.1%. The complication rate for patients with only one EAC parameter at the point of protocol was 34.3%, which was higher than other groups (p = 0.041). Patients who had IPR did not have significantly more complications (31.8%) than those who had FPR (22.6%; p = 0.078). Regression analysis showed male gender and injury severity score to be independent predictors of complications. CONCLUSIONS: This study highlights important trends in the IPR and FPR groups, suggesting that differences in resuscitation parameters may guide care in certain patients; further study is, however, required. We advocate the use of the existing protocol, while research is continued for high-risk subgroups. Cite this article: Bone Joint J 2017;99-B:122-7.

Is Early Appropriate Care of axial and femoral fractures appropriate in multiply-injured elderly trauma patients?

BACKGROUND: Previous work established resuscitation parameters that minimize complications with early fracture management. This Early Appropriate Care (EAC) protocol was applied to patients with advanced age to determine if they require unique parameters to mitigate complications. METHODS: Between October 2010 and March 2013, 376 consecutive skeletally mature patients with unstable fractures of the pelvis, acetabulum, thoracolumbar spine, and/or proximal or diaphyseal femur fractures were treated at a level I trauma center and were prospectively studied. Patients aged ≤30 years (n = 114), 30 to 60 years (n = 184), and ≥60 years (n = 37) with Injury Severity Scores (ISS) ≥16 and unstable fractures of the pelvis, acetabulum, spine, and/or diaphyseal femur were treated within 36 h, provided they showed evidence of adequate resuscitation. ISS, Glasgow Coma Scale (GCS), and American Society of Anesthesiologists (ASA) classification were determined. Lactate, pH, and base excess (BE) were measured at 8-h intervals. Complications included pneumonia, pulmonary embolism (PE), acute renal failure, acute respiratory distress syndrome (ARDS), multiple organ failure (MOF), deep vein thrombosis, infection, sepsis, and death. RESULTS: Patients ≤30 years old (y/o) were more likely to sustain gunshot wounds (p = 0.039), while those ≥60 y/o were more likely to fall from a height (p = 0.002). Complications occurred at similar rates for patients ≤30 y/o, 30 to 60 y/o, and ≥60 y/o. There were no differences in lactate, pH, or BE at the time of surgery. For patients ≤30 y/o, there were increased overall complications if pH was <7.30 (p = 0.042) or BE <-6.0 (p = 0.049); patients ≥60 y/o demonstrated more sepsis if BE was <-6.0 (p = 0.046). CONCLUSIONS: EAC aims to definitively manage axial and femoral shaft fractures once patients have been adequately resuscitated to minimize complications. EAC is associated with comparable complication rates in young and elderly patients. Further study is warranted with a larger sample to further validate EAC in elderly patients. LEVEL OF EVIDENCE: level II prospective, comparative study.

The design and synthesis of artificial photosynthetic antennas, reaction centres and membranes.

Artificial antenna systems and reaction centres synthesized in our laboratory are used to illustrate that structural and thermodynamic factors controlling energy and electron transfer in these constructs can be modified to optimize performance. Artificial reaction centres have been incorporated into liposomal membranes where they convert light energy to vectorial redox potential. This redox potential drives a Mitchellian, quinone-based, proton-transporting redox loop that generates a Deltamu H(+) of ca. 4.4 kcal mol(-1) comprising DeltapH ca. 2.1 and Deltapsi ca. 70 mV. In liposomes containing CF(0)F(1)-ATP synthase, this system drives ATP synthesis against an ATP chemical potential similar to that observed in natural systems.

Mimicking photosynthetic solar energy transduction.

Increased understanding of photosynthetic energy conversion and advances in chemical synthesis and instrumentation have made it possible to create artificial nanoscale devices and semibiological hybrids that carry out many of the functions of the natural process. Artificial light-harvesting antennas can be synthesized and linked to artificial reaction centers that convert excitation energy to chemical potential in the form of long-lived charge separation. Artificial reaction centers can form the basis for molecular-level optoelectronic devices. In addition, they may be incorporated into the lipid bilayer membranes of artificial vesicles, where they function as components of light-driven proton pumps that generate transmembrane proton motive force. The proton gradient may be used to synthesize adenosine triphosphate via an ATP synthase enzyme. The overall energy transduction process in the liposomal system mimics the solar energy conversion system of a photosynthetic bacterium. The results of this research illustrate the advantages of designing functional nanoscale devices based on biological paradigms.

Role of T- and B-lymphocytes in pulmonary host defences.

Pulmonary infectious diseases cause significant morbidity and mortality in both industrialized and developing countries. Adaptive immune responses are required to defend the lung against pathogens that survive in normal macrophages and extracellular organisms that evade phagocytosis. Microbes initiate both innate immune responses and specific adaptive immune responses. Innate immune response molecules regulate T-lymphocyte differentiation. Activated T-lymphocytes provide cytokines, which activate macrophages and lytic signals that lyse infected antigen-presenting cells. Antibodies produced by plasma cells facilitate microbial clearance through diverse effector mechanisms including opsonization, complement fixation and antibody-dependent cytotoxicity. Lymphocytes determine the specificity of the immune response and orchestrate effector limbs of the immune response.

Surgery for cervical spinal cord compression in patients with multiple sclerosis.

OBJECTIVE: The goal of this study was to investigate the clinical and paraclinical features, treatment, and outcomes of patients with multiple sclerosis (MS) and coexisting spinal cord compression secondary to either cervical spondylosis or cervical disc disease. Patients with MS commonly experience neurological disabilities that present as myelopathy associated with bladder dysfunction. For some patients with MS, however, this neurological deterioration may result from coexisting spinal cord compression attributable to either spondylosis or a herniated disc. Overlapping symptoms of the two conditions do not allow clear clinical determination of the underlying cause of worsening. METHODS: Patients with MS who underwent cervical decompression surgery were selected. Medical records were retrospectively reviewed, to collect data on their pre- and postoperative clinical courses. RESULTS: Nine women and five men with definite MS were selected for cervical decompression surgery to treat neurological deterioration considered to be at least partially attributable to spinal cord compression. The most common symptoms were progressive myelopathy (n = 13), neck pain (n = 11), and cervical radiculopathy (n = 10). Bladder dysfunction was notably absent among these patients with MS with moderate disabilities. Surgical intervention was frequently delayed because the neurological deterioration was initially thought to be attributable to MS. The majority of patients experienced either improvement or stabilization of their preoperative symptoms in the immediate postoperative period; three subjects (21%) maintained this improvement after a mean follow-up period of 3.8 years. No MS relapses, permanent neurological worsening, or serious complications resulting from surgery or general anesthesia were noted. CONCLUSION: Carefully selected patients with MS and cervical spinal cord compression secondary to either spondylosis or disc disease may benefit from surgical decompression, with minimal associated morbidity. Clinical features (especially neck pain and cervical radiculopathy) and magnetic resonance imaging may assist clinicians in differentiating between the two conditions and may guide appropriate treatment without undue delay.

Gamma delta-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia.

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR beta- and/or delta-chain were intratracheally inoculated with Klebsiella pneumoniae. Gamma delta T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only alpha beta-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in gamma delta-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-gamma and TNF-alpha gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from gamma delta-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking gamma delta-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that gamma delta-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-gamma and TNF-alpha.

Pharmacokinetics of ICG and HPPH-car for the detection of normal and tumor tissue using fluorescence, near-infrared reflectance imaging: a case study.

We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg-1 ICG or 0.3 mg kg-1 HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5-1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.

Localisation and accumulation of a new carotenoporphyrin in two primary tumour models.

We have investigated the tumour-localising properties and in vivo fluorescence kinetics of a hexamethoxylated carotenqporphyrin (CP6) in two primary tumour models: UV-B-induced early skin cancer in hairless mice and chemically induced mucosal dysplasia in the rat palate. CP6 fluorescence kinetics are investigated by measuring in vivo fluorescence spectra and images of the mouse skin and the rat palate at different time points after injection. For the tumour-localising properties, microscopic phase-contrast and fluorescence images are recorded. The in vivo fluorescence kinetics in the mouse skin show localization of CP6 in the tumours. However, fluorescence microscopy images show that CP6 localises in the dermis and structures that are not related to the malignant transformation of the mouse skin. The fluorescence kinetics in the rat palate show a significant correlation between the degree of malignancy and the CP6 fluorescence build-up time in the palate. The microscopic images show that CP6 fluorescence localises in the connective tissue and not in the dysplastic epithelium. In conclusion, CP6 does not localise preferentially in (pre-) cancerous tissue in the two primary tumour models studied here, in contrast to reports about localisation of carotenoporphyrins in transplanted tumours. However, the CP6 build-up time in rat palates correlates with the degree of malignancy and this might possibly be a useful parameter in tumour detection.

Bacterial clearance and survival are dependent on CXC chemokine receptor-2 ligands in a murine model of pulmonary Nocardia asteroides infection.

Survival from murine pulmonary nocardiosis is highly dependent on CXC chemokine receptor-2 (CXCR2) ligand-mediated neutrophil chemotaxis and subsequent clearance of the infectious agent Nocardia asteroides. Intratracheal inoculation of N. asteroides rapidly up-regulated the CXC chemokines macrophage inflammatory protein-2 (MIP-2) and KC within 24 h, with levels remaining elevated through day 3 before returning to near baseline levels by day 7. Coinciding with elevated MIP-2 and KC were the rapid recruitment of neutrophils and clearance of the organism. Anti-Ly-6G Ab-mediated neutrophil depletion before bacterial challenge resulted in strikingly increased mortality to N. asteroides infection. The relative contribution of MIP-2 in neutrophil recruitment was examined by anti-MIP-2 Ab treatment before nocardial infection. MIP-2 neutralization had no detrimental effects on survival, neutrophil recruitment, or bacterial clearance, suggesting the usage of additional or alternative CXCR2-binding ligands. The importance of the CXC family of chemokines was determined by the administration of an anti-CXCR2 Ab capable of blocking ligand binding in vivo. Anti-CXCR2 treatment greatly increased mortality by preventing neutrophil migration into the lung. Paralleling this impaired neutrophil recruitment was a 100-fold increase in lung bacterial burden. Combined, these observations indicate a critical role for neutrophils and CXC chemokines during nocardial pneumonia. These data directly link CXCR2 ligands and neutrophil recruitment and lend further support to the concept of CXC chemokine redundancy. For infections highly dependent on neutrophils, such as nocardial pneumonia, this is of critical importance.

Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice.

Acrylonitrile (AN) and acrylamide (AM) are commonly used in the synthesis of plastics and polymers. In rodents, AM and AN are metabolized to the epoxides glycidamide and cyanoethylene oxide, respectively. The aim of this study was to determine the role of cytochrome P450 in the metabolism of AM and AN in vivo. Wild-type (WT) mice, WT mice pretreated with aminobenzotriazole (ABT, 50 mg/kg ip, 2 h pre-exposure), and mice devoid of cytochrome P450 2E1 (P450 2E1-null) were treated with 50 mg/kg [(13)C]AM po. WT mice and P450 2E1-null mice were treated with 2.5 or 10 mg/kg [(13)C]AN po. Urine was collected for 24 h, and metabolites were characterized using (13)C NMR. WT mice excreted metabolites derived from the epoxides and from direct GSH conjugation with AM or AN. Only metabolites derived from direct GSH conjugation with AM or AN were observed in the urine from ABT-pretreated WT mice and P450 2E1-null mice. On the basis of evaluation of urinary metabolites at these doses, these data suggest that P450 2E1 is possibly the only cytochrome P450 enzyme involved in the metabolism of AM and AN in mice, that inhibiting total P450 activity does not result in new pathways of non-P450 metabolism of AM, and that mice devoid of P450 2E1 do not excrete metabolites of AM or AN that would be produced by oxidation by other cytochrome P450s. P450 2E1-null mice may be an appropriate model for the investigation of the role of oxidative metabolism in the toxicity or carcinogenicity of these compounds.

CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression, which occurs in association with neutrophil dysfunction or deficiency. ELR+ CXC chemokines are a subfamily of chemokines that play a critical role in neutrophil chemotaxis and activation both in vitro and in vivo. We hypothesized that interaction of these ligands with CXC chemokine receptor-2 (CXCR2), their sole murine receptor, is a major component of neutrophil-dependent pulmonary host defense against Aspergillus fumigatus. In immunocompetent animals, neutrophils were recruited to the lung in response to intratracheally administered A. fumigatus conidia. In a model of transient in vivo depletion of neutrophils, animals developed invasive pulmonary aspergillosis, associated with delayed influx of neutrophils into the lung. In both normal and neutrophil-depleted animals, the ELR+ CXC chemokines MIP-2 and KC were induced in response to intratracheal administration of conidia. Ab-mediated neutralization of the common ELR+ CXC chemokine receptor, CXCR2, resulted in development of invasive disease indistinguishable from the disease in neutrophil-depleted animals, while control animals were highly resistant to the development of infection. CXCR2 neutralization was associated with reduced lung neutrophil influx and resulted in a marked increase in mortality compared with controls. In contrast, animals with constitutive lung-specific transgenic expression of KC were resistant to the organism, with reduced mortality and lower lung burden of fungus. We conclude that CXCR2 ligands are essential mediators of host defense against A. fumigatus, and may be important targets in devising future therapeutic strategies in this disease.

Intrapulmonary tumor necrosis factor gene therapy increases bacterial clearance and survival in murine gram-negative pneumonia.

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity in bacterial pneumonia. To augment the expression of TNF within the lung, a recombinant adenoviral vector containing the murine TNF cDNA (Ad5mTNF) has been developed, and the intratracheal administration of this vector resulted in the dose- and time-dependent expression of TNF in the lung, but not systemically. Administration of Ad5mTNF resulted in significant airspace and peribronchial inflammation, with a predominant neutrophil influx by 2 days, and mononuclear cell infiltrates by 4 to 7 days posttreatment. Importantly, the administration of Ad5mTNF at a dose of 1 x 10(8) PFU significantly improved the survival of animals challenged concomitantly with Klebsiella pneumoniae, which occurred in association with enhanced clearance of bacteria from the lung and decreased dissemination of K. pneumoniae to the bloodstream. However, the delivery of higher doses of Ad5mTNF (5 x 10(8) PFU) was not beneficial and in fact the intratracheal administration of a similar dose of control vector (Ad5LacZ) actually enhanced Klebsiella-induced lethality by impairing clearance of K. pneumoniae from the lung. Our studies suggests that the transient transgenic expression of TNF within the lung dose dependently augments antibacterial host defense in murine Klebsiella pneumonia.

Carotenohematoporphyrins as tumor-imaging dyes. Synthesis and in vitro photophysical characterization.

Multichromophoric dyes for use in tumor imaging have been synthesized and photophysically characterized. Structurally, these dyes are dyads and triads that consist of one or two carotenoid polyenes covalently attached to hematoporphyrin (HP) or hematoporphyrin dimethyl ester (HPDME) moieties via ester linkages. The ground-state absorption of each compound shows that the electronic interaction between the chromophores is small. The fluorescence quantum yield for the dyad monocaroteno-HPDME is 0.033 and the dicaroteno-HPDME triads have yields between 0.016 and 0.007, all of which are reduced with respect to the parent compound HPDME (0.09). Global analysis of the transient fluorescence decays of the dyads and triads requires two exponential components (approximately 5-6 ns and approximately 1-2 ns) to fit the data, while a single exponential component with a lifetime of 9.3 ns describes the decay data of the parent HPDME. Possible mechanisms for the observed porphyrin fluorescence quenching by the nearby carotenoid are discussed. Nanosecond transient absorption reveals a carotene triplet with maximum absorption at 560 nm and a 5.0 microsecond lifetime. No transient was detected at 450 nm, indicating rapid (< or = 10 ns) triplet energy transfer from the hematoporphyrin to the carotenoid moieties in fluid as well as in rigid media. The yield of triplet energy transfer from the porphyrin to the carotenoid moiety is unity. Singlet oxygen (O2(1 delta g), studies support the transient absorption data, as none of these compounds is capable of sensitizing O2(1 delta g). Liposome vesicles were used to study the photophysical characteristics of the dyes in phospholipid membranes. Singlet oxygen was not sensitized by the dyads and triads in liposomes. Transient absorption measurements suggest that the triads are substantially aggregated within the phospholipid bilayer, whereas aggregation in the dyads is less severe.

Acquisition of CD24 expression by Lin-CD43+B220(low)ckit(hi) cells coincides with commitment to the B cell lineage.

The present study describes three subsets of bone marrow-derived Lin CD43+B220(low) (B220(low)) progenitor cells which represent distinct stages in hematopoietic development. These populations differ in their expression of CD24 and ckit and the occurrence of IgH gene rearrangement. B220(low) CD24-ckit(hi) progenitors have their IgH loci in germ-line configuration and are multipotent since they can give rise to B cells, T cells and myeloid cells. B220lowckit(hi) cells which have acquired CD24 expression have retained IgH loci in germ-line configuration and the ability to generate B cells, however, they have lost the ability to generate T cells and myeloid cells. Thus acquisition of CD24 by B220(low) cells occurs concurrently with the transition from a multipotent to a lineage-restricted progenitor population. B220(low)CD24+ cells expressing low levels of ckit are also lineage restricted, giving rise to only B cells and have begun D-J(H) rearrangement, implying that initiation of IgH rearrangement coincides with the down-regulation of ckit expression.

Intrapulmonary delivery of tumor necrosis factor agonist peptide augments host defense in murine gram-negative bacterial pneumonia.

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity in Klebsiella pneumonia. Recently, a TNF agonist peptide consisting of the 11-amino-acid TNF binding site (TNF70-80) has been shown to possess many of the leukocyte-activating properties of TNF without the associated toxicity when administered locally or systemically. Given the beneficial effects of TNF in gram-negative pneumonia, we hypothesize that the intratracheal (i.t.) administration of TNF70-80 would augment lung innate immunity in mice challenged with intrapulmonary Klebsiella pneumoniae. The administration of TNF70-80 i.t. to CBA/J mice 7 days prior to, but not concomitantly with, the i.t. delivery of 3 x 10(3) CFU of K. pneumoniae resulted in a marked increase in survival compared to that of animals receiving a control peptide i.t. In addition, pretreatment with TNF70-80 resulted in improved bacterial clearance, which occurred in association with enhanced lung myeloperoxidase activity (as a measure of lung polymorphonuclear leukocyte influx), and increased expression of the important activating cytokines TNF, macrophage inflammatory protein-2, interleukin-12, and gamma interferon compared that for animals receiving control peptide. Finally, the administration of TNF70-80 intraperitoneally resulted in enhanced rather than decreased lethality of Klebsiella pneumonia compared to that for animals receiving either TNF70-80 or control peptide i.t. Our studies suggest that the intrapulmonary, but not systemic, administration of the TNF agonist peptide may serve as an important immunoadjuvant in the treatment of murine Klebsiella pneumonia.

Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane.

Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.)--an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposome's internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of F0F1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of approximately 12 kcal mol(-1), with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.

Generation of lytic natural killer 1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates.

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)-, c-kit+, Sca2+, interleukin (IL)-2/15Rbeta (CD122)- marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5-6 d followed by IL-15 alone for an additional 4-5 d expanded the starting population 30-40-fold and gave rise to a virtually pure population of NK1.1+, CD3- cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9-11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin-, CD44+, CD25-, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I- syngeneic targets, suggesting the existence of novel class I receptors.

Differential effects of Flk-2/Flt-3 ligand and stem cell factor on murine thymic progenitor cells.

Most primitive thymic progenitors, termed CD4(low) cells (CD25- CD44+ CD117+), retain the ability to generate multiple lymphoid lineages. T cell lineage commitment occurs as CD4(low) cells differentiate into pro-T cells (CD25+ CD44+ CD117+). We previously reported that the in vitro cytokine responses of CD4(low) and pro-T cells differ. While Flt-3 ligand (Flt-3L) has been shown to be involved in early bone marrow hemopoiesis, its role in thymopoiesis has not been thoroughly examined. Here, we report that Flt-3L has no significant effect on pro-T cells, either by in vitro proliferation or in fetal thymic organ culture repopulation. In contrast, CD4(low) cells cultured in vitro for 3 days in IL-3 + IL-6 + IL-7 + Flt-3L generated a twofold increase in cell number 21 days after transfer into fetal thymic organ culture that increased to sixfold by day 35 when compared with the corresponding CD4(low) cells cultured in IL-3 + IL-6 + IL-7 + stem cell factor. Additionally, the Flt-3L-cultured CD4(low) cells displayed fetal thymic organ culture repopulation kinetics that more closely approximated those seen with freshly isolated CD4(low) cells. These data suggest that Flt-3L serves as a self-renewal or proliferation/expansion signal for CD4(low) cells, while the effect of stem cell factor is more likely to transduce a differentiation signal, resulting in more rapid repopulation at the expense of cell expansion.