The BMP2 variant L51P restores the osteogenic differentiation of human mesenchymal stromal cells in the presence of intervertebral disc cells.

Spinal fusion is hampered by the presence of remaining intervertebral disc (IVD) tissue and leads to spinal non-union. While the exact mechanism remains unknown, we hypothesise that factors preventing disc ossification, such as antagonists of the bone morphogenetic proteins (BMP), could be responsible for this process. The objective of this study was to investigate spinal non-union using an in vitro human model with a focus on the BMP signalling components and to identify factors contributing to the incomplete and delayed ossification. Human bone marrow-derived mesenchymal stromal cells (MSC) were cocultured with IVD cells in the presence of L51P, a BMP2 variant with osteoinductive potential. The ossification of MSC was evaluated by quantitative reverse transcription polymerase chain reaction (qPCR), alkaline phosphatase (ALP) activity and alizarin red staining. Endogenous expression of major BMP antagonists, namely Gremlin (GREM1), Noggin (NOG) and Chordin (CHRD) was detected in IVD-derived cells, with abundance in nucleus pulposus cells. Osteogenesis of MSC was hindered by IVD cells as shown by reduced alizarin red staining, ALP activity and qPCR. L51P, added to the cocultures, restored mineralisation, blocking the activity of the BMP antagonists secreted by IVD cells. It is possible that the BMP antagonists secreted by IVD cells are responsible for spinal non-unions. The inhibition of BMP antagonists with L51P may result in an efficient and more physiological osteoinduction rather than delivery of exogenous osteogenic factors. Therefore, L51P might represent an attractive therapeutic candidate for bone healing.

The complex biology and contribution of Staphylococcus aureus in atopic dermatitis, current and future therapies.

Atopic dermatitis (AD) is a complex, chronic inflammatory skin disorder affecting more than 10% of U.K. children and is a major cause of occupation-related disability. A subset of patients, particularly those with severe AD, are persistently colonized with Staphylococcus aureus and exacerbation of disease is commonly associated with this bacterium by virtue of increased inflammation and allergic sensitization, aggravated by skin barrier defects. Understanding the complex biology of S. aureus is an important factor when developing new drugs to combat infection. Staphylococcus aureus generates exoproteins that enable invasion and dissemination within the host skin but can also damage the skin and activate the host immune system. Antibiotics are often used by dermatologists to aid clearance of S. aureus; however, these are becoming less effective and chronic usage is discouraged with the emergence of multiple antibiotic-resistant strains. New ways to target S. aureus using monoclonal antibodies and vaccines are now being developed. This review will attempt to evaluate the key biology of S. aureus, current treatment of S. aureus infections in AD and recent advances in developing new anti-S. aureus therapies that have potential in severe AD.

Structural Characterisation Reveals Mechanism of IL-13-Neutralising Monoclonal Antibody Tralokinumab as Inhibition of Binding to IL-13Rα1 and IL-13Rα2.

Interleukin (IL)-13 is a pleiotropic T helper type 2 cytokine frequently associated with asthma and atopic dermatitis. IL-13-mediated signalling is initiated by binding to IL-13Rα1, which then recruits IL-4Rα to form a heterodimeric receptor complex. IL-13 also binds to IL-13Rα2, considered as either a decoy or a key mediator of fibrosis. IL-13-neutralising antibodies act by preventing IL-13 binding to IL-13Rα1, IL-4Rα and/or IL-13Rα2. Tralokinumab (CAT-354) is an IL-13-neutralising human IgG4 monoclonal antibody that has shown clinical benefit in patients with asthma. To decipher how tralokinumab inhibits the effects of IL-13, we determined the structure of tralokinumab Fab in complex with human IL-13 to 2 Å resolution. The structure analysis reveals that tralokinumab prevents IL-13 from binding to both IL-13Rα1 and IL-13Rα2. This is supported by biochemical ligand-receptor interaction assay data. The tralokinumab epitope is mainly composed of residues in helices D and A of IL-13. It is mostly light chain complementarity-determining regions that are driving paratope interactions; the variable light complementarity-determining region 2 plays a key role by providing residue contacts for a network of hydrogen bonds and a salt bridge in the core of binding. The key residues within the paratope contributing to binding were identified as Asp50, Asp51, Ser30 and Lys31. This study demonstrates that tralokinumab prevents the IL-13 pharmacodynamic effect by binding to IL-13 helices A and D, thus preventing IL-13 from interacting with IL-13Rα1 and IL-13Rα2.

Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation.

How the immune system senses aeroallergens and triggers an aberrant inflammation is poorly understood. Dectin-2 is a house dust mite (HDM)-sensing pattern recognition receptor. In a 3-week mouse model of repeated intranasal HDM challenge, anti-Dectin-2 potently attenuated the characteristic allergic inflammation and airway hyper-responsiveness. Anti-Dectin-2 also prevented neutrophil influx following a single HDM challenge. Interestingly, cysteinyl leukotrienes, but not chemokine and cytokine levels were inhibited by anti-Dectin-2 in this acute model, and in ex vivo challenge of cultured alveolar macrophages with HDM. Furthermore in the single-challenge model, zileuton, an inhibitor of leukotriene production, produced a similar effect as Dectin-2 blockade. Together these data suggest alveolar macrophage sensing of HDM by Dectin-2 elicits the production of cysteinyl leukotrienes, and this axis is key for the initiation of airway inflammation to this aeroallergen. Finally, we found Dectin-2-positive infiltrating cells present in bronchial biopsies from asthmatic subjects.

Preclinical development of CAT-354, an IL-13 neutralizing antibody, for the treatment of severe uncontrolled asthma.

BACKGROUND AND PURPOSE: IL-13 is a pleiotropic Th2 cytokine considered likely to play a pivotal role in asthma. Here we describe the preclinical in vitro and in vivo characterization of CAT-354, an IL-13-neutralizing IgG4 monoclonal antibody (mAb), currently in clinical development. EXPERIMENTAL APPROACH: In vitro the potency, specificity and species selectivity of CAT-354 was assayed in TF-1 cells, human umbilical vein endothelial cells and HDLM-2 cells. The ability of CAT-354 to modulate disease-relevant mechanisms was tested in human cells measuring bronchial smooth muscle calcium flux induced by histamine, eotaxin generation by normal lung fibroblasts, CD23 upregulation in peripheral blood mononuclear cells and IgE production by B cells. In vivo CAT-354 was tested on human IL-13-induced air pouch inflammation in mice, ovalbumin-sensitization and challenge in IL-13 humanized mice and antigen challenge in cynomolgus monkeys. KEY RESULTS: CAT-354 has a 165 pM affinity for human IL-13 and functionally neutralized human, human variant associated with asthma and atopy (R130Q) and cynomolgus monkey, but not mouse, IL-13. CAT-354 did not neutralize human IL-4. In vitro CAT-354 functionally inhibited IL-13-induced eotaxin production, an analogue of smooth muscle airways hyperresponsiveness, CD23 upregulation and IgE production. In vivo in humanized mouse and cynomolgus monkey antigen challenge models CAT-354 inhibited airways hyperresponsiveness and bronchoalveolar lavage eosinophilia. CONCLUSIONS AND IMPLICATIONS: CAT-354 is a potent and selective IL-13-neutralizing IgG4 mAb. The preclinical data presented here support the trialling of this mAb in patients with moderate to severe uncontrolled asthma.

Distinct roles for S100a8 in early embryo development and in the maternal deciduum.

S100a8 is a cytosolic protein expressed in myeloid cells where it forms a stable heterodimer with another S100 protein family member, S100a9. The S100a9(-/-) mouse is viable and phenotypically normal, whereas the S100a8(-/-) condition is embryonic lethal. We present evidence that S100a8, without S100a9, has a previously unrecognized role in embryo development between fertilization and the 8-cell stage at embryonic day (E) 2.5. S100a8 also has a second role in the maternal deciduum, where expression is associated with the vasculature from the E8.5 stage to the formation of mature placenta. Uterine natural killer cells that have a role in vascular remodelling colocalise with the S100a8 vascular expression in the metrial triangle. In inflammatory responses in peripheral tissues, S100a8 is a potent chemoattractant and also an anti-oxidant. Both roles may be important in the developing placenta. Thus we highlight two new S100a9-independent roles for S100a8 in early embryo development.

Altered immunomodulating and toxicological properties of degraded Quillaja saponaria Molina saponins.

Quillaja saponins are readily hydrolyzed under physiological conditions, yielding deacylated forms that are significantly less toxic than their precursors. Yet, deacylated saponins are unable to stimulate a strong primary immune response. Although deacylated saponins elicit a strong total IgG response, their capacity to stimulate a Thl type IgG isotype profile (i.e. high levels of IgG2a and IgG2b) has been significantly diminished. Instead, an IgG profile closer to that of a Th2 immune response is stimulated (i.e. high IgG1 levels). Deacylated saponins have also lost their capacity to elicit an effective T cell immunity, as shown by their stimulation of a marginal lymphoproliferative response and their inability to elicit the production of cytotoxic lymphocytes (CTL). Modification of the immune-modulating properties brought by the degradation of quillaja saponins during vaccine storage may change the intended immune response from a Th1 to a Th2 type. This alteration would have negligible effects on vaccines depending on Th2 immunity mediated by neutralizing antibodies. However, the performance of vaccines directed against intracellular pathogens as well as therapeutic cancer vaccines may be seriously affected by the loss of their capacity to stimulate both a Th1 immune response and the production of CTL.

Quantum-cascade laser measurements of stratospheric methane and nitrous oxide.

A tunable quantum-cascade (QC) laser has been flown on NASA's ER-2 high-altitude aircraft to produce the first atmospheric gas measurements with this newly invented device, an important milestone in the QC laser's future planetary, industrial, and commercial applications. Using a cryogenically cooled QC laser during a series of 20 aircraft flights beginning in September 1999 and extending through March 2000, we took measurements of methane (CH(4)) and nitrous oxide (N(2)O) gas up to ~20 km in the stratosphere over North America, Scandinavia, and Russia. The QC laser operating near an 8-mum wavelength was produced by the groups of Capasso and Cho of Bell Laboratories, Lucent Technologies, where QC lasers were invented in 1994. Compared with its companion lead salt diode lasers that were also flown on these flights, the single-mode QC laser cooled to 82 K and produced higher output power (10 mW), narrower laser linewidth (17 MHz), increased measurement precision (a factor of 3), and better spectral stability (~0.1 cm(-1) K). The sensitivity of the QC laser channel was estimated to correspond to a minimum-detectable mixing ratio for methane of approximately 2 parts per billion by volume.

Development of semisynthetic triterpenoid saponin derivatives with immune stimulating activity.

Aldehyde-containing triterpene saponins have adjuvant properties, but only those from Quillaja saponaria Molina stimulate the production of cytotoxic T lymphocytes (CTL) against exogenous antigens. Quillaja saponins have two normonoterpene ester moieties, linked linearly to their fucosyl residue, that play a critical role in the stimulation of CTL. These ester moieties are also responsible for these saponins' instability and toxicity. Based on the structure-activity relationships for the different groups of Q. saponaria saponins, new semi-synthetic analogs were developed that have the adjuvanticity of quillaja saponins, yet with less toxicity and greater stability in aqueous solutions. The quillaja saponin analogs were prepared by replacing their hydrolytically unstable ester groups with another lipophilic chain linked by a stable amide bond on these saponins' glucuronic acid residue. One of these analogs, GPI-0100, is a dodecylamide saponin derivative that stimulates an antibody isotype profile that corresponds to a Th1 type immune response, as well as CTL production against exogenous antigens.

Airborne laser infrared absorption spectrometer (ALIAS-II) for in situ atmospheric measurements of N2O, CH4, CO, HCl, and NO2 from balloon or remotely piloted aircraft platforms.

The Airborne Laser Infrared Absorption Spectrometer II (ALIAS-II) is a lightweight, high-resolution (0.0003-cm(-1)), scanning, mid-infrared absorption spectrometer based on cooled (80 K) lead-salt tunable diode laser sources. It is designed to make in situ measurements in the lower and middle stratosphere on either a balloon platform or high-altitude remotely piloted aircraft. Chemical species that can be measured precisely include long-lived tracers N(2)O and CH(4), the shorter-lived tracer CO, and chemically active species HCl and NO(2). Advances in electronic instrumentation developed for ALIAS-I, with the experience of more than 250 flights on board NASA's ER-2 aircraft, have been implemented in ALIAS-II. The two-channel spectrometer features an open cradle, multipass absorption cell to ensure minimal contamination from inlet and surfaces. Time resolution of the instrument is

Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro.

PURPOSE: 20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. METHODS: Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). RESULTS: Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. CONCLUSIONS: The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

Carcinogenicity of 2',3'-dideoxycytidine in mice.

2',3'-dideoxycytidine (ddC) is a synthetic pyrimidine nucleoside analogue approved for treatment of HIV-positive patients. Previous studies indicated that ddC has the potential to cause thymic lymphoma in C57BL/6 x C3H F1 (hereafter called B6C3F1) mice. In this study, we evaluated the carcinogenic potential of ddC in two different mouse models. B6C3F1 hybrid mice carry ecotropic endogenous proviral sequences that may be activated to cause lymphoma, whereas NIH Swiss mice lack proviral sequences that can be expressed. The mice were treated with ddC by gavage at 500 and 1000 mg/kg/day for up to 6 months (human dose, 2.25 mg/day) and evaluated for toxicity, plasma levels of ddC, and pathological changes. Lymphocyte cell markers from the thymic lymphomas were assessed by immunophenotyping. Expression of p53 protein was evaluated using immunohistochemical staining. Treatment-related thymic lymphomas were present in both mouse models with a higher incidence in NIH Swiss than in B6C3F1 mice. The lymphomas were more prevalent in females than in males of both mouse models. Most mice with thymic lymphoma died during the course of the study. In addition to the thymus, lymphoma was often present in lymph nodes, spleen, and other organs. Lymphomas arose more frequently in mice that lack endogenous ecotropic retroviral sequences and thus were not due to activation of endogenous provirus. During the third month of the study, a few NIH Swiss mice that died had granulosa cell tumors of the ovary. Treatment-related but reversible thymic atrophy was observed in both mouse models. There was a very high correlation between the internal dose of ddC and the incidence of thymic lymphoma in both mouse models. Most of the lymphocytes from control thymuses and ddC-induced lymphomas were positive for Thy-1.2 (pan-T), heat stable antigen, and CD4 and CD8 markers, with no marked differences in the lymphocyte markers of the tumors between sexes or dose groups. p53 protein was detected in only 20% (23/115) of the ddC-induced lymphomas with mostly minimal expression in scattered cells. Because ddC induced lymphomas in two different mouse models, the potential carcinogenic risk should be considered in long-term treatment of HIV-positive patients, especially children and adolescent patients treated with ddC.

Removal of Stratospheric O3 by Radicals: In Situ Measurements of OH, HO2, NO, NO2, ClO, and BrO.

Simultaneous in situ measurements of the concentrations of OH, HO(2), ClO, BrO, NO, and NO(2) demonstrate the predominance of odd-hydrogen and halogen free-radical catalysis in determining the rate of removal of ozone in the lower stratosphere during May 1993. A single catalytic cycle, in which the rate-limiting step is the reaction of HO(2) with ozone, accounted for nearly one-half of the total O(3) removal in this region of the atmosphere. Halogen-radical chemistry was responsible for approximately one-third of the photochemical removal of O(3); reactions involving BrO account for one-half of this loss. Catalytic destruction by NO(2), which for two decades was considered to be the predominant loss process, accounted for less than 20 percent of the O(3) removal. The measurements demonstrate quantitatively the coupling that exists between the radical families. The concentrations of HO(2) and ClO are inversely correlated with those of NO and NO(2). The direct determination of the relative importance of the catalytic loss processes, combined with a demonstration of the reactions linking the hydrogen, halogen, and nitrogen radical concentrations, shows that in the air sampled the rate of O(3) removal was inversely correlated with total NOx, loading.

Aircraft (ER-2) laser infrared absorption spectrometer (ALIAS) for in-situ stratospheric measurements of HCI, N(2)O, CH(4), NO(2), and HNO(3).

The Aircraft Laser Infrared Absorption Spectrometer (ALIAS) instrument is a high-resolution (0.0003 cm-(1)) scanning tunable-diode-laser spectrometer designed, tested, and flown more than 30 times on the National Aeronautics and Space Administration's high-altitude ER-2 aircraft in the Airborne Arctic Stratospheric Expedition of 1991-1992. Using long-path infrared laser absorption spectroscopy to detect optical absorptions as small as 10-(5), ALIAS provides fast, continuous in-situ measurements of key atmospheric gases, with gas detection sensitivities of tens of parts in 10(12). With four lasers and detectors in a single liquid-nitrogen Dewar, simultaneous measurements of HCI, NO(2), HNO(3), CH(4), and N(2)O are made using laser sources at 3.4-8 µm, injected into a 1-m-long, 80-pass Herriott cell.

Chlorine chemistry on polar stratospheric cloud particles in the arctic winter.

Simultaneous in situ measurements of hydrochloric acid (HCl) and chlorine monoxide (ClO) in the Arctic winter vortex showed large HCl losses, of up to 1 part per billion by volume (ppbv), which were correlated with high ClO levels of up to 1.4 ppbv. Air parcel trajectory analysis identified that this conversion of inorganic chlorine occurred at air temperatures of less than 196 +/- 4 kelvin. High ClO was always accompanied by loss of HCI mixing ratios equal to (1/2)(ClO + 2Cl(2)O(2)). These data indicate that the heterogeneous reaction HCl + ClONO(2) --> Cl(2) + HNO(3) on particles of polar stratospheric clouds establishes the chlorine partitioning, which, contrary to earlier notions, begins with an excess of ClONO(2), not HCl.

Chemical loss of ozone in the arctic polar vortex in the winter of 1991-1992.

In situ measurements of chlorine monoxide, bromine monoxide, and ozone are extrapolated globally, with the use of meteorological tracers, to infer the loss rates for ozone in the Arctic lower stratosphere during the Airborne Arctic Stratospheric Expedition II (AASE II) in the winter of 1991-1992. The analysis indicates removal of 15 to 20 percent of ambient ozone because of elevated concentrations of chlorine monoxide and bromine monoxide. Observations during AASE II define rates of removal of chlorine monoxide attributable to reaction with nitrogen dioxide (produced by photolysis of nitric acid) and to production of hydrochloric acid. Ozone loss ceased in March as concentrations of chlorine monoxide declined. Ozone losses could approach 50 percent if regeneration of nitrogen dioxide were inhibited by irreversible removal of nitrogen oxides (denitrification), as presently observed in the Antarctic, or without denitrification if inorganic chlorine concentrations were to double.

Cancer dormancy: studies of the murine BCL1 lymphoma.

Dormancy in the murine BCL1 lymphoma can be induced by several strategies including cytoreductive therapy of mice with large tumor burdens and challenge of allogeneic chimeric mice or idiotype-immunized mice with BCL1 tumor. Dormant tumor cells were isolated from the spleens of the chimeric mice and the majority were shown to be noncycling. In idiotype-immunized mice that had lost dormancy, tumor growth occurred at a relatively rapid rate. A proportion of idiotype-immunized mice that had lost dormancy spontaneously regressed and then again relapsed; in these mice, the serum antiidiotypic levels were inversely related to the tumor burden.

Intracellular routing rather than cross-linking or rate of internalization determines the potency of immunotoxins directed against different epitopes of sIgD on murine B cells.

Several variables influence the potency of an immunotoxin (IT) prepared with a monoclonal antibody (mAb) and ricin A chain (IT-A). These include the affinity of the mAb, the nature and density of the target antigen (Ag), the epitope on the target Ag bound by the mAb, the type of cell target, and the rate of endocytosis and route of internalization of the bound IT-A. In a previous report, we demonstrated that anti-delta mAbs directed against epitopes which are putatively more proximal to the plasma membrane make more effective IT-As than those directed against epitopes that are putatively more distal from the plasma membrane. It is known that the latter mAbs cross-link sIgD less effectively than the former. Therefore, in the present study, we determined whether the differential cytotoxicity of IT-As directed against these epitopes is related to their ability to cross-link their specific surface antigen (sIgD). We further determined whether they were internalized at different rates by normal B cells. Our results show that neither cross-linking nor rate of internalization account for the different potencies of anti-Fc vs anti-Fd IT-As. However, when these IT-As were used in the presence of the lysosomotropic agent chloroquine, the less potent IT-A became 100-fold more potent and was as cytotoxic as the effective anti-Fc IT-A. Taken together with the results of other studies, these findings further support the hypothesis that the epitope specificity of a given mAb may be an important factor in determining the intracellular routing of an IT-A after internalization.

Evaluation of ricin A chain-containing immunotoxins directed against different epitopes on the delta-chain of cell surface-associated IgD on murine B cells.

Over the past decade, immunotoxins (IT) composed of mAb covalently coupled to toxins or their subunits have been developed for the treatment of malignancies and autoimmune diseases. Despite specific binding to target cells, not every mAb makes a therapeutically potent ricin A chain-containing IT (IT-A). A number of variables influence the potency of a mAb as an IT-A, including the affinity of the mAb, the nature and density of the cell surface Ag, and the type of target cell used. The present report investigates the influence of the epitope specificity of a mAb on the effectiveness of that mAb as an IT-A. Seven mAb directed against different regions of the mouse delta H chain of surface IgD, were conjugated to deglycosylated ricin A chain, and tested for their ability to kill murine B cells. The panel of IT-A had similar A chain activities and similar binding avidities. However, the mAb directed against epitopes in the Fc portion of surface IgD made more effective IT-A than those directed against epitopes in the Fd region. Overall, the anti-Fc-A were approximately 60- to 150-fold more toxic than the anti-Fd-A. Taken together with previous studies, these findings suggest that the epitope on a target Ag recognized by a given mAb is an important variable in determining the potency of a mAb as an IT-A.

Immunoconjugates containing ricin A chain and either human anti-gp41 or CD4 kill H9 cells infected with different isolates of HIV, but do not inhibit normal T or B cell function.

We have previously reported that immunoconjugates composed of deglycosylated ricin A chain coupled to either recombinant (r) CD4 or two different monoclonal human anti-gp41 antibodies (rCD4-dgA and anti-gp41-dgA, respectively) are specifically toxic to HIV-infected lines of human T cells (H9) and monocytes (U937). In order to further evaluate these immunoconjugates as potential therapeutic reagents for killing HIV-infected cells, H9 cells infected with five different isolates of HIV were used as target cells in vitro. All three HIV-specific immunoconjugates were toxic to H9 cells infected with each HIV isolate, but were virtually nontoxic to uninfected cells. Chloroquine markedly potentiated the specific toxicity of all three conjugates, particularly the anti-gp41-dgAs. None of the conjugates affected the ability of normal peripheral blood B cells to respond to mitogen or the ability of normal T cells to respond to alloantigens.