Mesenchymal stem/stromal cell-based therapies for severe viral pneumonia: therapeutic potential and challenges.

Severe viral pneumonia is a significant cause of morbidity and mortality globally, whether due to outbreaks of endemic viruses, periodic viral epidemics, or the rarer but devastating global viral pandemics. While limited anti-viral therapies exist, there is a paucity of direct therapies to directly attenuate viral pneumonia-induced lung injury, and management therefore remains largely supportive. Mesenchymal stromal/stem cells (MSCs) are receiving considerable attention as a cytotherapeutic for viral pneumonia. Several properties of MSCs position them as a promising therapeutic strategy for viral pneumonia-induced lung injury as demonstrated in pre-clinical studies in relevant models. More recently, early phase clinical studies have demonstrated a reassuring safety profile of these cells. These investigations have taken on an added importance and urgency during the COVID-19 pandemic, with multiple trials in progress across the globe. In parallel with clinical translation, strategies are being investigated to enhance the therapeutic potential of these cells in vivo, with different MSC tissue sources, specific cellular products including cell-free options, and strategies to 'licence' or 'pre-activate' these cells, all being explored. This review will assess the therapeutic potential of MSC-based therapies for severe viral pneumonia. It will describe the aetiology and epidemiology of severe viral pneumonia, describe current therapeutic approaches, and examine the data suggesting therapeutic potential of MSCs for severe viral pneumonia in pre-clinical and clinical studies. The challenges and opportunities for MSC-based therapies will then be considered.

Purified ß-glucans from the Shiitake mushroom ameliorates antibiotic-resistant Klebsiella pneumoniae-induced pulmonary sepsis.

Bacterial infection remains the main cause of acute respiratory distress syndrome and is a leading cause of death and disability in critically ill patients. Here we report on the use of purified ß-glucan (lentinan) extracts from Lentinus edodes (Shiitake) mushroom that can reduce infection by a multidrug-resistant clinical isolate of Klebsiella pneumoniae in a rodent pneumonia model, likely through immunomodulation. Adult male Sprague-Dawley rats were subjected to intra-tracheal administration of K. pneumoniae to induce pulmonary sepsis and randomized to three groups; vehicle control (Vehicle, n = 12), commercial lentinan (CL, n = 8) or in-house extracted lentinan (IHL, n = 8) were administered intravenously 1 h postinfection. Physiological parameters and blood gas analysis were measured, bacterial counts from bronchoalveolar-lavage (BAL) were determined, along with differential staining of white cells and measurement of protein concentration in BAL 48 h after pneumonia induction. Use of IHL extract significantly decreased BAL CFU counts. Both CL and IHL extractions reduced protein concentration in BAL. Use of IHL resulted in an improvement in physiological parameters compared to controls and CL. In conclusion, administration of lentinan to treat sepsis-induced lung injury appears safe and effective and may exert its effects in an immunomodulatory manner.

The Impact of Therapists' Responses to Resistance to Change: A Sequential Analysis of Therapist Client Interactions in Motivational Interviewing.

AIMS: The study aims to examine how therapists trained in motivational interviewing (MI) respond to resistance and whether this has an impact on subsequent client speech. METHODS: Fifty recorded Motivational Enhancement Therapy sessions were examined using a sequential behavioural coding method for speech. Client counter-change talk formed the baseline for coding and categorizing subsequent therapist speech and the following client speech. Transitional analysis identified the probable occurrence of specific therapist and client utterances at each stage. RESULTS: Following client expressed resistance or counter-change talk, MI consistent therapist utterances were most commonly observed. A moderate to strong predictive relationship was found between MI-consistent therapist speech and subsequent client change talk. A moderate predictive relationship was found between therapist MI-consistent behaviours and client ambivalence. A moderate to strong predictive relationship was found between MI-inconsistent therapist speech and subsequent client counter-change talk and a weak negative predictive relationship was found between MI-inconsistent therapist speech and client expressed ambivalence. CONCLUSIONS: In the face of initial expressed resistance to change, MI-consistent therapist speech appears to increase subsequent client utterances regarding intentions to change drinking behaviour.

Mesenchymal stromal cell therapies: potential and pitfalls for ARDS.

Mesenchymal stem/stromal cells (MSCs) offer considerable promise as a novel therapeutic strategy for acute respiratory distress syndrome (ARDS). MSCs may be able to "reprogramme" the immune response to reduce destructive inflammatory elements while preserving the host response to pathogens. In addition, MSCs may be able to enhance the repair and resolution of lung injury. Resolution of ARDS is impeded by destruction of the integrity of the epithelial barrier, which inhibits alveolar fluid clearance and depletes surfactant. MSCs appear to restore epithelial and endothelial function, via both paracrine and cell contact dependent effects. ARDS is frequently a component of a generalized process resulting in dysfunction and failure of multiple organs. MSCs have been demonstrated to decrease injury and/or restore function in other organs, including the kidney, liver and heart. MSCs may directly attenuate bacterial sepsis, the commonest and most severe cause of ALI/ARDS. The fact that MSCs are in clinical studies for a wide range of disease processes is a clear advantage for translating MSCs to clinical testing in patients with ARDS. However, some important knowledge gaps exist that may impede clinical translation. The ultimate success of MSCs as a therapy for patients with ARDS will likely be dependent on a greater knowledge of their mechanisms of action and the determination of the optimal strategies for their use in the clinical setting.

Pulmonary overexpression of inhibitor κBα decreases the severity of ventilator-induced lung injury in a rat model.

BACKGROUND: Activation of the nuclear factor-κB (NF-κB) pathway is central to the pathogenesis of lung injury and inflammation. We determined whether targeted overexpression of inhibitor-κBα (IκBα) in the lung could decrease the severity of ventilator-induced lung injury (VILI). METHODS: Anaesthetized adult male Sprague-Dawley rats were randomly allocated to undergo intratracheal instillation of: (i) vehicle alone (surfactant, n=10); (ii) 1×10(10) adeno-associated virus encoding IκBα (AAV-IκBα, n=10); (iii) 5×10(10) AAV-IκBα (n=10); and (iv) 1×10(10) AAV-Null (n=5). This was followed by 4 h of injurious mechanical ventilation. Subsequent experiments examined the effect of IκBα overexpression in animals undergoing 'protective' mechanical ventilation. RESULTS: IκBα overexpression increased survival duration at both the lower [3.8 h (0.4)] and higher [3.6 h (0.7)] doses compared with vehicle [2.7 h (1.0)] or the null transgene [2.2 h (0.8)]. IκBα overexpression reduced the alveolar-arterial oxygen gradient (kPa) at both the lower [53 (21)] and higher [52 (19)] doses compared with vehicle [75 (8.5)] or the null transgene [70 (15)], decreased alveolar neutrophil infiltration, and reduced alveolar concentrations of interleukin (IL)-1ß and IL-10. The lower IκBα dose was as effective as the higher dose. IκBα overexpression had no effect in the setting of protective lung ventilation. CONCLUSIONS: Inhibition of pulmonary NF-κB activity by IκBα overexpression reduced the severity of VILI in a rat model.

Oxidative stress promotes hypertension and albuminuria during the autoimmune disease systemic lupus erythematosus.

Several lines of evidence suggest that essential hypertension originates from an autoimmune-mediated mechanism. One consequence of chronic immune activation is the generation of oxygen-derived free radicals, resulting in oxidative stress. Renal oxidative stress has direct prohypertensive actions on renal microvascular and tubular function. Whether oxidative stress contributes to the prevalent hypertension associated with autoimmune disease is not clear. We showed previously that female NZBWF1 mice, an established model of the autoimmune disease systemic lupus erythematosus (SLE), develop hypertension associated with renal oxidative stress. In the present study we tested the hypothesis that oxidative stress contributes to autoimmune-mediated hypertension by treating SLE and control (NZW/LacJ) mice with tempol (2.0 mmol/L) and apocynin (1.5 mmol/L) in the drinking water for 4 weeks. Although the treatment did not alter SLE disease activity (assessed by plasma double-stranded DNA autoantibodies), blood pressure and renal injury (urinary albumin) were reduced in the treated SLE mice. Tempol plus apocynin-treated SLE mice had reduced expression of nitrosylated proteins in the renal cortex, as well as reduced urinary and renal cortical hydrogen peroxide, suggesting that treatment reduced renal markers of oxidative stress. These data suggest that renal oxidative stress plays an important mechanistic role in the development of autoimmune-mediated hypertension.

Placental ischemia impairs middle cerebral artery myogenic responses in the pregnant rat.

One potential mechanism contributing to the increased risk for encephalopathies in women with preeclampsia is altered cerebral vascular autoregulation resulting from impaired myogenic tone. Whether placental ischemia, a commonly proposed initiator of preeclampsia, alters cerebral vascular function is unknown. This study tested the hypothesis that placental ischemia in pregnant rats (caused by reduced uterine perfusion pressure [RUPP]) leads to impaired myogenic responses in middle cerebral arteries. Mean arterial pressure was increased by RUPP (135±3 mm Hg) compared with normal pregnant rats (103±2 mm Hg) and nonpregnant controls (116±1 mm Hg). Middle cerebral arteries from rats euthanized on gestation day 19 were assessed in a pressure arteriograph under active (+Ca(2+)) and passive (0 Ca(2+)) conditions, whereas luminal pressure was varied between 25 and 150 mm Hg. The slope of the relationship between tone and pressure in the middle cerebral artery was 0.08±0.01 in control rats and was similar in normal pregnant rats (0.05±0.01). In the RUPP model of placental ischemia, this relationship was markedly reduced (slope=0.01±0.00; P<0.05). Endothelial dependent and independent dilation was not different between groups, nor was there evidence of vascular remodeling assessed by the wall:lumen ratio and calculated wall stress. The impaired myogenic response was associated with brain edema measured by percentage of water content (RUPP P<0.05 versus control and normal pregnant rats). This study demonstrates that placental ischemia in pregnant rats leads to impaired myogenic tone in the middle cerebral arteries and that the RUPP model is a potentially important tool to examine mechanisms leading to encephalopathy during preeclamptic pregnancies.

Treatment of mast cells with carbon dioxide suppresses degranulation via a novel mechanism involving repression of increased intracellular calcium levels.

BACKGROUND: Intranasal noninhaled delivery of carbon dioxide (CO2) is efficacious in the symptomatic treatment of seasonal allergic rhinitis. The goal of this study was to determine whether and how 100% CO2 inhibits mast cell degranulation, thereby possibly contributing to the reduction of symptoms in seasonal allergic rhinitis. METHODS: Peritoneal mast cells isolated from rats and labelled with sulforhodamine-B (SFRM-B) were used to determine whether CO2 treatment could block mast cell degranulation and histamine release in response to 48/80. In addition, the effect of CO2 on intracellular calcium levels in unstimulated and stimulated mast cells was determined by fluorescent microscopy. RESULTS: Treatment with 48/80 caused >90% of mast cells containing SFRM-B to degranulate, resulting in a marked decrease in the fluorescent intensity within the mast cells, and simultaneously causing a significant increase in histamine release. Significantly, the stimulatory effect of 48/80 on fluorescent intensity and histamine levels was greatly inhibited (>95%) to near control levels by pretreatment with 100% CO2. Treatment with 48/80 also caused a robust transient increase in intracellular calcium, whereas pretreatment with CO2 repressed the increase in calcium (>70%) in response to 48/80. CONCLUSIONS: Results from this study provide the first evidence of a unique regulatory mechanism by which CO2 inhibits mast cell degranulation and histamine release by repressing stimulated increases in intracellular calcium. Thus, our data provide a plausible explanation for the reported therapeutic benefit of noninhaled intranasal delivery of 100% CO2 to treat allergic rhinitis.

Effects on lung function, symptoms, and bronchial hyperreactivity of low-dose inhaled beclomethasone dipropionate given with HFA-134a or CFC propellant.

The aim of this study was to compare the efficacy of BDP 200 microg bid via metered dose inhaler, using HFA-134a (Chiesi Farmaceutici S.p.A., Parma, Italy) versus CFC (Becotide, Allen & Hanburys, U.K.) as a propellant. 172 adult patients (86 in each group) with stable mild persistent asthma who completed a 7-day run-in period were randomized to receive a 6-week treatment in a double-blind, double dummy, parallel-group design; 164 patients completed the study. Morning and evening PEFR, use of rescue salbutamol, number of day-time and night-time asthma attacks, number of night-time awakenings and clinical symptoms were recorded daily on a diary card. Pulmonary function tests (FEV(1), FVC, PEFR, and MEF(50)) were measured at the clinic before and after the 1-week run-in period, and after 3 and 6 weeks of treatment. A challenge test with inhaled methacholine was completed at baseline and at the end of the treatment period to assess potential bronchial hyper-reactivity in a subgroup of subjects (n = 65; 34 HFA, 31 CFC). In accordance with asthma of mild severity (FEV(1) predicted over 90% in both groups), a small improvement in lung function compared to baseline was seen for both treatments, significantly for FEV(1) in BDP HFA and MEF(50) in both groups. The two formulations of BDP had similar efficacy for the primary outcome variable morning PEFR (ITT population mean difference 5.8 L/min; C.I. -4.9 to +16.5) as well as for the secondary outcomes of evening PEFR and clinic FEV(1). There were small improvements in methacholine PD(20) and PC20 in both groups, with no significant difference between treatments. A total of 22 and 19 drug-related adverse events were reported in the BDP HFA and CFC groups, respectively; most events were of seasonal nature or were local effects due to the use of inhaled corticosteroids. It can be concluded that the newly developed formulation of BDP given via HFA-134a seems to provide similar asthma control, compared with the same low daily dose of the active drug delivered via CFC. Further studies are needed using higher doses in moderate to severe asthma to confirm these preliminary findings.

General health status of children with D-transposition of the great arteries after the arterial switch operation.

BACKGROUND: To study the long-term impact on general health status of D-transposition of the great arteries (D-TGA) after the arterial switch operation (ASO) during infancy, we asked parents to complete the Child Health Questionnaire, Parent Form-50 when their children were 8 years old. METHODS AND RESULTS: Of 160 eligible patients, questionnaires were completed for 155 subjects (96%). Median age at surgery was 6 days (range 1 to 67 days), and median age at completion of the Child Health Questionnaire was 8.1 years (7.6 to 10.0 years). Subsequent to questionnaire completion, children underwent psychometric testing. Mean Physical Health Summary and Psychosocial Summary scores were 54.0+/-6.1 and 49.7+/-9.9, respectively, which were similar to those of normal subjects. Compared with the normative sample, parents of D-TGA patients reported more problems with attention, learning, and speech, as well as greater frequency of developmental delay (P<0.001 for each). Worse Psychosocial Summary scores were significantly associated with lower full-scale IQ (P=0.001) and lower achievement in reading (P=0.005) and math (P=0.007). Worse Physical Health Summary scores were associated with longer hospital stay after the ASO (P=0.02). General health status scores were not significantly related to presence of ventricular septal defect, age at surgery, perfusion variables during the ASO, sex, or history of cardiac reoperation. CONCLUSIONS: At age 8 years, children with D-TGA after ASO have an overall physical and psychosocial health status similar to that of the general population. Lower IQ and academic achievement are associated with worse psychosocial health status, whereas longer hospital course after initial surgery is associated with worse physical health status.

Accumulation of atmospheric and sedimentary PCBs and toxaphene in a Lake Michigan food web.

Seston, sediment, settling organic matter, and food web members were collected from Grand Traverse Bay, Lake Michigan, between April 1997 and September 1998 to examine PCB and toxaphene biomagnification. Stable isotopes of nitrogen and carbon were analyzed in samples and used to establish trophic structure of the food web and to determine the importance of atmospheric versus sedimentary sources in delivering PCBs to the food web. Nitrogen isotopes were confounded by multiple variables in this system, particularly seasonal variation, and did not display a simple pattern of enrichment among trophic levels. However, delta13C displayed little seasonal variation and was positively correlated with PCB concentrations among food web members (r2 = 0.69). Plots of delta13C vs PCBs separate food web members into three distinct groupings comprised of invertebrates, primary forage fish, and predatory fish. Stable isotope values of the primary organic sources indicate that the atmosphere, and not the sediment, is the most likely source of PCBs to the food web of Lake Michigan. Additionally, we suggest that seston may be important in delivering PCBs to pelagic food web members and species that receive a majority of their nutrition through pelagic sources. In contrast, settling particles are implicated in delivering PCBs to benthic organisms and Mysis relicta.

Mitochondrial and peroxisomal beta-oxidation capacities of organs from a non-oilseed plant.

Until recently, beta-oxidation was believed to be exclusively located in the peroxisomes of all higher plants. Whilst this is true for germinating oilseeds undergoing gluconeogenesis, evidence demonstrating mitochondrial beta-oxidation in other plant systems has refuted this central dogma of plant lipid metabolism. This report describes a comparative study of the dual mitochondrial and peroxisomal beta-oxidation capacities of plant organs. Oxidation of [1-(14)C] palmitate was measured in the cotyledons, plumules and radicles of Pisum sativum L., which is a starchy seed, over a 14 day period from the commencement of imbibition. Respiratory chain inhibitors were used for differentiating between mitochondrial and peroxisomal beta-oxidation. Peroxisomal beta-oxidation gave a steady, baseline rate and, in the early stages of seedling development, accounted for 70-100% of the beta-oxidation observed. Mitochondrial beta-oxidation gave peaks of activity at days 7 and 10-11, accounting for up to 82% of the total beta-oxidation activity at these times. These peaks coincide with key stages of seedling development and were not observed when normal development was disrupted by growth in the dark. Peroxisomal beta-oxidation was unaffected by etiolation. Since mitochondrial beta-oxidation was overt only during times of intense biosynthetic activity it might be switched on or off during seedling development. In contrast, peroxisomes maintained a continuous, low beta-oxidation activity that could be essential in removing harmful free fatty acids, e.g. those produced by protein and lipid turnover.

Comparison of pharmacokinetics and systemic effects of inhaled fluticasone propionate in patients with asthma and healthy volunteers: a randomised crossover study.

BACKGROUND: Inhaled corticosteroids are currently the cornerstone of asthma treatment. Some studies of high-dose fluticasone propionate in patients with no or mild asthma have, however, suggested substantial systemic absorption. We investigated the pharmacokinetics of fluticasone propionate in patients with asthma receiving appropriate doses for severity. METHODS: We did a double-blind, randomised, crossover study in 11 patients with asthma and 13 matched healthy controls (age 20-65 years; asthma patients forced expiratory volume in 1 s <75% and stable on high-dose inhaled corticosteroids). Patients received one 1000 microg intravenous dose or 1000 microg daily for 7 days inhaled (via spacer device) fluticasone propionate. In the 12 h after dosing, we monitored plasma fluticasone propionate and cortisol concentrations by mass spectrometry and competitive immunoassay with use of direct chemiluminescence. Analysis was by intention to treat. FINDINGS: After inhalation, geometric mean values were significantly lower in the asthma group than in controls for fluticasone propionate plasma area under curve (1082 [95% CI 850-1451] vs 2815 pg mL(-1) h(-1) [2262-3949], -62% difference [45-72]; p<0.001), maximum concentrations (117 [91-159] vs 383 pg/mL [302-546], -68% [-50 to -81]; p<0.001), and systemic bioavailability (10.1 [7.9-14.0] vs 21.4% [15.4-32.2], -54% [-27 to -70]; p=0.001). Intravenous-dose clearance, volume of distribution at steady state, plasma half-life, and mean residence time, were similar in the two groups. Less suppression of plasma cortisol concentrations was seen in the asthma group than in controls 4-12 h after inhaled fluticasone propionate. INTERPRETATION: Systemic availability of fluticasone propionate is substantially less in patients with moderate to severe asthma than in healthy controls. Inhaled corticosteroids that are absorbed through the lungs need to be assessed in patients who are receiving doses appropriate for disease severity, and not in normal volunteers.

Pea chloroplast carnitine acetyltransferase.

The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is sufficient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA <--> short-chain acylcarnitine + CoASH. CAT activity was detected in thc chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate. activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial purification of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is sufficient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate specificity, handling C2-C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts.

Contribution of mitochondria and peroxisomes to palmitate oxidation in pea tissues.

Total, mitochondrial and peroxisomal palmitate oxidation capacities were compared in pea, from the dry seed to 14 days after imbibition. Total beta-oxidation varied over the measured time period and showed four peaks of activity at day 2, days 5-6, day 10 and days 12-13. The contribution of peroxisomal and mitochondrial beta-oxidation to this overall beta-oxidation varied. Over the first 48 h of seed germination, peroxisomal beta-oxidation accounted for 80-100% of the total observed beta-oxidation. The larger peaks of beta-oxidation at days 5-6, day 10 and days 12-13 were due primarily to mitochondrial beta-oxidation activity, which accounted for 70-90% of the observed total beta-oxidation at these times. The peaks of activity are related to observed stages in seedling development.

Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth.

Acyl-CoA dehydrogenase activity has been measured in homogenates of post-imbibition to 14-day-old hydroponically grown pea seeds at daily intervals, using C(4), C(12) and C(16) acyl-CoA substrates. The activity peaks of the different chain-length acyl-CoA dehydrogenases did not transpose at all points and the ratios of the chain-length activities were not constant. It therefore has to be concluded that more than one dehydrogenase is present in pea mitochondria. There was a post-imbibition initial surge of activity with short- and mid-chain-length substrates. The C(16)-handling enzyme first peaked at 3-4 days, which coincided with the onset of plumule unfurling and greening. Further peaks were observed with all three substrates, coinciding with secondary root formation and leaf enlargement and later with cotyledon degeneration. Overall activity showed that the long-chain acyl-CoA dehydrogenase was much more active than the short-chain acyl-CoA dehydrogenase.

Simultaneous X-Ray and TeV Observations of a Rapid Flare from Markarian 421.

Mrk 421 was observed for about 2 days with BeppoSAX in 1998 April as part of a worldwide multiwavelength campaign. A large, well-defined flare was observed in X-rays. The same flare was observed simultaneously at TeV energies by the Whipple Observatory gamma-ray telescope. These data provide (1) the first evidence that the X-ray and TeV intensities are well correlated on timescales of hours and (2) the first exactly simultaneous X-ray and TeV spectra. The results imply that the X-ray and TeV photons derive from the same region and from the same population of relativistic electrons. The physical parameters deduced from a homogeneous synchrotron self-Compton model for the spectral energy distribution yield electron cooling times close to the observed variability timescales.

Bronchodilator response to salbutamol after chronic dosing with salmeterol or placebo.

It has been hypothesized that regular inhaled beta2-agonist therapy causes desensitization of beta2-receptors. The aim of this study was to define whether beta2-receptor desensitization occurs after treatment with the long-acting beta2-agonist salmeterol, assessed by measuring the bronchodilator response to cumulative repeated doses of inhaled salbutamol before and after treatment. Forty nine stable adult patients with asthma were randomized to receive either salmeterol 50 microg b.d. or placebo b.d. from an Accuhaler for 4 weeks after an initial 2 week run-in period without beta2-agonists. All patients were receiving inhaled corticosteroids. Bronchodilator responsiveness to cumulative repeated doses of inhaled salbutamol were measured before and 12 and 36 h after the last dose of study treatment. The primary efficacy endpoint was the peak forced expiratory volume in one second (FEV1) response before and after treatment. There were no significant differences between the two treatment groups in the absolute peak FEV1 or maximal peak expiratory flow (PEF) results 12 or 36 h after the last dose of study treatment. Significantly higher clinic lung function and diary card parameters were noted in the salmeterol group when compared to the placebo-treated patients, demonstrating the beneficial effects of regular salmeterol. Regular salmeterol usage did not lead to reduced efficacy of usual or higher than usual doses of salbutamol.

Synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptors for Leishmania major alpha-D-mannosylphosphate transferase.

Protozoan parasites of the genus Leishmania synthesise lipophosphoglycans, phosphoglycans and proteophosphoglycans that contain phosphosaccharide-repeat units of [-6Gal beta 1-4Man alpha 1-P-]. In this study, a GDP-Man-dependent alpha-mannosylphosphate-transferase activity was detected in washed Leishmania major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. The divalent-cation-dependent alpha-mannosylphosphate-transferase activity had an apparent K(m) for GDP-Man of about 15-20 microM and a pH optimum of 7.0. The activity showed a requirement for a non-reducing terminal beta Gal residue and for one or more phosphodiester units preceding the acceptor site. Based on these results, the activity may be defined as a GDP-Man: Gal beta 1-4Man alpha 1-P-R alpha-mannosylphosphate-transferase. This acceptor specificity is consistent with a role for the alpha-mannosylphosphate transferase in the elongation of phosphosaccharide-repeat domains of Leishmania glycoconjugates rather than in the priming of these domains. An identical or similar activity must exist in the amastigote forms of the Leishmania that produce and secrete proteophosphoglycan material and the activity therefore represents a feasible target for the development of chemotherapeutics.