Research report - quark

S c i e n t i f i c R e p o r t s
I m m u n o p a t h o l o g y
Email Address: immunosec@mail.wch.sa.gov.au
Head: Professor Antonio Ferrante
1 Intracellular signalling in neonatal T lymphocytes
CST Hii, A Ferrante, M Costabile, D Roberton (University Dept of Paediatrics),AW Murray (School of Biological Science, Flinders University); Immunological responses in the newborn are poor, which in part explains the increased susceptibility of the newborn,especially when born prematurely, to bacterial and viral infections. A contributing factor is the poor response ofT lymphocytes. The reasons behind the decreased T cell responses are poorly understood. Our studies have concentratedon the ability of pharmacological agents, A23187 and phorbol ester, or phytohaemagglutinin and phorbol ester, tostimulate the activities of crucial signalling molecules that regulate T cell responses. These signalling molecules includethe extracellular signal-regulated protein kinase (ERK) and c-jun N terminal kinase (JNK) families of the mitogen-activated protein (MAP) kinases. In previous studies, we demonstrated that MAP kinases play central roles in regulating T cell cytokine production. Our results demonstrate that the above T cell agonists were unable to stimulate the activities ofthe MAP kinases in neonate T cells to the same extent as in adult T cells. These differences were unlikely to be due todifferences in the levels of surface receptor expression between neonate and adult cells since some of the agonists that wehave chosen bypass surface receptors. We have also excluded differences in the levels of MAP kinase expression as a causesince both populations of cells were found to express similar levels of these kinases.
Our hypothesis is that an over-active MKP1 (MAP kinase phosphatase-1), a phosphatase which dephosphorylates/inactivatesERK and JNK, in neonatal T lymphocytes may limit the degree to which the MAP kinases can be stimulated. Western blotanalyses confirmed that the activating tyrosine and threonine residues of ERK in neonatal T cells were phosphorylated toa lower degree than in adult cells.
Interestingly, we have now demonstrated that orthovanadate, a known phosphotyrosine phosphatase inhibitor, was able todirectly stimulate neonatal T cell proliferation even when the neonatal T cells were unresponsive to phytohaemagglutininand phorbol ester, established T cell mitogens. We are currently exploring whether low levels of orthovanadate cantransform mitogen-unresponsive neonatal T cells into mitogen responsive cells.
2 Intracellular signalling pathways involved in cartilage injury in arthritis
CST Hii, LA Marin, G Parashakis, D Halliday with A Ferrante, D Roberton (University Department of Paediatrics),and AW Murray (School of Biological Science, Flinders University); Arthritic diseases such as chronic juvenile arthritis remain a major burden to our community. The prominent feature ofthese diseases is inflammation of the joint and the resultant destruction of cartilage. Neutrophils have been proposed toplay a prominent role in the acute exacerbation of rheumatoid arthritis. Our previous studies demonstrated that bothadherence of neutrophils to cartilage and the subsequent secretion of elastase are mandatory for the degradation ofproteoglycan. Studies in numerous cell-types have demonstrated that adherence of cells to extracellular matrices activatesa number of signalling molecules, including protein kinase C (PKC), tyrosine kinases, phosphatidylinositol 3 kinase (PI-3-kinase), the extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase. Each of these kinases havebeen reported to positively modulate one or more neutrophil responses. The aims of this study were therefore to determinewhether interfering with the activities of these kinases would affect the ability of neutrophils to degrade cartilage proteoglycan.
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We demonstrated that wortmannin or LY294002, specific inhibitors of PI-3-kinase, and SB203580, a specific inhibitor ofp38 MAP kinase, suppressed the ability of neutrophils to degrade proteoglycan. Proteoglycan degradation was not affectedby either GF109203X, a specific inhibitor of PKC or by PD98059, a specific inhibitor of MAPK/ERK kinase. More importantly,the degradation of cartilage by neutrophils isolated from the joints of chronic juvenile rheumatoid arthritis patients wasalso inhibited by wortmannin. These results suggest that PI-3-kinase and p38, but not PKC or ERK, regulate the cartilagedegrading properties of neutrophils. Neither wortmannin nor SB203580 inhibited the adherence of neutrophils to cartilagepieces, demonstrating that a step(s) post adherence was inhibited. Consistent with this, we have found that neutrophildegranulation and respiratory burst were inhibited by wortmannin and SB503250. We have also confirmed that incubationof neutrophils with cartilage resulted in activation of PI-3-kinase and p38 MAP kinase. The time courses of neutrophiladherence and increases in kinase activities are consistent with adherence-induced activation of these kinases. These dataidentify PI-3-kinase and p38 as being involved in regulating neutrophil-mediated degradation of proteoglycan.
3 Tumour necrosis factor (TNF) and TNF mimetic peptides in infection and immunity
A Ferrante, CST Hii, C Haddad, AM Tan, DA Rathjen, N Moghaddami with R Flower (University of SA) The cytokine TNF is produced by the host in response to an infection and is involved in increasing the antimicrobialactivity of phagocytic cells such as the neutrophil. We have demonstrated both in vitro and in vivo that TNF increasedresistance to a range of medically important bacteria; Staphylococcus aureus, Haemophilus influenzae and Pseudomonasaeruginosa. Since this cytokine is also considered to be highly pathogenic, we have been trying to understand the mechanismsby which TNF primes neutrophils for increased antimicrobial activity in an effort to develop immunomodulators whichmay be useful to enhance immunity against infection in immunocompromised individuals and the very young whoexperience physiological immaturity. Our goal is to separate the pathophysiological effects of the molecule from theantimicrobial function. In studying the intracellular signals stimulated by TNF, we have found that TNF induces quite aselective group of intracellular signals in neutrophils, compared to those induced in other cell types such as endothelialcells. In neutrophils, TNF fails to stimulate the activities of sphingomyelinase and phosphatidylcholine specific phospholipaseC, the extracellular signal-regulated protein kinase and the jun N-terminal kinase. The cytokine activated phospholipaseA2 and p38 MAP kinase. It is known that p38 regulates the activity of phospholipase A2 which in turn regulates theexpression of complement receptor Type 3, a receptor involved in phagocytosis of bacteria.
In attempts to harness the useful properties of TNF without its associated pathological properties we have engineered a
series of small peptides based on the TNF primary structure. One of these, corresponding to amino acid residues 70-80,
which had an isoleucine substitution (H-Pro-Ser-Thr-His-Val-Le-Ile-Thr-His-Thr-Ile-Oh), of the TNF monomer
(TNF70-80), was found to mimic TNF in its ability to enhance the antimicrobial activity of neutrophils but lacked the
associated pathology-inducing properties. This 11-mer peptide retains the ability to stimulate neutrophils and binds to
both the p55 and p75 kDa TNF-α receptors, but unlike TNF fails to stimulate expression of adhesion molecules on
endothelial cells or exhibit in vivo toxicity. Because of this selective action, we anticipate that TNF70-80 will serve as a
unique tool to identify the signalling pathways that TNF uses to prime neutrophils for enhanced microbicidal activity.
We are currently examining the ability of TNF70-80 to stimulate the activities of the signalling molecules listed above.
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4 Cross talk between endogenous and exogenous mediators in lung inflammation
HPA Jersmann, A Ferrante, CST Hii, JV Ferrante Inflammation in the lung is characterised by neutrophil accumulation and activation and increased adhesiveness of theendothelium and epithelial cells. At infection foci, both exogenous tissue-stimulating mediators of bacterial origin andendogenous mediators such as cytokines are released. In order to understand the way this mixed mediator networkoperates to promote the inflammatory reaction, we have examined the cross talk between the cytokine TNF and thebacterial product lipopolysaccharide (LPS) on the above functions. The data showed that human neutrophils, pretreatedwith TNF displayed significantly enhanced superoxide production in response to LPS (from either E coli K-235 or E coli0127:B8). The TNF-induced increase in the LPS response was paralleled by an increase in LPS binding to the neutrophils,which could be abrogated by an anti-CD14 monoclonal antibody. The results demonstrate that TNF significantly increasedthe LPS-induced release of oxygen radicals in neutrophils through the upregulation of cell-surface CD14.
While TNF and LPS individually upregulated the expression of E-selectin, intercellular adhesion molecule-1 and vascularcell adhesion molecule-1 in human umbilical vein endothelial cells (HUVEC) in vitro, the two mediators were synergisticin this activity. The response was characterised by a parallel increase in adhesion molecule mRNA expression and in theamount of Nuclear Factor-kappa B (NF-KB) that had been translocated into the nucleus. Furthermore, TNF and LPS weresynergistic in the activation of p38 mitogen-activated protein kinase (p38), but not of extracellular signal-regulated kinase(ERK) or c-jun N-terminal kinase (JNK). The results show that endogenous and exogenous mediators have synergisticaction on the expression of adhesion molecules probably by promoting p38 activation and the accumulation of NF-KB inthe nucleus. Preliminary results have also shown that the synergism between TNF and LPS occur also in lung epithelial cells.
5 Pathogenesis of preterm birth in women with a history of preterm labour and delivery
H McDonald, M Annells; with J O’Loughlin (Obstetrics), R Vigneswaran (Neonatology), B Rowan-Kelly, A Ferrante(Immunopathology) Please refer to Microbiology and Infectious Diseases.
6 Malaria vaccine: Targeting the extracellular asexual blood stage (merozoite) of Plasmodium falciparum
LM Kumaratilake, A Ferrante with CM Rzepczyk (Australian Centre for International and Tropical Health and Nutrition,Queensland Institute of Medical Research) and R Anders (Walter and Eliza Hall Institute for Medical Research, Melbourne) A high proportion of villagers in the Solomon Islands and Papua New Guinea, where Plasmodium falciparum is prevalent,have antibodies to the merozoite surface protein MSP-2 (to both allelic forms FC-27 and 3D7). When the distribution ofthe IgG subclass nature of these antibodies was examined it was found, unexpectedly, that these were predominantly of theIgG3 subclass. This contrasted with antibodies to whole schizont antigens where there was a predominance of IgG1 andIgG3 subclass as well as an increased representation of IgG2 and IgG4. When the serum IgG subclasses to MSP-2 wereexamined in children of 6 months to 5 years of age living in the Solomon Islands, high titres were observed by age 5 andthe antibodies were also confined mainly to the IgG3 subclass.
Using affinity columns containing MSP-2 antigen as well as those containing anti IgG subclass specific antibodies it hasbeen possible to fractionate anti MSP-2 antibodies into their respective IgG subclass to high purity and to characterise theproperties of these antibody types in relation to their anti-merozoite activity and immunity to malaria. The activity ofthese was examined in relation to inhibition of merozoite invasion of erythrocytes and ability to promote phagocytosis.
All four IgG subclass anti MSP-2 antibody fractions inhibited merozoite invasion of erythrocytes. Between 5 x 106 to 5 x 107molecules of IgG subclasses per merozoite were required to inhibit their invasion. Phagocytosis of merozoites was studiedby both a flow cytometry analysis and examination of Giemsa stained smears. We found that IgG subclasses promoted Wo m e n’s a n d C h i l d r e n’s Ho s p i t a l R e s e a r c h R e p o r t 1 9 9 8 Pa g e 104 S c i e n t i f i c R e p o r t s
phagocytosis by neutrophils to different degrees. Using purified IgG subclass specific anti MSP-2 antibodies, only 1,200molecules of IgG1 and 50,000 molecules of IgG3 per merozoite but 5 x 105 molecules of IgG2 and 1 x 106 molecules ofIgG4 were required to promote phagocytosis. It is evident that this is many-fold lower than the amount of antibodyrequired to directly inhibit invasion of merozoites. The cellular-mediated damage to merozoites was indeed even moreeffective when a combination of cytokine treatment and antibody were used. Furthermore, IgG4 anti MSP-2 antibodiesinhibited the ability of IgG1 and IgG3 types to promote adhesion and phagocytosis of merozoites. Serum in which theratio of IgG1-IgG3: IgG2-IgG4 was high was markedly more opsonic than serum in which this ratio was low. While antiMSP-2 antibodies can promote phagocytosis, it was evident from these studies that phagocytosis is also promoted throughantibodies against other surface antigens since the addition of excess MSP-2 antigen to immune serum did not affect itsability to promote phagocytosis of merozoites.
7 Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause
granulomatous amoebic encephalitis
Free-living amoebae cause three well-defined disease entities: a rapidly fatal primary meningoencephalitis, a chronicgranulomatous amoebic encephalitis (GAE), and a chronic amoebic keratitis. GAE occurs in immuno-compromisedpersons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. Thefinding that this amoeba has caused infection in some healthy children has raised the possibility that humans may lackimmunity to B mandrillaris. Human serum was examined for the presence of surface antibodies specific for this amoebaby immunofluorescence. Sera from adults contained titres of 1/64 - 1/256 of anti-B mandrillaris antibodies (IgM and IgGclasses), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similar tothose in adults were seen in serum of 1 - 5-year-old children.
Initial studies with serum from a child who died of B mandrillaris GAE showed an increase in antibody titre of 1/1024(IgM = 1/64, IgG = 1/1024). This is a 4-fold increase in antibody titre relative to the titre found in control children ofsimilar age, suggesting that the child responded to the amoeba antigens. Whether this represents a protective response ornot remains to be established. The rise in the titre, which is well above the range in non-infected children, could provide ameans of diagnosing the disease.
8 Changes in immunological function during chemotherapy for small cell lung cancer (SCLC)
A Ferrante, DP Harvey, B Rowan-Kelly, M Busuttil, AM Tan with I Olver (Cancer Services, Royal Adelaide Hospital) Chemotherapy induced changes in immunological functions were studied. A cohort of 5 patients treated for SCLC withcarboplatin 135 mg/m2/day each for 3 days every 28 days had blood sampled on the day prior to chemotherapy, then days4, 14 and 28 of each 6 courses. Examination of the functional activation of circulating mononuclear leucocytes showedthat while chemotherapy had no effect on mitogen (PHA, ConA, PWM, S. aureus) induced lymphoproliferation, it affectedcytokine production by lymphocytes and other mononuclear cells. There was a decrease in the ability of lymphocytes toproduce IL-2 and IFN-γ but not lymphotoxin (LT) in response to PHA, ConA and PWM. The production of cytokinesTNFα and IFN-1β, produced mainly by monocytes, was decreased in mononuclear cells stimulated with the 4 mitogens.
The chemotherapy caused WHO grade 3 and 4 neutropenia but had no effect on the key neutrophil functions of adherence,chemotaxis, respiratory burst, degranulation and bactericidal activity. Chemotherapy had no effect on serum complementactivity and immunoglobulin class and subclass levels remained normal. There were no effects on the IgE and allergenspecific IgG levels to a panel of allergens. The data show that chemotherapy, besides causing neutropenia, leads to changesin lymphocyte and monocyte responses as reflected in their ability to produce cytokines. This suggests that patientsreceiving chemotherapy may benefit from immunostimulants or cytokine therapy.
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9 Engineered polyunsaturated fatty acids which target asthma
BS Robinson, A Ferrante, DA Rathjen Inflammation associated with the overproduction of leukotrienes by leukocytes plays a central role in the pathogenesis ofasthma. We have chemically synthesised a group of unusual polyunsaturated fatty acids (PUFA) which have more selectiveimmunosuppressive properties [in particular β-oxa-derivatives; β-oxa-21:3(n-3), β-oxa-23:4(n-6) and β-oxa-21:3(n-6)]than their natural PUFA counterparts and therefore may be useful for asthma therapy. Pretreatment of human neutrophilswith these engineered β-oxa-PUFA substantially suppressed the production of leukotriene B4 (LTB4) in a time- andconcentration- dependent manner when the cells were challenged with calcium ionophore A23187. In contrast, naturally-occurring PUFA only slightly inhibited (in the case of eicosapentaenoic (20:5(n-3)) and docosahexaenoic (22:6(n-3) acids)or increased (in the case of eicosatetraenoic acid (arachidonic acid, 20:4 (n-6) the formation of LTB4. β-oxa-21:3(n-3)caused the greatest attenuation of LTB4 synthesis (which was comparable with commercially available leukotrieneinhibitors such as zileuton and nordihydroguaiaretic acid. β-oxa-21:3(n-3) inhibited neutrophils prelabelled with[3H]20:4(n-6) to produce [3H]5-hydroxyeicosatetraenoic acid (5-HETE) and [3H]LTB4 in the presence of calciumionophore, indicating that 5-lipoxygenase and possibly LTA4 hydrolase enzymes in the leukotriene pathway wereinactivated. Preliminary work has indicated that β-oxa-21:3(n-3) is taken up by neutrophils and incorporated intophospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes converted a marginal amount of β-oxa-21:3(n-3) to two oxygenated metabolites, tentatively identified as hydroxylated derivatives of β-oxa-21:3(n-3). β-oxa-21:3(n-3) did not affect the total uptake of exogenous [3H]20:4(n-6) by the cells, but greatly suppressed incorporation ofthe unesterified [3H]20:4(n-6) into triacylglycerol and slightly enhanced its incorporation into phospholipids.
Concomitantly, pretreatment of the leukocytes with β-oxa-21:3(n-3) inhibited the development of lipid bodies (lipid-richcytoplasmic inclusions which play a role in enhanced leukotriene synthesis) by 20:4(n-6).
We are continuing to investigate the inhibitory mechanism of β-oxa-21:3(n-3) on the leukotriene pathway in neutrophils.
The above data indicate that β-oxa-21:3(n-3) has the hallmarks of an anti-asthmatic agent. In vivo studies using β-oxa-21:3(n-3) in an asthma animal model are planned. To this end, we have administered β-oxa-21:3(n-3) to rats by gavage orintraperitoneal injection and found it to be incorporated into the lipids of tissues, including important target sites such asthe lungs, spleen, blood cells and plasma. Rats given this fatty acid maintained normal body weight, retained normal liverand kidney function (assessed by measuring blood parameters and histological examination) and showed no signs ofdebility, indicating that the compound was not toxic to the animals.
10 Chemically engineered polyunsaturated fatty acids as inhibitors of chronic inflammation
M Costabile, CST Hii, M Pitt, C Easton, BS Robinson, A Poulos, DA Rathjen, A Ferrante Diets rich in ω-3 fatty acids, while generally perceived to be anti-inflammatory, have not produced the expected degree ofdisease alleviation. This may be explained by the fact that while ω-3 fatty acids inhibit production of cytokines and eicosanoids,they also increase leukocyte oxygen radical production, adherence and degranulation. Our studies have shown that differentactivities of fatty acids are governed by specific fatty acid structural elements. On this basis, we have synthesised a seriesof novel fatty acids (β-oxa 21:3 ω-3, β-thia 23:4 ω-6 and b-thia 21:3 ω-6) which inhibited the PHA-TPA inducedproliferation of purified T lymphocytes and production of IL-2, TNF-β and IFN-γ in a dose and time dependent manner.
However, all three fatty acids were very poor in stimulating oxygen radical production.
The effects of these fatty acids were independent of their metabolism via the lipoxygenase and cyclooxygenase pathwaybut were related to alterations in the PHA-PMA activation of intracellular signalling molecules such as mitogen activatedprotein kinases. Preliminary results from animal studies show that at least one of these significantly inhibits the delayedtype of hypersensitivity reaction, is readily absorbed following oral administration and is non-toxic in terms of liver andkidney functions.
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11 Regulation of cytokine mRNA stability by unsaturated fatty acids
Cytokines including TNF, IL-1 and IL-6 are mediators of pathogenesis in chronic inflammatory diseases such as asthma,rheumatoid arthritis and cystic fibrosis. Attempts to manage these diseases using monoclonal antibodies or solublereceptors have met with major limitations. An approach which may prove more successful is to target the cytokinemessenger RNA. We are studying the interaction between PUFAs and cytokine mRNA in relation to effects at the level ofmRNA stability. Preliminary work has yielded the interesting discovery that some modified PUFAs can prevent cytokinesynthesis by significantly increasing the rate of degradation of TNF mRNA.
12 Antimalarial properties of chemically engineered fatty acids
A Ferrante, N Trout, M Pitt, L Kumaratilake, DA Rathjen, CST Hii with C Easton (School of Chemistry, Australian National University, Canberra) and R Prager (Flinders University) Our findings that leukocytes use the generation of polyunsaturated fatty acids in their armament to kill intraerythrocyticstages of the blood stage of Plasmodium falciparum which causes malaria, lead us to propose a new strategy fordeveloping antimalarial drugs. A series of novel polyunsaturated fatty acids has been generated as part of a strategy tochemically engineer polyunsaturated fatty acids which have selective antimalarial activity but lack immunostimulatoryproperties seen with the natural polyunsaturated fatty acids. Four classes of polyunsaturated fatty acids (PUFA) or relatedcompounds were generated. These are the β-oxa, β-thia, γ-thia and amino acid conjugated compounds. Compoundswithin these groups were found to display antimalarial activity. β-oxa PUFA tested showed strong activity and displayedeither reduced or complete lack of ability to stimulate neutrophil oxygen radical production, thus eliminating the mostundesirable characteristic of the natural fatty acids.
Another approach was to make protected hydroperoxy compounds which would act as pro-drugs. Two of these weresynthesised: 15-00C Me3 OCH3 20:4n-6 and 15-00C Me2 OCH3 OCH:β-oxa 23:4n-6 and when added to P falciparumcultures, these two compounds showed little anti-parasite activity. But when these were converted to the hydroperoxyderivative prior to adding to parasites, they were extremely active. The amino acid conjugated PUFA also ranged fromthose displaying poor to those with strong antimalarial activity.
Preliminary evidence showed that the engineered PUFA were also active against the chloroquine resistant P falciparum K1 isolate, although no synergism between the fatty acids and chloroquine was observed. Investigations with the nitrocompounds showed that a number of these also had antimalarial activity.
The in vivo activity of some of these compounds was demonstrated in mouse malaria where these significantly increasedsurvival time of mice infected with P berghei anka and significantly depressed the parasitaemia in mice infected with P berghei. Thus the studies are progressing well towards achieving the general objective of identifying fatty acid basedantimalarial agents which target selectively the parasite and not the host.
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13 IgG subclass antibodies to glutamic acid decarboxylase and risk for progression to clinical
insulin-dependent diabetes
A Ferrante, with A Pollard and J Couper (Paediatrics) Type-1 diabetes is the second most common disease of childhood and a major cause of renal failure, cardiovasculardisease and blindness in the community. Damage to the beta cells in the pancreatic islets is a function of cell mediatedimmunity and cytotoxic T lymphocytes which recognise the islet autoantigens insulin, IA2 and GAD. Alternatively, damageto this tissue is mediated via complement fixing and/or opsonic antibodies in co-operation with leukocytes to deliverADCC reactions. It is perceived that antibodies and cytotoxic T lymphocytes will antagonise each other’s activity. However,antagonising the action of cytotoxic T lymphocytes via complement and opsonic promoting IgG subclasses is not conducivewith protection but merely precipitates tissue damage via ADCC or complement-mediated mechanisms. We thereforehypothesise that tissue damage is prevented when patients have a predominance of nonopsonic/non-complement activatingIgG subclass autoantibodies (namely IgG2 and IgG4). If GAD is a pathogenic autoantigen then progression to clinicaldiabetes is associated with an IgG1/IgG3 response to GAD and an IgG2/IgG4 response with low risk of clinical disease.
In our preliminary work we have measured IgG subclass antibodies to GAD in 34 newly diagnosed type-1 diabetespatients and in 28 at-risk, first-degree relatives of people with type-1 diabetes. In the newly-diagnosed patients, total IgGantibodies to GAD were detected in 74% (25/34); IgG1and/or IgG3 were significantly more frequent than IgG4 orIgG4/IgG2 (14/34 versus 5/34, p=0.01). GAD antibody-negative patients were significantly younger (p=0.01). In 15 at-riskrelatives who had not progressed to clinical diabetes after a median of 4.5 years, 10 had IgG2 and/or IgG4 antibodiescompared to only 3/13 progressors (p=0.02). Total IgG and IgG2 antibodies were higher in non-progressors.
Non-progressors were older than progressors (p = 0.01), and relatives with IgG2 and/or IgG4 responses were also older(p = 0.01). These results suggest that measurement of IgG subclass antibodies to GAD may contribute to diabetes riskassessment in islet antibody relatives.
14 Engineered polyunsaturated fatty acids which depress cytokine-induced inflammation in the endothelium
BS Robinson, A Ferrante, CST Hii, G Parashakis, JV Ferrante, A Poulos, DA Rathjen, with AW Murray (Flinders University) A novel polyunsaturated fatty acid, β-oxa-23:4(n-6), was found to be highly active in suppressing tumour necrosis factor-α(TNF-α), lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA)-induced upregulation of leukocyte adhesionto human umbilical vein endothelial cells (HUVEC) and expression of the adhesion molecules E-selectin, intercellularadhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (at both the protein and mRNA level).
This fatty acid was found to poorly activate the neutrophil oxidase and release of oxygen radicals. The inhibitory action ofβ-oxa-23:4(n-6) was reduced when the cells were pretreated with nordihydroguaiaretic acid (lipoxygenase inhibitor) butnot with indomethacin (cyclooxygenase inhibitor). This infers that conversion of β-oxa-23:4(n-6) to an oxygenatedproduct(s) via the lipoxygenase pathway is required for activity. The β-oxa-PUFA was taken up by HUVEC andincorporated into phospholipids (predominantly phosphatidylcholine) and neutral lipids (mainly triacylglycerol andcholesterol ester). The β-oxa-23:4(n-6) recovered in the major HUVEC phospholipids (phosphatidylcholine,phosphatidylethanolamine and phosphatidylinositol) was found to be almost exclusively located in the sn-2 position of themolecule. HUVEC did not modify β-oxa-23:4(n-6) by chain elongation, desaturation or b-oxidation. In the presence ofTNF-α, HUVEC converted a small amount of β-oxa-23:4(n-6) to a 15-monohydroxylated fatty acid derivative by thelipoxygenase pathway. We are currently investigating whether this 15-monohydroxylated derivative of β-oxa-23:4(n-6)plays a role in inhibiting HUVEC adhesion molecule expression. Administration of β-oxa-23:4(n-6) to rats and mice bygavage or intraperitoneal injection resulted in the incorporation of β-oxa-23:4(n-6) into the lipids of various tissues,including liver, kidneys, lungs, heart, spleen, brain and blood. It is noteworthy that animals given the β-oxa-PUFA Wo m e n’s a n d C h i l d r e n’s Ho s p i t a l R e s e a r c h R e p o r t 1 9 9 8 Pa g e 108 S c i e n t i f i c R e p o r t s
maintained normal body weight, retained normal liver and kidney function (assessed by measuring blood biochemicalparameters and histological examination) and showed no signs of debility, indicating that the compound was not toxic.
Collectively the data suggest that this β-oxa-PUFA could be explored as an agent to decrease inflammation in blood vessels.
Staff par ticipating in research
Prof A Ferrante, FRCPath, PhD, Director
Dr DA Rathjen, PhD, Deputy Director
Mr G Harvey, Dip Med Lab Sci, Laboratory Manager (University Department of Paediatrics)
Ms R Bachmayer, Technical Assistant
Ms MF Busuttil, BApplSci, MedLabSci, Technical Officer
Ms K Carman, Adv Cert Med Lab Sci, Technical Assistant
Mr M Costabile, BSc(Hons), PhD candidate
Mrs JV Ferrante, BSc(Hons), Research Assistant
Mr D Goh, BSc(Hons), SC-1
Ms C Haddad,BSc(Hons), candidate
Ms DP Harvey, Technical Assistant
Dr CST Hii, PhD, Senior Research Officer
Dr H Jersmann, MBBS, PhD candidate
Dr LM Kumaratilake, BVSc, PhD, Senior Research Fellow
Ms LA Marin, BSc, Technical Officer
Ms N Moghaddami, MSc Pathobiology, PhD candidate
Ms T Nilsson, BAppl Sci(Hons), PhD candidate
Ms G Parashakis, Adv Cert Med Lab Sci, Technical Assistant
Dr M Pitt, PhD, Senior Research Officer
Dr BS Robinson, BAgSci, PhD, Senior Research Officer
Mr B Rowan-Kelly, BSc (Hons), SC-1
Ms E Southcott, BSc(Hons), PhD candidate
Dr R Staugas, MBBS, FRCP, MD candidate
Mr RJ Storer, BSc(Hons) PhD candidate
Ms A-M Tan, BSc, Research Assistant
Dr N Trout , BSc(Hons) PhD, Research Officer
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Inte r national and Inte rstate Travel and Prese ntation s
M Costabile
28th Annual Conference of the Australasian Society for Immunology, Melbourne, 1998
• Inhibition of lymphocyte proliferation and cytokine production by β-oxa and β-thia fatty acids.
CST Hii
28th Annual Conference of the Australasian Society for Immunology, Melbourne, 1998
• Inadequate activation of MAP kinases in Neonatal T lymphocytes.
• Arachidonyl co-enzyme A: A novel neutrophil ligand/second messenger?
L Kumaratilake
IXth International Congress of Parasitology, Makuhari Masse, Chiba, Japan, 1998
• Plasmodium falciparum merozoite invasion inhibition by cytokine primed human neutrophils. L Marin
28th Annual Conference of the Australasian Society for Immunology, Melbourne, 1998
• Involvement of phosphatidylinositol-3-kinase and p38 MAP kinase in neutrophil mediated-degradation of proteoglycan.
Inte r national and Inte rstate Vi sitors
Dr Robin Anders,
Walter and Eliza Hall Institute, Victoria, Australia.
Professor Channing Der,
University of North Carolina at Chapel Hill, North Carolina, USA
Comme rcial D e velopme nts
PCT/AU92/00313
Patent: Methods and compositions for treating malaria and other diseases
Granted - Australia, USA and Europe
Pending - Europe, Canada and Japan, Singapore
PCT/AU94/00607
Patent: Polyunsaturated fatty acids and uses thereof
Granted - Australia and USA
PCT/AU95/00677
Patent: Modified polyunsaturated fatty acid analogues
Granted - China
Pending - Australia, Europe, USA, Canada, Japan, Vietnam, Korea
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PCT/AU95/00717
Patent: Synthetic fatty acid analogues
Granted - Australia and China
Pending - Europe, USA, Canada, Japan, Vietnam and Korea
PCT AU97/00231
Patent: Method of treating immunopathologies using polyunsaturated fatty acidsPending - Australia, Europe, USA, Canada, Japan, China, Vietnam and Korea.
Othe r Research-related Ac tiv it y
A Ferrante
Editorial board of the following international journals: Immunology and Asian Journal of Allergy and Immunology;
Member, Research Grants Subcommittee, Royal Adelaide Hospital Foundation; Member Medvet Biotechnology Grant Assessment Committee; Member, National Scientific Advisory Board of Rotarians Against Malaria L Kumaratilake
SA regional Officer for the Australian Society of Parasitology
M Costabile
Teaches Immunology course to diploma and degree students at the University of South Australia.
Grants
Britton, W, Briscoe, H, Rathjen DA
Control of tuberculosis infection with tumour necrosis factor-like synthetic peptides.
Australian Research Council
Ferrante A
Antimalarial activity of engineered polyunsaturated fatty acids
UNDP/World Bank/World Health Organisation Special Program for Research and Training in Tropical Diseases
Ferrante A
The immunopharmacology of GMDP in cancer patients.
Peptech Ltd
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Ferrante A, Costabile M
Inhibition of cytokine-induced demyelination by fatty acids
Australian Research Council Postgraduate Award (Industry) with Peptech Limited
Ferrante A, Hii CST, Rathjen DA
Promotion of neutrophil antimicrobial function by tumour necrosis factor
Bl Funding University of Adelaide
Ferrante A, Kumaratilake L
Malaria vaccine: targeting Plasmodium falciparum merozoite by phagocytes.
World Health Organisation - TDR/UNDP/World Bank/WHO Special Program for Research and training in
Tropical Diseases
Ferrante A, Robinson BS, Hii CST, Murray AW
Engineered PUFA which depress cytokine-induced inflammation in the endothelium
National Heart Foundation
Rathjen DA, Ferrante A, Gibson, R
Enhancement of non-specific immunity against non-typable Haemophilus influenzae by dietary fatty acid manipulations.
Channel 7 Children’s Research Foundation
Publication s
Britton WJ, Meadows N, Rathjen DA, Roach DR, Briscoe H
A tumour necrosis factor memetic peptide activates a murine macrophage cell line to inhibit mycobacterial growth in a
nitric oxide-dependent fashion. Infect & Immun (1998) 2122-27.
Couper JJ, Harrison LC, Aldis JE, Coleman PG, Honeyman MC and Ferrante A
IgG Subclass antibodies to glutamic acid decarboxylase and risk for progression to clinical insulin-dependent diabetes.
Human Immunology (1998) 59; 493-499
Hii CST, Huang ZH, Bilney A, Costabile M, Murray AW, Rathjen DA, Der CJ, Ferrante A
Stimulation of p38 phosphorylation/activation by arachidonic acid in HeLa cells, HL60 promyelocytic leukaemic cells and
human neutrophils: evidence for cell-type specific activation of MAP kinases. J Biol Chem (1998) 273 (30):19277-19282.
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Jersmann HPA, Rathjen DA, Ferrante A
Enhancement of lipopolysaccharide-induced neutrophil oxygen radical production by tumor necrosis factor α.
Infect Immun (1998) 66:1744-1747
Kumaratilake, LM
(1998) Host’s Response to Malaria. In: Celebration: Fifty years of Sri Lankan/Australian interactions.
Ed C Vanden Drieson, Colombo Government Press. P519-526.
Pitt MJ, Easton CJ, Ferrante A, Poulos A, Rathjen DA
Synthesis of polyunsaturated β-thia and β-thia fatty acids from naturally derived polyunsaturated fatty alcohols and in
vitro evaluation of their susceptibility to β-oxidation. Chem. Phys. Lipids (1998) 92:63-69
Pitt MJ, Easton CJ, Robertson TA, Kumaratilake LM, Ferrante A, Poulos A and Rathjen DA
Synthesis of hydroperoxide and perketal derivatives of polyunsaturated fatty acids as potential antimalarial agents.
Tetrahedron Letter. (1998) 39:4401-4404.
Robinson BS, Hii CST, Ferrante A
Activation of phospholipase A2 in human neutrophils by polyunsaturated fatty acids and its role in stimulation of
superoxide production. Biochem J (1998) 336, 611-17.
Storer RJ, and Ferrante A
Hydrogen peroxide assay for amine oxidase activity. In: Polyamine Protocols - Methods in Molecular Biology Series.
DML Morgan (Ed), Humana Press Inc. NJ. (1998) 79:81-90
Storer, RJ and Ferrante A
Radiochemical assay of diamine oxidase - In: Polyamine Protocols - Methods in Molecular Biology Series.
DML Morgan (Ed), Humana Press Inc. NJ. (1998) 79:91-95.
Xia, P, Gamble JR, Rye KA, Wang L, Hii CST, Cockerill P, Khew-Goodall Y, Bert AG, Barter PJ, Vadas MA
Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway.
Proc Natl Acad Sci USA (1998) 95:14196-14201.
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Source: http://www.wch.sa.gov.au/research/publications/report/research98/pdfs/immuno.pdf