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E Handman

Publications and source records attributed to E Handman.

At least 19 recordsLinked to original sources
PubMed2005

A simple method allowing DIC imaging in conjunction with confocal microscopy.

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Animals↗
PubMed2004

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.

Animals↗
PubMed2004

Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway.

The severity of disease caused by infection with Leishmania major depends critically on the genetics of the host. Early induction of T helper (Th)1-type immune responses in the resistant C57BL/6 mice and Th2-type responses in the susceptible BALB/c mice are thought to determine cure or disease, respectively. We have previously mapped three host response loci in a genetic cross between C57BL/6 and BALB/c mice, and here we show definitively the involvement of these loci in disease severity using animals congenic for each of the loci. Surprisingly, in the late stage of infection when the difference in disease severity between congenic and parental mice was most pronounced, their cytokine profile correlated with the genetic background of the mice and not with the severity of disease. This indicates that the loci that we have mapped are acting by a mechanism independent of Th phenotype.

Animals↗
PubMed2004

Dissociation of disease susceptibility, inflammation and cytokine profile in lmr1/2 congenic mice infected with Leishmania major.

Severity of disease caused by Leishmania major depends on the genetics of the host. Early induction of T helper cell type 1 (Th1)-type responses in resistant C57BL/6 mice and T helper cell type 2 (Th2) in susceptible BALB/c mice is thought to determine cure or disease respectively. We have mapped three loci that confer susceptibility or resistance upon congenic mice on the C57BL/6 or BALB/c backgrounds. Here we examine the histopathology and production of interleukin 4 (IL-4) and interferon gamma (IFN-gamma) in the skin and draining lymph nodes in the congenic and parental mice. We show an evolving granuloma with a staged infiltration of inflammatory cells, but no difference between the groups. As an indication of an early-polarised Th1/Th2 response we measured IFN-gamma and IL-4 in the lymph nodes and found no difference between any of the mice during the first 48 h. During infection, the level of IL-4 correlated with the lesion size, indicating that IL-4 reflects the disease severity rather than controls it. Considering this effect, B6.C(lmr1,lmr2) mice had similar cytokine levels to the parental C57BL/6 mice despite increased susceptibility and C.B6(lmr1,lmr2) were similar to BALB/c despite increased resistance. We conclude that the lmr loci affect disease severity by a mechanism independent of conventional helper T-cell responses.

Animals↗
PubMed2001

Molecular and immunological characterisation of the glucose regulated protein 78 of Leishmania donovani(1).

To identify novel potential Leishmania vaccine antigens, antibodies from patients with visceral leishmaniasis (VL) were used to isolate clones from a cDNA expression library of L. donovani amastigotes. Glucose Regulated Protein (GRP78), a member of the 70 kDa heat-shock protein family was identified and characterised. The GRP78 gene was localised to chromosome 15 in L. donovani, L. major, and L. mexicana by pulse-field gel electrophoresis. The Leishmania GRP78 protein contain a carboxy-terminal endoplasmic reticulum retention signal sequence (MDDL) as does the Trypanosoma cruzi GRP78. Immunofluorescence using antibodies to the recombinant DNA-derived GRP78 protein showed staining localised to reticular material throughout the cytoplasm and in the perinuclear region of promastigotes, suggesting that the protein is localised in the endoplasmic reticulum. The protective efficacy of GRP78 was assessed in mice vaccine experiments. A GRP78 DNA vaccine primed for an immune response that protected C57Bl/6 and C3H/He mice against infection with L. major. Similarly vaccination with a recombinant form of GRP78 purified from Escherichia coli and administered with Freund's as adjuvant induced protective immunity in C57Bl/6 mice.

Amino Acid Sequence↗
PubMed2001

Paradoxical effects of IL-12 in leishmaniasis in the presence and absence of vaccinating antigen.

Protective immunity against Leishmania major requires parasite-specific CD4+T helper cells, the development of which is promoted by interleukin 12 (IL-12). In this study we investigated the use of IL-12 DNA to enhance the protective immunity induced by prophylactic vaccination with the L. major Parasite Surface Antigen 2 (PSA-2) DNA. A plasmid was constructed in which the two murine IL-12 subunits p35 and p40 were secreted as a biologically active single chain cytokine. The immunomodulatory effects of this IL-12 DNA were examined by codelivery with PSA-2 DNA in susceptible BALB/c and resistant C3H/He mice and subsequent infection with L. major promastigotes. Surprisingly, administration of IL-12 DNA alone had a protective effect, while coadministration of IL-12 with PSA-2 DNA abrogated protection. This effect of IL-12 DNA was dose dependent and affected by the timing of administration in relation to PSA-2 DNA. The effect of IL-12 on protection was associated with a reduced number of INF-gamma-producing T cells early in infection. A further understanding of this paradoxical effect of IL-12 and possibly other cytokines on protective immunity may be important for their use as adjuvants for Leishmania DNA vaccines.

Adjuvants, Immunologic↗
PubMed2001

The dendritic cell populations of mouse lymph nodes.

The dendritic cells (DC) of mouse lymph nodes (LN) were isolated, analyzed for surface markers, and compared with those of spleen. Low to moderate staining of LN DC for CD4 and low staining for CD8 was shown to be attributable to pickup of these markers from T cells. Excluding this artifact, five LN DC subsets could be delineated. They included the three populations found in spleen (CD4(+)8(-)DEC-205(-), CD4(-)8(-)DEC-205(-), CD4(-)8(+)DEC-205(+)), although the CD4-expressing DC were of low incidence. LN DC included two additional populations, characterized by relatively low expression of CD8 but moderate or high expression of DEC-205. Both appeared among the DC migrating out of skin into LN, but only one was restricted to skin-draining LN and was identified as the mature form of epidermal Langerhans cells (LC). The putative LC-derived DC displayed the following properties: large size; high levels of class II MHC, which persisted to some extent even in CIITA null mice; expression of very high levels of DEC-205 and of CD40; expression of many myeloid surface markers; and no expression of CD4 and only low to moderate expression of CD8. The putative LC-derived DC among skin emigrants and in LN also showed strong intracellular staining of langerin.

Animals↗
PubMed2001

Cloning, expression and antigenicity of the L. donovani reductase.

The protozoan parasite Leishmania undergoes a morphological and biochemical transformation from the promastigote to the amastigote form during its life cycle, which is reflected in the expression of stage-specific proteins. One of these proteins shows homology to a superfamily of reductase proteins. We have cloned the reductase gene from L donovani and have shown that it differs in only one nucleotide from the L. major homologue, resulting in one amino acid change. A cytosine (C) to guanine (G) transposition in the coding sequence leads to a nonconserved substitution of asparagine (N) for lysine (K). Only 2 of 22 plasma samples from patients with visceral leishmaniasis were found to have detectable anti-reductase antibodies and peripheral blood mononuclear cells (PBMC) from one of three individuals previously infected with visceral leishmaniasis proliferated in the presence of recombinant reductase protein. Interestingly, 6 of 10 PBMC isolated from Danish controls proliferated in the presence of the reductase protein. Intracellular IFNgamma was found in a significant percentage of cells in all the tested PBMC cultures from Danes, whereas IL4 was only found in a small proportion of cells, or not at all. The results indicate the presence of cross-reacting CD45R0 memory T-cells in individuals not exposed to Leishmania. Several previous studies have shown that T-cells from nonexposed individuals often respond to crude Leishmania antigen preparations. The present study suggests that this reactivity is partly caused by T-cells recognising L. donovani reductase.

Amino Acid Sequence↗
PubMed2001

Neutralization of interferon-gamma in neonatal SOCS1-/- mice prevents fatty degeneration of the liver but not subsequent fatal inflammatory disease.

Mice lacking the suppressor of cytokine signalling-1 (SOCS1) die within weeks of birth with extensive fatty degeneration of the liver, consistent with acute hepatic toxicity to interferon-gamma (IFN-gamma), and inflammation of multiple organs. We show here that treatment for 1 week from birth with neutralizing antibody to IFN-gamma rescues SOCS1-/- mice from lethal liver disease but the mice subsequently succumb to chronic inflammatory lesions characterized by T-lymphocyte infiltration of skeletal muscle, pancreas, lung, liver and skin. Elevated blood levels of eosinophils, neutrophils and platelets were also observed and the thymic lymphocyte population was depleted of CD4+ CD8+ T cells and showed a reduced CD4 : CD8 ratio. All T-cell populations in thymus, spleen and lymph node exhibited an increased proportion of cells bearing the activation marker CD44. These data suggest an important role for SOCS1 in T-lymphocyte regulation.

Animals↗
PubMed2001

Leishmaniasis: current status of vaccine development.

Leishmaniae are obligatory intracellular protozoa in mononuclear phagocytes. They cause a spectrum of diseases, ranging in severity from spontaneously healing skin lesions to fatal visceral disease. Worldwide, there are 2 million new cases each year and 1/10 of the world's population is at risk of infection. To date, there are no vaccines against leishmaniasis and control measures rely on chemotherapy to alleviate disease and on vector control to reduce transmission. However, a major vaccine development program aimed initially at cutaneous leishmaniasis is under way. Studies in animal models and humans are evaluating the potential of genetically modified live attenuated vaccines, as well as a variety of recombinant antigens or the DNA encoding them. The program also focuses on new adjuvants, including cytokines, and delivery systems to target the T helper type 1 immune responses required for the elimination of this intracellular organism. The availability, in the near future, of the DNA sequences of the human and Leishmania genomes will extend the vaccine program. New vaccine candidates such as parasite virulence factors will be identified. Host susceptibility genes will be mapped to allow the vaccine to be targeted to the population most in need of protection.

Animals↗
PubMed2000

Therapy of murine cutaneous leishmaniasis by DNA vaccination.

Prophylactic DNA vaccination protects mice against infection with Leishmania major by inducing an exclusive Th1 immune response dominated by the production of IFN-gamma. Here we show that DNA vaccines, initially designed to prevent infection, can also have a significant therapeutic effect. In L. major infected mice, vaccination with DNA encoding the Parasite Surface Antigen/gp46/M2 causes reduction in lesion size and promotes healing in both genetically resistant C3H/He mice and susceptible BALB/c mice. The therapeutic effect is underpinned by a shift in the T cell-derived cytokine environment with an increase in the IFN-gamma producing Th1 type cells. Application of such immunotherapy in conjunction with antiparasite drugs may result in faster or more certain cure of the disease in humans.

Animals↗
PubMed2000

Mice unresponsive to GM-CSF are unexpectedly resistant to cutaneous Leishmania major infection.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play a protective role in leishmanial infection. Mice with a null mutation in the gene for the beta common (beta c) chain of the receptors for GM-CSF, interleukin(IL)-3 and IL-5 (beta c-null mice) display normal steady state hemopoiesis and develop lung disease similar to the human condition, alveolar proteinosis, due to a lack of signaling by GM-CSF. We therefore expected to observe a heightened sensitivity to Leishmania major in the beta c-null mice. Surprisingly, the beta c-null mice were more resistant to cutaneous infection than wild-type (wt) mice. Upon intradermal injection of L. major promastigotes, fewer beta c-null mice developed cutaneous lesions than wt mice and these lesions were smaller and healed more rapidly than in wt mice. This resistance to disease was associated with a reduced percentage of in vitro infected beta c-null macrophages. Macrophages from beta c-null mice displayed a more activated phenotype and produced increased amounts of nitric oxide following infection with L. major, both in vivo and in vitro. Paradoxically, however, the parasite burden in the draining lymph nodes was similar in both beta c-null and wt mice, suggesting that at least a subpopulation of cells was susceptible to the parasite. The mechanism preventing normal lesion development remains to be elucidated.

Animals↗
PubMed2000

A new standard for the assessment of disease progression in murine cutaneous leishmaniasis.

Infection of mice with Leishmania major has been used both as a model for the cutaneous disease in humans and as a model for the more general control and function of helper T cells in immunity. In both cases, disease patterns and disease progression have been assessed by two complementary methods, lesion size and parasite burden in the draining lymph nodes. We propose a much improved method for the graphical representation of lesion development which conveys more information with better accuracy. We also describe a polymerase chain reaction method for determining parasite burden, which is faster and allows the analysis of larger numbers of experimental animals than the current limiting dilution analysis. Moreover, these methods are equally applicable to other infectious diseases, an obvious one being schistosomiasis.

Animals↗
PubMed1999

Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.

Amino Acid Sequence↗