Emerging tropical diseases in Australia. Part 1. Leptospirosis.

Human leptospirosis is a zoonotic disease of global importance that causes significant morbidity and mortality, particularly in developing nations. In this review, the history, epidemiology, transmission, clinical presentation and treatment of this disease, and its impact in Australia, are discussed. Central to this review is the delineation of diagnostic methods for the disease and the challenges that this disease presents for both the clinician and diagnostic laboratory. This information should furnish clinicians with an updated tool to help overcome a number of problems associated with the diagnosis of leptospirosis.

Three-dimensional structure of interleukin-2.

Interleukin-2 is an effector protein that participates in modulating the immune response; it has become a focal point for the study of lymphokine structure and function. The three-dimensional structure of the interleukin molecule has been solved to 3.0 angstrom resolution. Interleukin-2 has a novel alpha-helical tertiary structure that suggests one portion of the molecule forms a structural scaffold, which underlies the receptor binding facets of the molecule.

Leptospirosis and Goodpasture's syndrome: testing the aetiological hypothesis.

Leptospiral pathogens have a world-wide distribution and cause a spectrum of disease ranging from a mild, influenza-like illness to Weil's disease, which manifests itself in multi-organ failure. Recently, Leptospira-reactive sera from 40 leptospirosis patients were investigated in an ELISA designed to detect antibodies to the human glomerular basement membrane (GBM). The aim was to determine if host-derived leptospiral immunoglobulins cross-react with proteins in the human GBM, so facilitating the development of Goodpasture's syndrome. As all 40 sera were found negative in the anti-GBM ELISA, the hypothesis that, during the immune phase of leptospirosis, patients are at risk of developing Goodpasture's syndrome was not supported. Further work is required to determine if leptospirosis is a risk factor in the development of any other pulmonary-renal syndromes that are associated with auto-immune diseases, such as Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, Behçet's disease, IgA nephropathy and systemic lupus erythematosus.

Hypomagnesaemia in the first 10 days of severe leptospirosis.

Magnesium imbalance in leptospirosis has, for the most part, been neglected by the medical and leptospirosis communities. In a recent, retrospective study, serum concentrations of magnesium were followed in 15 patients with severe leptospirosis. The results revealed that 14 of the 15 patients developed hypomagnesaemia at some time during the first 10 days of their illness. In severely ill patients, such magnesium deficiency can worsen clinical outcome. Magnesium concentrations may affect a number of organ systems and mental status. Since altered mental status in leptospirosis is a poor prognostic indicator, it is suggested that serum concentrations of magnesium be monitored closely in patients with leptospirosis. Any hypomagnesaemia can then be treated promptly, in an effort to reduce the morbidity and mortality attributable to the disease.

Macrophage functions are regulated by murine decidual and tumor extracellular matrices.

Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Understanding the mechanisms which prevent maternal immunological rejection of the fetus remains a fundamental unsolved problem in immunology. We have previously reported that macrophages are inhibited by maternal decidual stromal cells residing at the maternal-fetal interface. In view of the central role of macrophages in cell-mediated immunity, this inhibition may contribute to preventing maternal antifetal responses. We now report that it was the solid phase signals embedded in the extracellular matrix (ECM) made by decidual cells which are responsible for inhibiting macrophage-mediated lysis of TNF-alpha-resistant P815 mastocytoma cells. The latter macrophage function is acquired after stimulation by interferon gamma and endotoxin. All these macrophage functions were also inhibited by ECM isolated from the Engelberth-Holm-Swarme (EHS) tumor. This tumor ECM has a similar biochemical composition to decidual ECM. This ECM inhibited the effector, as opposed to the stimulator, phase of macrophage-mediated tumor lysis. Laminin, type IV collagen, and heparan sulfate proteoglycans, the major known components of decidual and EHS ECMs, did not inhibit the above macrophage functions. Altogether these data indicate that macrophages were inhibited by solid phase signals embedded in decidual and EHS ECMs. Whether the solid phase signals in these two ECMs are biochemically identical remains to be determined. To our knowledge, such signals are a novel pathway of inhibiting macrophage functions which may be important in understanding the maternal-fetal immunologic relationship, and the pathogenesis of perinatal infections. Furthermore, the ability of EHS tumor ECM to inhibit macrophage functions may indicate that some tumors may defend themselves against host macrophage responses using solid phase signals. This may be important in understanding some host-tumor relationships.

Macrophage functions are regulated by the substratum of murine decidual stromal cells.

Because of their paternal antigens, the fetus and placenta may be considered an allograft in the maternal host. Local properties of the maternal-fetal interface, the placenta and decidua basalis, are important in preventing maternal immunologic rejection of the fetoplacental allograft. However, the exact nature of these local properties remains a fundamental unsolved problem in immunology. We now report that three macrophage functions were inhibited by the substratum formed by monolayers of decidual stromal cells via a novel pathway. Solid-phase inhibitors blocked macrophage adhesion, spreading, and lysis of tumor necrosis factor-alpha-resistant P815 mastocytoma tumor cells. Inhibition was not solely attributable to an inability of macrophages to adhere to decidual substratum because there were differences in macrophage functions on this surface versus polyhema where no adherence occurred. Because macrophages play a central role in cell-mediated immunity, including allograft rejection, inhibiting their function in the decidua basalis may help prevent maternal antifetal responses.

Structure of the universal stress protein of Haemophilus influenzae.

BACKGROUND: The universal stress protein UspA is a small cytoplasmic bacterial protein whose expression is enhanced several-fold when cellular viability is challenged with heat shock, nutrient starvation, stress agents which arrest cell growth, or DNA-damaging agents. UspA enhances the rate of cell survival during prolonged exposure to such conditions, suggesting that it asserts a general "stress endurance" activity. However, neither the structure of UspA nor the biochemical mechanism by which it protects cells from the broad spectrum of stress agents is known. RESULTS: The crystal structure of Haemophilus influenzae UspA reveals an asymmetric dimer with a tertiary alpha/beta fold similar to that of the Methanococcus jannaschi MJ0577 protein, a protein whose crystal structure revealed a novel ATP binding motif. UspA differs significantly from the MJ0577 structure in several details, including the triphosphate binding loop of the ATP binding motif; UspA shows no ATP binding activity. CONCLUSIONS: Within the universal stress protein family that is delineated by sequence similarity, UspA is the only member which has been correlated with a cellular activity, and MJ0577 is the only member which has been assigned a biochemical activity, i.e., ATP binding. UspA has a similar fold to the MJ0577 protein but does not bind ATP. This suggests that members of this protein family will segregate into two groups, based on whether or not they bind ATP. By implication, one subset of the universal stress proteins presumably has an ATP-dependent function, while another subset functions in ATP-independent activities.

Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay.

Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations.

Effects of a cytosolic protein on the interaction of rat pancreatic zymogen granules in vitro.

Photon correlation spectroscopy has been used to study the kinetics of aggregation of isolated rat pancreatic zymogen granules in vitro by monitoring time-dependent changes in mean particle size derived from the photon count autocorrelation function, g2(tau). Isolated granules were stable in isotonic sucrose (pH 5.4-7.0). At pH 6.0 they maintained a mean diameter of 1225 +/- 18 nm with a polydispersity index of 0.199 +/- 0.007. The mean granule diameter showed a limited decrease (approx. 20%) with increasing pH within the range 5.4-7.0, but the polydispersity index was unaltered. At pH greater than 7.0 granule instability was indicated by a rapid reduction in total photon counts. In solutions of monovalent cations ([M+] greater than 10 mM) and divalent cations ([M2+] greater than 0.5 mM) zymogen granules aggregated at a rate dependent upon both ion and granule concentration. These effects were consistent with the bimolecular nature of the interaction mechanism and were clearly distinguishable from the limited size changes associated with osmolarity. At concentrations of Na+ or K+ salts greater than 50 mM granule aggregation was accompanied by anion-dependent solubilisation. A soluble protein fraction separated from the pancreatic acinar cell cytosol by gel filtration reduced the mean diameter and polydispersity index of zymogen granules suspended in isotonic sucrose, inhibited cation-induced aggregation and stabilised granules to solubilisation induced by raising pH greater than 7.0 or exposure to high ionic strength media. The inhibitory effects of this protein were apparent at concentrations less than or equal to 10 micrograms X ml-1 (i.e. at inhibitor: granule protein ratios less than 1:20) and could not be mimicked by bovine serum albumin, the Ca2+-binding proteins calmodulin and troponin C (less than or equal to 100 micrograms X ml-1), nor the highly negatively charged polymer polyglutamate (less than or equal to 10 micrograms X ml-1). Inhibitory activity was also absent from fractions of rat liver cytosol prepared identically to pancreatic acinar cytosol. These observations are consistent with the presence in pancreatic acinar cells of a specific cytosolic granule stabilisation factor (or factors) that normally restricts zymogen granule interaction and may therefore play an important role in the regulation of granule mobility and exocytosis.

Biochemical and biophysical characterization of insulin granules isolated from rat pancreatic islets by an iso-osmotic gradient.

The size and polydispersity of insulin granules isolated from rat pancreatic islets by centrifugation on a linear iso-osmotic gradient (300 mosM) have been characterized by quasi-elastic light scattering and photon correlation spectroscopy. The separation of granules by the linear gradient technique was compared directly to isolation on discontinuous gradients of hypertonic sucrose (300-1950 mosM) and the greater efficiency of separation assessed by parallel measurements of protein, insulin, cytochrome oxidase and beta-glucuronidase. Granules isolated from pancreatic beta-cells had a mean particle diameter of 342 nm, buoyant density of 1.104, hydrated mass of 23 femtograms and maximal insulin content of 8-9.6 . 10(5) molecules per granule.

Crystals of an ATPase fragment of bovine clathrin uncoating ATPase.

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).

Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity.

Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication.

The metzincins--topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases.

The three-dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable "Met-turn." This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and astacin and a little further away from adamalysin II (17% identity). The pairs astacin/adamalysin II, astacin/human neutrophil collagenase, and adamalysin II/human neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation "metzincins" has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin-like enzymes, which share the truncated zinc-binding motif HEXXH and, moreover, similar topologies in their N-terminal domains.

A gene, cobA + hemD, from Selenomonas ruminantium encodes a bifunctional enzyme involved in the synthesis of vitamin B12.

Coenzymes derived from vitamin B12 (cyanocobalamin) are particularly important for core metabolism in ruminant animals. Selenomonas ruminantium, a Gram-positive obligate anaerobe isolated from cattle, is the main contributor of vitamin B12 to such ruminant animals. In nature, there are both aerobic and anaerobic pathways for B12 synthesis - the latter is only partly elucidated. Until now, there has been no investigation of B12 synthesis in S. ruminantium, which must use an anaerobic pathway. This paper reports the cloning of the chromosomal operon from S. ruminantium which is responsible for the first committed steps in corrinoid synthesis. Five open reading frames were found in the cloned fragment. All deduced amino acid sequences had similarity to defined proteins in the databases that are involved in porphyrin and corrin synthesis. Of particular interest is the gene designated cobA + hemD, which encodes a single polypeptide possessing two catalytic functions - uroporphyrinogen III synthase and uroporphyrinogen III 2,7-methyltransferase. This enzyme converts hydroxymethylbilane to precorrin-2. The functions of the protein coded by cobA + hemD were established by heterologous expression in Escherichia coli. The CobA activity has been demonstrated for three distinct types of proteins - monofunctional, bifunctional with siroheme formation and, this report, bifunctional with uroporphyrinogen III synthesis. The type found in S. ruminantium (cobA + hemD) is probably restricted to obligately anaerobic fermentative bacteria.

Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in Azotobacter chroococcum.

We report the cloning and analysis of a gene and its cognate regulatory element from a member of the Azotobacteriaceae which are involved in the breakdown of an aromatic compound. The genes from Azotobacter chroococcum encoding p-hydroxybenzoate hydroxylase (pobA) and its regulatory protein (pobR) were cloned from a genomic library and sequenced. Sequence analysis of pobA revealed homology with other bacterial p-hydroxybenzoate hydroxylase enzymes. Residues essential to the structure and function of the enzyme have been conserved. The pobR gene encodes a DNA binding regulatory protein with similarity to proteins from the AraC/XylS family of transcriptional activators. A fragment containing both pobA and pobR was cloned into pUC19 and p-hydroxybenzoate hydroxylase activity was induced in Escherichia coli by the addition of p-hydroxybenzoate. A frame-shift mutation introduced into the pobR gene prevented expression of p-hydroxybenzoate hydroxylase, indicating that PobR is the protein required for transcription of pobA. Interestingly, A. chroococcum PobR has no homology to the PobR protein that is the transcriptional activator of pobA in Acinetobacter strain ADP1, a protein that is homologous to the IclR family of transcriptional regulators. However, PobR from A. chroococcum is homologous to several other proteins, suggesting that these proteins will also function as transcriptional activators of pobA.

Renal parenchymal malakoplakia. Histologic spectrum and its relationship to megalocytic interstitial nephritis and xanthogranulomatous pyelonephritis.

We report five cases of renal parenchymal malakoplakia. They represent a histologic spectrum of this uncommon inflammatory process as it involves the renal parenchyma. Only one of these five cases presented the classical histologic picture of malakoplakia ("diagnostic stage"). Two cases--although with similar cellular infiltrate--had a marked paucity of Michaelis-Gutmann bodies and thus might have been classified as megalocytic interstitial nephritis were it not for the identification of such calcific intracellular inclusions under the electron microscope. The fourth case presented a pseudosarcomatous morphology and is similar to what has been described as a late or fibrous stage in malakoplakia of the urinary bladder. The last case had a destructive, frankly granulomatous histologic picture with a preponderance of foamy histiocytes reminiscent of xanthogranulomatous pyelonephritis, but with clusters of von Kossa-positive Michaelis-Gutmann bodies. The last two forms of the disease have not been previously reported to occur in the kidney. We believe that these cases represent various stages of development of the disease.

Pregnancy as a natural model of allograft tolerance. Interactions between adherent macrophages and trophoblast populations.

From an immunologic viewpoint, the fetus with its paternal antigens may be considered a successful allograft in the maternal host. Understanding the basis of this host-allograft relationship remains a fundamental unsolved problem in transplantation immunobiology. We have previously demonstrated that local immunoregulation in the murine placenta prevented macrophage activities necessary for an effective response against the intracellular bacterium Listeria monocytogenes. Given the central role of macrophages both as antigen-presenting and cytolytic effector cells, such local immuno-regulation may ordinarily help prevent rejection of the fetoplacental unit with its paternal alloantigens by the maternal immune system. We therefore examined two types of interaction between macrophages and the placental cells that populate the maternal-fetal interface. (1) Upon activation to kill listeria efficiently, macrophages also acquire cytolytic capacities against some tumor and embryonic cells. We tested the hypothesis that macrophage activation in the placenta was inhibited to prevent macrophages from lysing fetal trophoblasts. We found, however, that trophoblasts isolated by dispase dispersion, differential isopyknic centrifugation, and adherence were not lysed by three different populations of cytolytic macrophages: (a) those activated in vivo during listeriosis, (b) peptone-elicited macrophages activated in vitro by recombinant interferon gamma and other lymphokines, and (c) the macrophage cell line RAW 264.7 activated in vitro. (2) Previous studies had demonstrated that cells from the placental region and their conditioned media inhibited a variety of lymphocyte functions. However, we found that these did not inhibit activation of adherent macrophages as assessed by induction of cell-surface Ia and acquisition of tumoricidal activity. In addition, under conditions where placental cells inhibited the proliferative response of a cloned CD4+ anti-Listeria T cell line to fixed, antigen-pulsed macrophages, the secretion of macrophage-activating lymphokines was not affected. These studies are important because they indicate that previously described suppressor systems in the murine placental region do not account for the profound local deficits in macrophage function seen during listeriosis.

A randomized prospective trial of anti-Tac monoclonal antibody in human renal transplantation.

Patient entry is now complete in a prospective trial of anti-Tac, a murine IgG2a monoclonal antibody directed against the p55 chain of the human IL-2 receptor, for the prevention of renal allograft rejection. Recipients of primary cadaver allografts were randomized to receive either anti-Tac (20 mg q.d. x 10 days beginning POD 1) plus low-dose CsA (4 mg/kg/day), azathioprine (2 mg/kg/day), and prednisone (30 mg q.d.), or conventional triple therapy with CsA (8 mg/kg/day), azathioprine, and prednisone. Forty patients were entered in each group, with current followup from 6 to 26 months. The results show a significant reduction in early rejection episodes in the anti-Tac-treated patients. During the 10-day treatment, 5 of 40 anti-Tac patients had rejection episodes, compared with 21 of 40 control patients (P less than 0.001). Anti-Tac significantly delayed the time to the first rejection (12.5 +/- 6.3 vs. 7.6 +/- 6.7 days) (P less than 0.05). Despite these effects, there were no differences in either actual or actuarial graft or patient survival between the two groups. Pneumonia, primarily CMV, developed in 5 treated and 4 control patients. In patients with functioning grafts mean serum creatinine at 3 months was 1.8 +/- 0.7 in the anti-Tac group and 2.0 +/- 0.8 in the control group (P = NS); at 12 months the values were 2.3 +/- 1.5 and 1.8 +/- 0.5, respectively (P = NS). The peak expression of IL-2 receptors on circulating T-cells was significantly lower in anti-Tac patients (15.1 +/- 3.6%) than in controls (21.9 +/- 4.5%) (P less than 0.05). Seven of 10 patients tested to date developed antimouse immunoglobulin antibodies, with antiidiotype shown in 6. These antibodies do not preclude subsequent treatment with OKT3. Five patients in this and previous anti-Tac protocols have received OKT3 for acute rejection despite known pretreatment antimouse antibodies, with resolution of rejection in all cases.