Are viruses associated with disc herniation? A clinical case series.

BACKGROUND: There is some limited evidence for the presence of viruses in herniated disc material including a previous case series that claimed to provide "unequivocal evidence of the presence of herpes virus DNA in intervertebral disc specimens of patients with lumbar disc herniation suggesting the potential role of herpes viruses as a contributing factor to the pathogenesis of degenerative disc disease". This study has not been replicated. The objective of our study was to determine if viruses were present in herniated disc fragments in participants with a prior history of back pain. METHODS: We recruited fifteen participants with a history of prior low-back pain prior to undergoing disc herniation surgery in the lumbar spine. Harvested disc samples were subject to next generation sequencing for detection of both RNA and DNA viral pathogens. Additionally, samples were analysed by a broadly reactive PCR targeting herpesviral DNA. Ethics approval was granted by the Human Research Ethics Committees of both Murdoch University, and St John of God Hospital, Western Australia. RESULTS: Of the fifteen research participants, 8 were female. Mean age was 49.4 years (SD 14.5 yrs) with a range of 24-70 years. All participants had prior back pain with mean time since first ever attack being 8.8 years (SD 8.8 yrs). No samples contained significant DNA sequences relating to known human viral agents. Inconsequential retroviral sequences were commonly found and were a mixture of putative animal and human retroviral protein coding segments. All samples were negative for herpesvirus DNA when analysed by pan-herpesvirus PCR. CONCLUSIONS: This study found no viral pathogens in any intervertebral disc fragments of patients who had previous back pain and underwent discectomy for disc herniation and thus it is unlikely that viruses are associated with disc herniation, however given the contradiction between key studies enhanced replication of this experiment is recommended.

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer(®) peptides.

Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function.

Efficacy of oryzalin and associated histological changes in Cryptosporidium-infected neonatal rats.

This paper reports the anti-cryptosporidial effects of, and concomitant amelioration of the histological changes in the gut of neonatal rats with intestinal cryptosporidiosis treated with the dinitroaniline, oryzalin. The ED50 was determined to be 7 mg/kg using twice daily doses administered for 3 consecutive days. A maximum inhibition of 85.5% was achieved at 25 mg/kg and this inhibition remained constant despite increasing the oryzalin dose to 200 mg/kg. Cryptosporidiosis significantly decreased the intestinal villus/crypt (VC) ratio by approximately 50% (duodenum = 2.3, jejunum = 2.5 and ileum = 1.7) when compared to uninfected untreated controls (duodenum = 4.3, jejunum = 5.9 and ileum = 4.5). Treatment with oryzalin doubled the VC ratio in the duodenum, jejunum and ileum following doses of 5 mg, 50 mg and 200 mg/kg respectively. Oryzalin concentrations in the small intestine contents and plasma were determined, using HPLC, at 0.5, 1 and 2 h after dosing. The much greater dose required to return VC ratios to normal in the ileum (200 mg/kg) compared to the duodenum (6.25 mg/kg) appeared to reflect the decreased concentration of the drug in the distal small intestine. Concentrations of oryzalin equivalent to the in vitro IC50 were maintained for 2 h in the first half of the small intestine following a single dose of 100 mg/kg.

Plasmodium: assessment of the antimalarial potential of trifluralin and related compounds using a rat model of malaria, Rattus norvegicus.

A rodent model of malaria, Plasmodium berghei was used to assess the antimalarial potential of dinitroaniline herbicides. Trifluralin, pendimethalin, oryzalin, and benfluralin were all active against P. berghei in vitro at, or close to, submicromolar concentrations, with a rank order of potency similar to that against other protozoa. The dinitroanilines did not elicit a cytotoxic effect against a mammalian cell line at concentrations 100-fold higher than those for activity against P. berghei. Neither trifluralin nor oryzalin exhibited any antimalarial activity in vivo after oral administration at the maximum dose tolerated by the host. In a pharmacokinetic study, it was found that the lack of in vivo antimalarial activity was due to poor absorption. Other DNs which have better absorption characteristics than either trifluralin or oryzalin may offer more scope for antimalarial activity in vivo.

Expression of Giardia duodenalis beta-tubulin as a soluble protein in Escherichia coli.

The beta-tubulin gene of the parasitic protozoan Giardia duodenalis has been expressed for the first time using a novel and direct method. The protein was expressed in both soluble and insoluble forms in an Escherichia coli-based expression system. The level of expression was found to be affected by several variables including the incubation temperature, length of time for which expression was carried out, and the E. coli culture volume. The protein expression system contributed no additional amino acids to the final fusion protein and the polyhistidine fusion sequence was easily removed from the beta-tubulin protein using a specific enterokinase enzyme. The expression system also provided a means of preparing a soluble protein and purifying it by a relatively straightforward affinity chromatography method to give a very high level of protein purity. This makes the protein suitable for a number of applications for characterization including beta-tubulin antibody assays, alpha-/beta-tubulin-binding regions, and beta-tubulin folding intermediates.

A comparison of the effects of a benzimidazole and the dinitroanilines against Leishmania infantum.

Leishmania infantum promastigotes and amastigotes were axenically cultured and exposed to the known tubulin binding compounds, the dinitroanilines, trifluralin, benfluralin, pendimethalin, oryzalin and the precursor of the dinitroanilines, chloralin, as well as isomers of chloralin and trifluralin and to the benzimidazole, albendazole. Drug induced inhibition was observed using [3H]thymidine uptake compared with untreated controls. In vitro analysis demonstrated a significant difference in the activity of five of the seven dinitroanilines between both life cycle stages of L. infantum. The amastigotes were 20-times more sensitive to chloralin and its isomer than to the dinitroanilines whereas the promastigotes were similar in sensitivity to the dinitroanilines and to chloralin and its isomer. This interesting finding suggests that the dinitroaniline precursors may have different target sites in the amastigotes to those within the promastigotes. Additionally, both chloralin and its isomer, and to a lesser extent benfluralin, caused a substantial stimulation of thymidine incorporation (up to 50%) at low concentrations. Dose response analysis suggests that the dinitroanilines may have more than one mode of action against L. infantum amastigotes and promastigotes. The inhibitory effects of the dinitroanilines against L. infantum vary from previous findings using the dinitroanilines against other Leishmania spp. The 348 base pair DNA sequence coding for beta-tubulin from amino acid residues 132 to 248 was obtained for L. infantum and used to compare the in vivo efficacy of albendazole with predicted activity based on beta-tubulin sequences of known benzimidazole sensitive protozoa. The use of beta-tubulin sequence as a predictive model of benzimidazole activity is discussed with particular reference to L. infantum.

A comparison of the effects of two dinitroanilines against Cryptosporidium parvum in vitro and in vivo in neonatal mice and rats.

The effects of two dinitroanilines, oryzalin and trifluralin, were compared against Cryptosporidium parvum, in vitro using HCT-8 cells and in vivo using neonatal Swiss ARC mice and Wistar neonatal rats. In vitro, oryzalin and trifluralin exhibited IC(50) values (concentration necessary to cause a 50% inhibition) of 750 and 800 nM, respectively. A viability assay showed that neither compound produced a cytotoxic effect on the host cells at concentrations as high as 1 microM. The in vivo component of this study consisted of inoculation of neonatal mice and neonatal rats with 10(5) viable oocysts of C. parvum per animal and the subsequent treatment of this infection with trifluralin and oryzalin administered via gastric intubation. At doses of 100 mg kg(-1) body weight administered twice daily for 3 consecutive days, trifluralin had no statistically significant effect on the number of oocysts recovered from the gut of either rats or mice compared with controls, whereas at the same concentration, oryzalin caused 90 and 79% inhibition of oocysts recovered from mice and rats, respectively.

Assessment of drugs against Cryptosporidium parvum using a simple in vitro screening method.

A rapid semi-quantitative screening method was devised for assessing the anticryptosporidial and cytotoxic effects of putative chemotherapeutic compounds. The method is suitable as an initial rapid screening procedure from which compounds demonstrating anticryptosporidial activity can be identified for further analysis. It has the advantages of speed, low cost and concurrent assessment of anticryptosporidial and cytotoxic effects and allows accurate determination of minimum lethal concentrations. Of the 71 compounds screened, six completely inhibited cryptosporidial growth at 1 microM (monensin, salinomycin, alborixin, lasalocid, trifluralin and nicarbazin) and a further eight showed significant anticryptosporidial activity at 1 or 20 microM (halquinol, bleomycin, suramin, mitomycin, doxycycline hydrochloride, toltrazuril, chloroquine phosphate and teniposide). Twelve compounds were found to have some degree of cytotoxicity at 1 microM and a further 12 at 20 microM.

Molecular and biological characterisation of Cryptosporidium in pigs.

OBJECTIVE: Genetic and biological characterisation of 12 isolates of Cryptosporidium from pigs and comparing them with Cryptosporidium isolates from humans and cattle. DESIGN: Cryptosporidium isolates from pigs were compared with those obtained from human and cattle using rDNA sequence analysis. The infectivity of two of the porcine isolates was determined in neonatal mice and the clinical history of the infected pigs recorded. RESULTS: Pig-derived isolates of Cryptosporidium exhibited two distinct genotypes; a porcine genotype and a bovine genotype, which is common to cattle and other livestock. The porcine genotype did not produce any infection in neonatal mice whereas the bovine genotype did. CONCLUSION: Two distinct genetically and biologically differing strains of Cryptosporidium appeared to be associated with acute diarrhoea in pigs. Whether Cryptosporidium was a primary or secondary pathogen is unclear but warrants further investigation. As the bovine genotype is known to infect humans, the results suggest that pigs can act as reservoirs of cryptosporidial infections for humans and other live-stock. The zoonotic potential of the pig-adapted genotype is uncertain and requires further study.

Murine strongyloidiasis: the effects of cyclosporin A and thiabendazole administered singly and in combination.

A single Cyclosporin A (CsA) dose of 30 mg kg-1 given orally at day 4 post-infection (p.i.) to Sprague-Dawley rats infected with Strongyloides ratti, reduced the faecal larval count by 46.8 +/- 1.2%. CsA was equally effective when the same dose rate was administered subcutaneously at day 4 p.i., reducing the faecal larval count by 41.6 +/- 8.6%. Thiabendazole (TBZ) given orally at 5 or 10 mg kg-1 (single dose at day 4 p.i.) reduced the faecal larval counts by 57.1 +/- 4.1% and 69.0 +/- 9.6%, respectively. Orally administered CsA was less effective than 5 mg TBZ kg-1 (at day 4 p.i.) Co-administration of 5 mg TBZ kg-1 and CsA did not elicit synergy or additive efficacy, indicating that CsA did not antagonise the anti-strongyloides activity of TBZ. The data suggests that for patients with current, historical or serological evidence of strongyloidiasis, CsA may be used where immunosuppressive therapy is required for other concurrent reasons or when TBZ is contraindicated.

Steady-state content of glycolytic/tricarboxylic acid-cycle intermediates, adenine nucleotide pools and the cellular redox-status in the infective (L3) larvae of (homogonic) Strongyloides ratti.

Infective (L3) larvae of Strongyloides ratti (homogonic strain) were freeze-clamped (-196 degrees C) and the steady-state content of the glycolytic, Krebs tricarboxylic acid (KTA)-cycle intermediates and adenine nucleotides analysed. Comparison of the mass-action ratios (MARs) of the glycolytic enzymes with their apparent equilibrium constants (K9eq) indicate that phosphoglucomutase, glucosephosphate isomerase, triosephosphate isomerase, phosphoglyceromutase and phosphopyruvate hydratase reactions were all at or near equilibrium, whilst hexokinase, phosphofructokinase and pyruvate kinase were displaced from equilibrium. The S. ratti aldolase and myokinase appear to be somewhat displaced from equilibrium and thus may have pseudoregulatory roles. The adenylate energy charge (AEC), ATP/ADP ratio and the available adenylate energy (AAE) indices were 0.9 +/- 0.04, 8.76 +/- 1.5 and 397 +/- 43, respectively. The free [NAD+]/[NADH+H+] ratio of the cytoplasmic compartment of S. ratti L3 larvae calculated employing the steady-state content of the oxidised and reduced substrates of lactate dehydrogenase (E.C. 1.1.1.27) and the combined glyceraldehyde 3-phosphate dehydrogenase (E.C. 1.2.1.12)/3-phosphoglycerate kinase (E.C. 2.7.2.3) system were ca. 523 and 1200, respectively. The free[NAD+]/[NADH+H+] ratio in the mitochondrial compartment of S. ratti L3 larvae calculated using the malate dehydrogenase (E.C. 1.1.1.37) equilibrium was found to be 1962:1. The data is discussed with respect to the predominantly aerobic nature of the energy metabolism of the L3 larvae.

Strongyloides ratti: mitochondrial enzyme activities of the classical electron transport pathway in the infective (L3) larvae.

Submitochondrial particles prepared from S. ratti L3 larvae exhibited NADH-oxidase (NOX), NADH-ferricyanide reductase (NFR), NADH-cytochrome-c-reductase (NCR), succinate-cytochrome-c-reductase (SCR), and cytochrome-aa3-oxidase activities of 2.1 +/- 0.3, 8.9 +/- 1.3, 0.6 +/- 0.1., 1.0 +/- 0.2 and 1.2 +/- 0.3 nm min-1 mg protein-1 respectively, at 37 degrees C. The NCR and NOX activities were 39.3% and 23.5% of the NFR activity, suggesting the occurrence of a rate-limiting step or bifurcation of the respiratory electron transport (RET) pathway on the oxygen-side of RET-Complex I. The NCR activity was 50% that of cytochrome-aa3-oxidase activity which suggests partitioning of electron flow at the level of RET-Complex III and/or the quinone-function. Antimycin A and rotenone but not 2-thenoyl trifluoroacetone (TTFA) inhibited NCR activity, the EC50 values were 3.6 x 10(-6) M, 3.7 x 10(-7) M, respectively. SCR activity was inhibited by antimycin A (EC50 = 3.8 x 10(-6) M) and TTFA (EC50 = 2.8 x 10(-5) M) but not by rotenone. The results suggest that presence of classical and alternate RET-pathways in S. ratti L3 larvae.

The effect of electron transport (ET) inhibitors and thiabendazole on the fumarate reductase (FR) and succinate dehydrogenase (SDH) of Strongyloides ratti infective (L3) larvae.

The fumarate reductase (FR) and succinate dehydrogenase (SDH) activities of isolated submitochondrial particles (SMPs) prepared from axenised L3 larvae of S. ratti were characterised with respect to their response to a selected range of inhibitors. Rotenone (a specific inhibitor of electron transport Complex I) inhibited the S. ratti FR (EC50 = 3.0 x 10(-7) M) but not SDH. This strongly suggests that the S. ratti FR is functionally linked with the S. ratti ET-Complex I. 2-Thenoyltrifluoroacetone (TTFA, an inhibitor of ET-Complex II) inhibited FR (EC50 = 2.6 x 10(-5) M) and SDH (EC50 = 2.8 x 10(-5) M) with similar effectiveness. Sodium malonate (substrate analogue of succinate) had a greater affinity for SDH (EC50 = 6.8 x 10(-4) M), than FR (EC50 = 1.9 x 10(-2) M). Sodium fumarate was ca. 8-fold more effective in inhibiting the S. ratti FR (EC50 = 6.0 x 10(-4) M) than SDH (EC50 = 4.8 x 10(-3) M). The S. ratti FR was more sensitive to inhibition by thiabendazole (TBZ; EC50 = 4.6 x 10(-4) M) than SDH (EC50 > 1.0 x 10(-3) M), suggesting that one of the sites-of-action of TBZ to be the FR of S. ratti mitochondria. More potent inhibitors of S. ratti FR, if developed, may prove to be effective chemotherapeutic agents in the management of human strongloidiasis.

Strongyloides ratti: fumarate reductase and succinate dehydrogenase activities of infective larvae.

Submitochondrial particles prepared from axenised infective (L3) larvae of S. ratti (homogonic-strain) were assayed spectrophotometrically for fumarate reductase (FR) and succinate dehydrogenase (SDH) and their kinetic properties characterised. The S. ratti FR (pH 8.2; 37 degrees C) exhibited a maximum specific activity of 3.45 nmol (min)-1 (mg protein)-1 at a sodium fumarate concentration of 0.3 mM. Interestingly, the FR activity declined at fumarate concentrations greater than 0.3 mM. The mechanism of this unusual inhibitory effect requires further study. The S. ratti SDH (pH 8.2; 37 degrees C) showed a Vmax of 17.4 nmol (min)-1 (mg protein)-1; the Kmsucc was 0.5 mM. Although the SDH:FR ratio cannot predicate vectorial electron flow as would occur in vivo, an in vitro ratio of 5.04:1 was observed for SMPs derived from S. ratti L3 larvae.

An alternative protocol for the serial passage and maintenance of a homogonic strain of Strongyloides ratti in female Sprague-Dawley rats.

A method which does not involve the tedious use of watch glass coprocultures for obtaining filariform infective (L3) larvae of Strongyloides ratti from faecal pellets of infected Sprague-Dawley rats is described. The alternative method utilises Baermannization (18 h) of faecal pellets to yield rhabditiform (L1) larvae of S. ratti and their subsequent culture for 72 h at 19 degrees C in tissue-culture-flasks containing only dechlorinated tap water to yield infective filariform (L3) larvae. The yields and infectivity of the L3 larvae obtained from the two methods were essentially similar.

The efficacy of two electron transport inhibitors (720C80 and 993C76) on murine strongyloidiasis: a comparison with albendazole.

The clinical efficacy of albendazole (ABZ) in the treatment of chronic uncomplicated strongyloidiasis has been reported to be highly variable. In our murine model of strongyloidiasis a single oral dose of 5 and 10 mg kg-1 ABZ reduced (at day 4 post infection) the faecal larval count (FLC) by 54.2 +/- 12.5% and 81.5 +/- 10.2%, respectively. 100 mg kg-1 ABZ reduced the FLC by 100%. Two inhibitors of protozoan and filarial electron transport (720C80 and 993C76) inhibited the endogenous O2 consumption of intact infective (L3) larvae of S. ratti by > 50% at 2 x 10(-5) M in vitro, and reduced the FLC by 72 +/- 9.3% and 62.0 +/- 10.3% respectively in vivo, at a dose of 70 mg kg-1. These results suggest that compounds designed as selective inhibitors of protozoan electron transport have significant efficacy against murine strongyloidiasis and may prove useful in the management of human strongyloidiasis.

Difference spectroscopic characterisation of the cytochrome complement in Acanthocheilonema viteae.

(Dithionite-reduced) minus (ferricyanide-oxidised) difference spectra of 600 x g and 12,000 x g subcellular pellet fractions of adult male Acanthocheilonema viteae exhibited alpha-absorption maxima (296 K) attributable to Cyt c555, Cyt b562 and aa3 (600-605 nm). The gamma(Soret) maximum of both fractions was evident at 427 nm, with a shoulder at 432-434 nm. 600 x g and 12,000 x g pellet fractions of adult female and mixed-sex adult A. viteae exhibited similar absorption maxima. (Succinate-reduced)--(ferricyanide-oxidised) difference spectra of the 12,000 x g pellet fraction of mixed-sex adult A. viteae showed absorption maxima at 555 and 562 nm, 600 and 630 nm, suggesting the reduction of Cyt c555, Cyt b562, Cyt aa3 (600 nm) and an unidentified species (630 nm peak) Antimycin A (10(-6) M) induced the disappearance of the maxima at 555, 600 and 630 nm corresponding to Cyt c555, Cyt aa3 and the unidentified species; the maximum at 562 nm prevailed in the presence of antimycin A. These antimycin A induced changes can be cited as classical evidence for the functional involvement of these a, b and c type cytochromes in respiratory electron transport. (Dithionite reduced + CO)--(dithionite reduced) difference spectra suggest that adult A. viteae may have one or more CO-binding-species, one of which appears to be a low-spin-haemoprotein with a b-type or c-type haem, which has essentially an electron carrier function rather than a ligand binding function.

The uptake and conversion of L-[U14C-] aspartate and L-[U14C-] alanine to 14CO2 by intact trophozoites of Giardia duodenalis.

1. Intact trophozoites of Giardia duodenalis (clone P1C10) took up and metabolised L-[U14C-] aspartate to 14CO2 at rates of 10.27 +/- 0.76 and 27.6 +/- 2.07 ng hr-1 10(-6) cells in a simple maintenance medium (MM) and in a complex bile supplemented (BIS-33) medium respectively. 2. Intact trophozoite of G. duodenalis (clone P1C10) also took up and metabolised L-[U14C-] alanine to 14CO2 at rates of 20.6 +/- 1.1 and 91.4 +/- 17.5 ng hr-1 10(-6) cells in the simple (MM) and complex (BIS-33) medium respectively. 3. trophozoite sonicates contained significant levels of aspartate-2-oxoglutarate transaminase (AST; EC 2.6.1.1) and alanine-2-oxoglutarate transaminase (ALT; EC 2.6.2.2.). Specific activities (at 23 degrees C) were 95.1 +/- 11.3 and 87.3 +/- 9.8 nmol (min)-1 (mg protein)-1 respectively. 4. These observations suggest that Giardia has the capacity to utilise aspartate and alanine and possibly other amino acids as alternative sources of energy. 5. The extrusion or uptake of alanine by Giardia trophozoites may be dictated by the intracellular redox-status of the protozoan parasite or components in the external mileu.

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport.

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO2) of 181.8 +/- 12.4 ng atoms min-1 mg dry weight-1 at 35 degrees C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 microM) produced 33 +/- 6.5% inhibition of the E-QO2. Unusually the rotenone-induced inhibition was not relieved by 5 mM-succinate. The E-QO2 of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 microM; 4 microM-antimycin inhibited less than or equal to 10% of the E-QO2. The electron donor couple ascorbate/TMPD augmented the E-QO2 in the presence of rotenone (2 microM) and antimycin A (4 microM) by 110%. Azide (1 mM) stimulated the antimycin A refractory QO2 by 36.6 +/- 7.2% which was only partially inhibited by 1.0 mM-KCN (IC50 = 0.8 mM). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.