Anti-tumour necrosis factor treatment increases circulating T helper type 17 cells similarly in different types of inflammatory arthritis.

We investigated changes in circulating T helper type 17 (Th17) cells following anti-tumour necrosis factor (TNF) in rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) patients. Peripheral blood mononuclear cells (PBMC) were isolated from 25 RA, 15 AS and eight PsA patients at baseline 4 and 12 weeks after treatment, and Th17 cell frequencies were analysed using interleukin (IL)-17 enzyme-linked immunospot (ELISPOT) and flow cytometry. A significant increase in IL-17-producing cells was observed by ELISPOT in RA and AS patients at 12 weeks. Flow cytometry confirmed significant increases in CD4(+) IL-17(+) cells at 12 weeks in RA and AS and 4 weeks in PsA patients. Anti-TNF treatment increases circulating Th17 cells in three different diseases.

Blockade of tumour necrosis factor-α in experimental autoimmune encephalomyelitis reveals differential effects on the antigen-specific immune response and central nervous system histopathology.

In various autoimmune diseases, anti-tumour necrosis factor (TNF)-α treatment has been shown to reduce both clinical disease severity and T helper type 1 (Th1)1/Th17 responses. In experimental autoimmune encephalomyelitis (EAE), however, the role of TNF-α has remained unclear. Here, C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 and treated with anti-TNF-α, control antibody or vehicle. The clinical disease course, incidence and severity were assessed. On day 20 after immunization the antigen-specific Th1/Th17 response was evaluated by enzyme-linked immunospot (ELISPOT) in spleen and central nervous system (CNS). Also, the extent of spinal cord histopathology was analysed on semi- and ultrathin sections. Our results demonstrate that anti-TNF-α treatment reduced the incidence and delayed the onset of EAE, but had no effect on disease severity once EAE had been established. Whereas anti-TNF-α treatment induced an increase in splenic Th1/Th17 responses, there was no effect on the number of antigen-specific Th1/Th17 cells in the spinal cord. Accordingly, the degree of CNS histopathology was comparable in control and anti-TNF-α-treated mice. In conclusion, while the anti-TNF-α treatment had neither immunosuppressive effects on the Th1/Th17 response in the CNS nor histoprotective properties in EAE, it enhanced the myelin-specific T cell response in the immune periphery.

Atmospheric freeze drying for the reduction of powder electrostatics of amorphous, low density, high surface area pharmaceutical powders.

Amorphous itraconazole (ITZ) was prepared by Thin Film Freezing (TFF) utilizing 1,4-dioxane as the solvent with subsequent solvent removal via conventional tray lyophilization (ITZ LYO) or atmospheric freeze drying (ITZ AFD). ITZ AFD was prepared under various drying conditions to assess the influence of drying parameters on powder properties. XRD analysis confirmed all products were amorphous and DSC analysis revealed both drying processes resulted in the formation of the nematic mesophase of ITZ. SEM revealed a larger pore size and agglomerate size with fewer fine particles (i.e. less than 10 microns in diameter) for ITZ AFD compared to ITZ LYO. Residual solvent analysis revealed a primary drying temperature of -10°C resulted in residual solvent levels above the acceptable limits set by the International Conference on Harmonization as a result of microcollapse. Primary drying temperatures of less than -10°C resulted in acceptable residual solvent levels. The extent of microcollapse did not alter the macrostructure of the resulting powder. Powder flowability was determined to be similar for ITZ AFD and ITZ LYO based on Carr's index and the Hausner ratio, as well as by dynamic angle of repose. All powders displayed poor flowability. Chargeability measurements demonstrated a lower charge transfer for ITZ AFD powders compared to ITZ LYO due to a combination of factors including differences in residual solvent level, particle size, pore size, surface area, and fine particles content. The reduction in chargeability as a result of AFD is highly desirable because it allows for improved powder handling and use post-production.

What have we learnt from targeted anti-TNF therapy?

Anti-tumour necrosis factor (anti-TNF) therapy of patients with rheumatoid arthritis dates back to 1992, when the first proof-of-principle trials were performed in London by Maini and Feldmann. Considerable studies of the mechanism of action were performed, and insights into the way in which anti-TNF therapy delivers its benefit were obtained. In this brief review, certain aspects of knowledge acquired and the many gaps will be reviewed. Focus will be on the TNF-dependent cytokine cascade and what it means, and potential new approaches to treatment. Finally, an entertaining challenge: might many or even all unmet clinical needs be dealt with through cytokine analysis?

Zinc requirement for DNA replication in stimulated human lymphocytes.

The requirement for Zn(++) in DNA replication by phytohemagglutinin-stimulated human lymphocytes was studied. When 6 microM o-phenanthroline, a chelator with a high affinity for Zn(++), is added to cultures of stimulated lymphocytes a nearly complete inhibition of thymidine incorporation results within a few hours. In contrast, the incorporation of uridine is only slightly reduced and the incorporation of leucine is unaffected. m-Phenanthroline, a nonchelating analogue, does not alter the rate of thymidine incorporation even when present in 10-fold greater amounts than o-phenanthroline. The inhibition of thymidine incorporation by o-phenanthroline could be entirely reversed by the addition of Zn(++) to the cultures, or could be prevented by the prior addition of either Zn(++) or Ni(++). All other divalent cations tested were incapable of reversing the o-phenanthroline inhibition of thymidine incorporation.

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.

Immunotherapy for rheumatoid arthritis.

Recently published studies confirm that the long-term use of biological agents targeting TNF-alpha in therapy for rheumatoid arthritis (RA) gives rise to sustained improvement in symptoms and signs of disease, and in the quality of life. Furthermore, it has emerged that anti-TNF therapy protects joints from structural damage, which unexpectedly is also observed in the patient population showing no apparent benefit in control of signs and symptoms. Therapeutic benefit is observed in established disease that is unresponsive to conventional DMARDS and in early DMARDS-naïve RA patients. Thus, for patients with RA, anti-TNF therapies set a new standard for symptom control and joint protection.

Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes.

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

Detection and characterization of DNA polymerase from Trypanosoma brucei.

The predominant DNA polymerase activity has been isolated from the parasitic flagellated protozoan, Trypanosoma brucei. Like mammalian DNA polymerase-alpha the trypanosome DNA polymerase is of large molecular weight (S, 6--8), is resistant to thermal denaturation, is sensitive to N-ethylmaleimide, and is inhibited by high ionic strength. However, specific antisera that cross-react with mammalian DNA polymerase-alpha from different species fail to cross-react with the trypanosome polymerase.

Development of a low background liquid scintillation counter for a shallow underground laboratory.

Pacific Northwest National Laboratory has recently opened a shallow underground laboratory intended for measurement of low-concentration levels of radioactive isotopes in samples collected from the environment. The development of a low-background liquid scintillation counter is currently underway to further augment the measurement capabilities within this underground laboratory. Liquid scintillation counting is especially useful for measuring charged particle (e.g., ß and α) emitting isotopes with no (or very weak) gamma-ray yields. The combination of high-efficiency detection of charged particle emission in a liquid scintillation cocktail coupled with the low-background environment of an appropriately designed shield located in a clean underground laboratory provides the opportunity for increased-sensitivity measurements of a range of isotopes. To take advantage of the 35m-water-equivalent overburden of the underground laboratory, a series of simulations have evaluated the scintillation counter's shield design requirements to assess the possible background rate achievable. This report presents the design and background evaluation for a shallow underground, low background liquid scintillation counter design for sample measurements.

Anti-type II collagen ELISA. Increased disease specificity following removal of anionic contaminants from salt-fractionated type II collagen.

The purification of type II collagen, for the detection of anti-type II collagen antibodies by ELISA procedures, involves removal of proteoglycans by guanidine-HCl, followed by pepsin solubilisation and salt fractionation. However, type II collagen purified in this way may contain contaminants, despite the apparent purity on SDS-polyacrylamide gels. In this paper we demonstrate how additional purification by DEAE chromatography reduces the degree of background binding in the type II collagen ELISA, leading to an increase in disease specificity. The contaminants included proteoglycan and bound serum IgG from both rheumatoid arthritis (RA) patients and healthy controls in ELISA. Furthermore, positive correlations were observed in the sera (n = 24) between degree of reactivity to the contaminants and to (1) purified proteoglycan (r = 0.50, P = 0.01) and (2) pepsin (r = 0.65, P = 0.001). Thus, inadequate purification of type II collagen produces false positive reactions in the collagen ELISA and gives rise to a high background. A lack of specificity has been frequently associated with this assay.

Cloning and analysis of Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG gene.

Genes encoding various Trypanosoma (Trypanozoon) brucei variable surface glycoproteins (VSGs) show considerable conservation among different members of this species, known as isotypes. The occurrence of isotypes in other salivarian trypanosomes has not been well documented. We have cloned sequences encoding Trypanosoma (Nannomonas) congolense ILNat 2.1 VSG, and used it in DNA blot hybridization analyses of this and other T. congolense clones originating from geographically separate regions of East Africa. The data indicate that the expression of ILNat 2.1 VSG gene proceeds by duplicative transposition resulting in the presence of an extra expression-linked copy in the expressing clones examined. Furthermore the ILNat 2.1 VSG gene sequence is absent or has greatly diverged, in all other T. (N.) congolense clones that belong to different serodemes. This suggests that some T. (N.) congolense VSGs may be limited to their respective antigen repertoires. The data are discussed in the light of their implications for antigenic variation in T. (N.) congolense, and parasite epidemiology.

Isolation and characterization of RNA from the intracellular parasite Theileria parva.

Using a rapid procedure to isolate schizonts of the intracellular parasite Theileria parva from infected bovine lymphocytes, we have prepared parasite RNA that is more than 90% pure. Characterization of this schizont RNA has revealed the presence of two ribosomal RNA species of 3.3 kb and 1.8 kb and a third non-adenylated abundant RNA species of 1.9 kb. In vitro translation of the isolated schizont mRNA has identified about 200 parasite specific polypeptides, only a few of which could be detected by translation of mRNA from infected host cells. By analysing the kinetics of liquid hybridization of schizont mRNA with its homologous complementary DNA the nucleotide sequence complexity of the abundant class of the parasite mRNA has been estimated to be 1.7 x 10(3) kb. Assuming a number average size of 2 kb per mRNA molecule this would represent 4000 transcripts for all abundance classes of the schizont mRNA. Using the same technique we estimate that approximately 10% of the mRNA isolated from infected lymphocytes were transcripts from the parasite genome. We conclude that the low number of parasite specific translation products in the mRNA from infected lymphocytes and the low number of parasite proteins detected in isolated schizonts reported previously is due to the low abundance of the parasite transcripts rather than a low number of expressed parasite genes.

Shared surface epitopes among trypanosomes of the same serodeme expressing different variable surface glycoprotein genes.

African trypanosomes evade the immune response of the mammalian host by undergoing antigenic variation, caused by sequence changes in a variable surface glycoprotein (VSG). The majority of trypanosome clones analyzed thus far are not known to share surface exposed epitopes or express appreciably homologous VSGs. We show here that four clones of Trypanosoma brucei from the same serodeme express different VSGs and share exposed epitopes to varying degrees, as defined by monoclonal antibodies. Rabbit antiserum against any one of the four VSGs recognizes epitopes present on all four trypanosomes in live cell immunofluorescence assay. The expressed VSGs are partially homologous at the N-terminus with multiple point substitutions of amino acids which distinguish each of the four VSGs. The genes coding for these VSGs are members of one gene family and an expression-linked copy with a unique restriction map is present in each trypanosome. Analysis of the ontogeny of the expressed genes should reveal mechanisms of evolution in trypanosome variable antigen repertoires.

The 5' flanking sequence of a Trypanosoma brucei variable surface glycoprotein gene.

The mechanism controlling transcription at several telomeric expression sites for variable surface glycoprotein (VSG) genes in Trypanosoma brucei is unknown. Most VSG genes in expression sites have a region 5' of the gene lacking restriction enzyme sites. This 'barren region' is involved in recombination events which replace the VSG gene with a copy of a different, non-telomeric, VSG gene leading to a switch in VSG expression. Alterations in the barren region have been considered as possible modulators of expression of the adjacent VSG gene in other switching events where no gene replacement occurs. The expressed copy of the ILTat 1.3 VSG gene remains in its expression site, on a 160 kilobase (kb) chromosome, in trypanosomes not expressing the ILTat 1.3 VSG. Here we report the complete sequence of the barren region adjacent to this gene, determined both from trypanosomes expressing the gene and from those that are not. The sequence is identical whether or not the ILTat 1.3 VSG gene is expressed. This confirms that alterations in the barren region are not involved in modulation of expression of the gene, as suggested by restriction enzyme mapping. Sequence data from the 5' flanking region of a second telomeric gene copy on an 80 kb minichromosome, and from the ILTat 1.3 expression site after replacement of the ILTat 1.3 gene by another gene from a minichromosome, provide evidence that telomeric VSG genes on minichromosomes are also flanked by long repeat arrays, and that these arrays are involved in inter-telomeric gene replacements as well as replacements by non-telomeric genes.

Expression of Trypanosoma brucei procyclin as a fusion protein in Escherichia coli.

Procyclin, a glycoprotein surface antigen of procyclic forms of Trypanosoma brucei, was expressed in Escherichia coli as a cro-beta-galactosidase fusion protein. Antibodies produced in rabbits immunised with gel-purified fusion protein bound to the surface of living procyclic culture forms in indirect immunofluorescence assays and were able to immunoprecipitate procyclin from lysates of trypanosomes biosynthetically labelled with tritiated proline. In addition, the antibodies recognised synthetic peptides corresponding to three different regions of the procyclin molecule, including a glutamic acid-proline dipeptide repeat. The results indicate that T. brucei procyclin expressed as a fusion protein is immunogenic and antigenically intact. In contrast, no rabbit antibodies could be produced against a 16-amino-acid synthetic peptide consisting of the dipeptide repeat, even when the peptide was coupled to carrier proteins.

Sequence and expression of a 90-kilodalton heat-shock protein family member of Theileria parva.

A Theileria parva specific full-length cDNA clone, T7, which encodes a protein with more than 60% homology to heat shock protein 90 (hsp90) of other organisms, has been identified. T7 appears to be a single copy gene. The gene is expressed as a protein of 87 kDa in both the sporozoite and schizont stages of T. parva. The protein was not found in the piroplasm stage, although the corresponding transcript was detected, suggesting post-transcriptional regulation of the gene. In the schizont stage the T7 protein is upregulated upon heat shock and localized in the cytoplasm.

Beam flatness perturbation effects of a perforated thermoplastic immobilization device on 6 and 12 MeV electron beams.

PURPOSE: Perforated thermoplastic masks are widely used in radiotherapy of head and neck malignancies. They provide for patient immobilization and increase setup reproducibility. Some oncology treatment centers cut mask portals (windows) for the beam to pass through; for those centers that do not, the mask affects beam fluence. The extent to which beam flatness is altered by such a mask is investigated. MATERIALS AND METHODS: The effects of perforated thermoplastic on 6 MeV and 12 MeV electron beams was described in terms of optical density differences in a comparative film study. RESULTS: Variations of beam flatness were documented of up to 11.8% at 5 mm depth for 6 MeV, and 8.1% for 12 MeV electrons. The depth at which this effect may be considered insignificant (mean optical density differences < 2%) is approximately 10 mm for both beam energies. CONCLUSIONS: For clinical situations where the target volume is superficial, some consideration should be given to beam inhomogeneity caused by the mask.

Trends in antifungal research.

With the increasing use of aggressive immunosuppressive therapies in the management of a variety of patient populations, the continuing presence of the AIDS pandemic and the therapeutic advances employed in critical care settings, an increasing number of serious fungal infections are being encountered by today's practicing clinicians. Traditionally, antifungal drug therapy has been delivered by means of intravenous infusion, oral administration, or topical application. Recently, a number of alternative routes of antifungal drug delivery have been developed and investigated, and the traditional means of antifungal administration have been improved to facilitate the therapeutic use of new and reformulated antifungal agents. Organized based on the route of administration, this chapter reviews these advances in antifungal drug delivery.

Resistance to collagen-induced arthritis in DBA/1 mice by intraperitoneal administration of soluble type II collagen involves both CD4+ and CD8+ T lymphocytes.

In this paper we report that intraperitoneal administration of type II collagen in a soluble form protects DBA/1 mice against collagen-induced arthritis (CIA) on subsequent arthritogenic challenge with soluble type II collagen in adjuvant. The degree of arthritis suppression, which is expressed in terms of reduced incidence of arthritis, delayed onset and reduced anti-collagen antibody titres, depends on the dose and timing of the pre-immunization collagen injection. In order to elucidate the rôles of CD4+ and CD8+ T lymphocytes subsets in arthritis resistance we administered monoclonal antibodies (mAb) to these antigenic determinants around the time of immunization with soluble type II collagen. Anti-CD4 mAb caused abrogation of arthritis resistance while anti-CD8 mAb was less effective. However, administration of anti-CD8 mAb two weeks after pre-immunization with soluble collagen was very effective in reversing arthritis resistance. From these findings we conclude that CD4+ and CD8+ T cells are involved in resistance to arthritis though the relative importance of each subset changes during the course of the process leading to resistance to CIA.