The Role of the Immune Response in the Pathogenesis of Thyroid Eye Disease: A Reassessment

Background

Although thyroid eye disease is a common complication of Graves’ disease, the pathogenesis of the orbital disease is poorly understood. Most authorities implicate the immune response as an important causal factor. We sought to clarify pathogenesis by using gene expression microarray.

Methods

An international consortium of ocular pathologists and orbital surgeons contributed formalin fixed orbital biopsies. RNA was extracted from orbital tissue from 20 healthy controls, 25 patients with thyroid eye disease (TED), 25 patients with nonspecific orbital inflammation (NSOI), 7 patients with sarcoidosis and 6 patients with granulomatosis with polyangiitis (GPA). Tissue was divided into a discovery set and a validation set. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays which include 54,000 probe sets.

Results

Principal component analysis showed that gene expression from tissue from patients with TED more closely resembled gene expression from healthy control tissue in comparison to gene expression characteristic of sarcoidosis, NSOI, or granulomatosis with polyangiitis. Unsupervised cluster dendrograms further indicated the similarity between TED and healthy controls. Heat maps based on gene expression for cytokines, chemokines, or their receptors showed that these inflammatory markers were associated with NSOI, sarcoidosis, or GPA much more frequently than with TED.

Conclusion

This is the first study to compare gene expression in TED to gene expression associated with other causes of exophthalmos. The juxtaposition shows that inflammatory markers are far less characteristic of TED relative to other orbital inflammatory diseases.     

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.     

Root cause investigation of a viral contamination incident occurred during master cell bank (MCB) testing and characterization – A case study

An adventitious agent contamination occurred during a routine 9 CFR bovine viral screening test at BioReliance for an Eli Lilly Chinese Hamster Ovary (CHO) cell-derived Master Cell Bank (MCB) intended for biological production. Scientists from the sponsor (Eli Lilly and Company) and the testing service company (BioReliance) jointly conducted a systematic investigation in an attempt to determine the root cause of the contamination. Our investigation resulted in the identification of the viral nature of the contaminant. Subsequent experiments indicated that the viral contaminant was a non-enveloped and non-hemadsorbing virus. Transmission electron microscopy (TEM) revealed that the viral contaminant was 25–30 nm in size and morphologically resembled viruses of the family Picornaviridae. The contaminant virus was readily inactivated when exposed to acidic pH, suggesting that the viral contaminant was a member of rhinoviruses. Although incapable of infecting CHO cells, the viral contaminant replicated efficiently in Vero cell with a life cycle of 16 h. Our investigation provided compelling data demonstrating that the viral contaminant did not originate from the MCB. Instead, it was introduced into the process during cell passaging and a possible entry point was proposed. We identified the viral contaminant as an equine rhinitis A virus using molecular cloning and DNA sequencing. Finally, our investigation led us to conclude that the source of the viral contaminant was the equine serum added to the cell growth medium in the 9 CFR bovine virus test.     

Synthesis, characterization, DNA binding, and cytotoxic studies of some mixed-ligand palladium(II) and platinum(II) complexes of alpha-diimine and amino acids

The syntheses of nine palladium(II) complexes of type [Pd(phen)(AA)]+ (where AA is an anion of glycine, L-alanine, L-leucine, L-phenylalanine, L-tyrosine, L-tryptophan, L-valine, L-proline, or L-serine) have been achieved. These palladium(II) complexes have been characterized by ultraviolet-visible, infrared, and 1H NMR spectroscopy. The binding studies of several complexes [M(NN)(AA)]+ (where M is Pd(II) as Pt(II), NN is 2,2'-bipyridine or 1,10-phenanthrodine, and AA is an anion of amino acid) with calf thymus DNA have been carried out using UV difference absorption and fluorescence spectroscopy. The mode of binding of the above complexes to DNA suggests the involvement of the hydrogen bonding between them. Several complexes [M(phen)(AA)]+ (where M is Pd(II) or Pt(II) and AA is an anion of amino acid) have also been screened for cytotoxicity in P388 lymphocytic cells. Of them, only two complexes, [Pd(Phen)(Gly)]+ and [Pd(phen)(Val)]+, show comparable cytotoxicity, as cisplatin does.     

Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides

Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.     

Brain metabolic markers reflect susceptibility status in cytokine gene knockout mice with murine cerebral malaria

Treatment of cerebral malaria, a complication of the world's most significant parasitic disease, remains problematic due to lack of understanding of its pathogenesis. Metabolic changes, along with cytokine expression alterations and blood cell sequestration in the brain, have previously been reported during severe disease in human infection and mouse models leading to the "cytopathic hypoxia" and "sequestration" theories of pathogenesis. Here, to determine the robustness of the metabolic changes and their relationship to disease development, we investigated changes in cerebral metabolic markers in a mouse model of cerebral malaria (CM) in wildtype (C57BL/6) and cytokine knockout (TNF(-/-), IFNgamma(-/-) and LTalpha(-/-)) mice using multinuclear magnetic resonance spectroscopy. Mice susceptible to CM (wildtype, TNF(-/-)) showed decreased cerebral glucose use, decreased Krebs cycle metabolism and decreased high-energy phosphates. Conversely, mice resistant to CM (IFNgamma(-/-), LTalpha(-/-)) showed little sign of these effects, despite identical levels of parasitemia. Previously reported changes in lactate were shown to be strain dependent. Elevated glutamine and decreased phosphorylation potential emerged as robust metabolic markers of susceptibility, further implicating the trytophan/NAD(+) pathway in disease development. Thus these metabolic changes are firmly linked both to the immune system response to malaria and to the occurrence of pathogenic changes in experimental CM.     

Mammalian cell gene expression: protein glycosylation.

Considerable advances have been made in identifying the factors determining the glycosylation pattern of glycoproteins secreted by mammalian cells. This has allowed a greater appreciation of the way in which recombinant proteins may be glycosylated after expression in a heterologous system. The studies reviewed herein extend the wider view that glycosylation of native and recombinant proteins is a complex event dependent on the protein moiety, the host cell, and also the environment in which transfected cells are cultured. The details of the way in which these factors combine to establish the glycosylation pattern of a secreted protein are now beginning to be unravelled.     

Inulinase (β-fructofuranosidase) production by Kluyeromyces marxianus in batch culture

Summary Production of inulinase in batch fermentation using various carbon sources with Kluyermoyces marxianus was examined. Inulinase synthesis in the culture proceeded parallel to cell growth. Glucose, fructose and sucrose were inferior carbon sources for inulinase broduction. Highest production (212 U/mL) was achieved on inulin based media.