Soluble interleukin-2 receptor is a thyroid hormone-dependent early-response marker in the treatment of thyrotoxicosis

Thyrotoxic patients exhibit increased levels of immune activation molecules (soluble interleukin-2 receptor [sIL-2R], intercellular adhesion molecule-1 [ICAM-1], and endothelial-leukocyte adhesion molecule-1 [ELAM-1]) in serum, although the clinical significance of these measurements remains unclear. In a randomized 4-week study, we have recently shown that in the treatment of hyperthyroidism, the combination of cholestyramine and methimazole (MMI) resulted in faster lowering of serum thyroid-hormone levels than did MMI alone. Stored serial serum samples from patients participating in this randomized treatment trial were analyzed for sIL-2R, soluble ICAM-1 (sICAM-1), and soluble ELAM-1 (sELAM-1). The levels of all three molecules were elevated in patients with hyperthyroidism. Although the levels of sICAM-1 and sELAM-1 remained elevated through the 4-week follow-up period in both groups of patients, the sIL-2R levels (normal levels, 1.0 to 4.2 ng/ml) decreased significantly in the 10 patients who received cholestyramine in addition to MMI (week 0, 14.2 +/- 1.5 ng/ml; week 2, 10.8 +/- 1.2 ng/ml; week 4, 8.9 +/- 1.5 ng/ml). In eight patients who received MMI alone, sIL-2R decreased less rapidly (week 0, 12.3 +/- 1.4 ng/ml; week 2, 12.3 +/- 1.3 ng/ml; week 4, 10.9 +/- 1.3 ng/ml). sICAM-1 and sELAM-1 were elevated at baseline but did not decrease during therapy. In the former group, free thyroxine and free triiodothyronine decreased faster. These data show that levels of sIL-2R in serum, but not those of sICAM-1 and sELAM-1, may be of clinical use in the early follow-up evaluation of medically treated patients.     

Effects of meloxicam, compared with other NSAIDs, on cartilage proteoglycan metabolism, synovial prostaglandin E2, and production of interleukins 1, 6 and 8, in human and porcine explants in organ culture

Some non-steroidal anti-inflammatory drugs (NSAIDs) can accelerate joint damage in osteoarthritis by enhancing the production of pro-inflammatory cytokines or inhibiting cartilage proteoglycan synthesis. Meloxicam, a new NSAID, was compared with standard NSAIDs for its effect on proteoglycan synthesis and degradation in human and porcine cartilage explants, as well as the production of prostaglandin E2 (PGE2) and interleukins 1 and 6 by human synovial tissue explants in-vitro. Meloxicam at submicromolar concentrations inhibited synovial PGE2 production but, up to therapeutic drug concentrations (< or = 4 microM), did not affect synovial production of the pro-inflammatory cytokine IL-1. In contrast, hydrocortisone, 10 microM, a positive control, inhibited release of this cytokine, and indomethacin, 100 microM, increased its production. The lack of effects of meloxicam were evident irrespective of intrinsic IL-1 bioactivity of the synovia, production of IL-1 inhibitors or time of incubation. Production of the part anti-inflammatory cytokine IL-6, was significantly increased by therapeutic concentrations of meloxicam, as well as by indomethacin. Another major pro-inflammatory cytokine, IL-8, was unaffected by therapeutic concentrations of meloxicam. Meloxicam, 0.1-4.0 microM, did not affect cartilage proteoglycan production whereas indomethacin, 100 microM, significantly reduced synthesis of these macromolecules. Thus meloxicam, at concentrations within the therapeutic range and at which pronounced inhibition of prostaglandin production is evident, affects neither cartilage proteoglycan production nor the production of those cytokines likely to be important in cartilage destruction.     

A 21-kDa C-terminal fragment of protein-disulfide isomerase has isomerase, chaperone, and anti-chaperone activities

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308-491), termed weePDI, comprises the C-terminal third of the molecule. The kcat for ribonuclease oxidative folding by weePDI is 0.26 +/- 0.02 min-1, 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains.     

Defective nonoxidative leucine degradation and endogenous leucine flux in cirrhosis during an amino acid infusion

The metabolic fate of leucine's first and second carbon may be different depending on the tissue in which leucine is metabolized, as well as the prevailing hormonal milieu of that tissue. However, previous studies of leucine kinetics in humans have used only leucine labeled (as tracer) at the first carbon position. Because cirrhosis is associated with factors (such as insulin resistance and altered fuel substrate utilization) that may influence how leucine is degraded, the kinetics of leucine's first and second carbon using a simultaneous infusion of [1-14C] leucine and [2-13C] leucine were studied in the postabsorptive state and during an amino acid infusion in 6 stable cirrhotic patients and 6 matched controls. The data were normalized for different body compartments that were quantified from the dilution of H2 [180] and bromide. The body cell mass, but not body weight or fat-free body mass, was decreased in cirrhosis (P < .001). In response to the amino acid infusion, total leucine appearance from proteolysis and leucine's incorporation into protein increased significantly in both groups, but were higher in cirrhotic patients. Endogenous protein breakdown decreased in normals but remained unchanged in cirrhosis. These alterations in leucine metabolism became more prominent when data were expressed based on the body cell mass rather than on body weight. The oxidation of leucine's first carbon (C1) was decreased in cirrhosis, but the oxidation of leucine's second carbon (C2) did not differ between groups during both the postabsorptive period and the amino acid infusion, while nonoxidative leucine degradation [the difference between the oxidation of leucine's (C1) and (C2)] was also decreased in cirrhosis. In addition, there was a positive correlation between nonoxidative leucine degradation (which represents leucine incorporation into fat), and the respiratory quotient obtained from indirect calorimetry (r = .87; P < .001). These data suggest that the extent of leucine carbon oxidation is dependent on whether fat or carbohydrate is the prevailing fuel substrate. In addition, cirrhotic patients have decreased nonoxidative leucine degradation and are unable to suppress endogenous protein breakdown normally in response to amino acid administration. These abnormalities may contribute to the diminished fat stores and body cell mass commonly observed in cirrhosis.     

Recurrent inguinal hernia: preferred operative approach

Inguinal herniorrhaphy remains one of the most common general surgical operations, with approximately 10 to 20 per cent performed for recurrence. Subsequent repairs provide considerable technical challenge, as well as substantially greater risk of developing further recurrence. Mesh repair is advocated by several specialized hernia centers, demonstrating re-recurrence rates less than 2 per cent. Detractors of this repair include cost, technical difficulty, and risk for infection. The purpose of this study was to compare results of mesh and nonmesh repairs for recurrent inguinal hernia, either using an anterior or posterior approach, at a large teaching institution. From January 1, 1985, to December 31, 1994, 146 patients underwent repair for recurrent inguinal hernia at the Veterans Administration Hospital at Memphis, Tennessee. Patients were stratified by type of repair: Lichtenstein (Mesh), open anterior (OA), Bassini, Marcy, McVay, Shouldice, and preperitoneal with or without mesh. Patient ages and weights were similar between groups. Mean operative time for Mesh repair (104 +/- 4 minutes) was longer than that for OA repairs (80 +/- 5 minutes, P < 0.05) or preperitoneal without mesh repairs (92 +/- 5 minutes, P < 0.05). Mesh-based posterior repairs had the longest operative times (116 +/- 5 minutes). Hospital stay averaged 2.8 +/- 0.3 days, similar among all groups. One wound infection (1.0%) occurred in patients undergoing Mesh repair, which required operative drainage. No patient required removal of mesh. Two patients in the Mesh group (5.9%) developed recurrence compared with four recurrences (18.0%) in patients undergoing OA repairs. Only one patient with a mesh-based posterior repair recurred (1.9%) compared to eight without mesh (21.6%, P < 0.01). Follow-up ranged from 2 to 12 years. Repair of recurrent inguinal hernia using either an anterior or posterior mesh repair technique, performed at a teaching facility, provides superior recurrence rates without increasing risk for infection or length of stay. Preperitoneal mesh based repair is the preferred technique.     

Evaluation of overall reproductive performance of dairy herds

Production of the bacteriocins enterocin A and enterocin B in Enterococcus faecium CTC492 was dependent on the presence of an extracellular peptide produced by the strain itself. This induction factor (EntF) was purified, and amino acid sequencing combined with DNA sequencing of the corresponding gene identified it as a peptide of 25 amino acids. The gene encodes a prepeptide of 41 amino acids, including a 16-amino-acid leader peptide of the double-glycine type. Environmental factors influenced the level of bacteriocin production in E. faecium CTC492. The optimal pH for bacteriocin production was 6.2. At pH 5.5, growth was slow, and very little bacteriocin was formed. The presence of NaCl or ethanol (EtOH) was also inhibitory to bacteriocin production, and at high concentrations of these solutes, no bacteriocin production was observed. The induction factor induced its own synthesis, and by dilution of the culture 106 times or more, nonproducing cultures were obtained. Bacteriocin production was induced in these cultures by addition of EntF. The response was linear, and low bacteriocin production could be induced by about 10(-17) M EntF. This response was attenuated by low pH or the presence of high concentrations of NaCl or EtOH, and 300 times more EntF was needed to induce detectable bacteriocin production in the presence of 6.5% NaCl. High levels of bacteriocin production in cultures grown at low pH or in the presence of high concentrations of NaCl or EtOH were obtained by addition of sufficient amounts of EntF.     

Protein farnesyltransferase from Trypanosoma brucei. A heterodimer of 61- and 65-kda subunits as a new target for antiparasite therapeutics

We have previously shown that protein prenylation occurs in the Trypanosomatids Trypanosoma brucei (T. brucei), Trypanosoma cruzi, and Leishmania mexicana and that protein farnesyltransferase (PFT) activity can be detected in cytosolic extracts of insect (procyclic) form T. brucei. A PFT that transfers the farnesyl group from farnesyl pyrophosphate to a cysteine that is 4 residues upstream of the C terminus of the Ras GTP-binding protein RAS1-CVIM has now been purified 60,000-fold to near homogeneity from procyclic T. brucei. By screening a mixture of hexapeptides SSCALX (X is 20 different amino acids), it was found that SSCALM binds to T. brucei PFT with sub-micromolar affinity, and affinity chromatography using this peptide was a key step in the purification of this enzyme. On SDS-polyacrylamide gel electrophoresis, the enzyme migrates as a pair of bands with apparent molecular masses of 61 and 65 kDa, and thus its subunits are approximately 30% larger than those of the mammalian homolog. The 61-kDa band was identified as the putative beta-subunit by photoaffinity labeling with a 32P-labeled analog of farnesyl pyrophosphate. Mimetics of the C-terminal tetrapeptide of prenyl acceptors have been previously shown to inhibit mammalian PFT, and these compounds also inhibit T. brucei PFT with affinities in the nanomolar to micromolar range, although the structure-activity relationship is very different for parasite versus mammalian enzyme. Unlike mammalian cells, the growth of bloodstream T. brucei is completely inhibited by low micromolar concentrations of two of the PFT inhibitors, and these compounds also block protein farnesylation in cultured parasites. These compounds also potently block the growth of the intracellular (amastigote) form of T. cruzi grown in fibroblast host cells. The results suggest that protein farnesylation is a target for the development of anti-trypanosomatid chemotherapeutics.     

Spontaneous intracranial hypotension: spinal MR findings

Several melanosome glycoproteins have been shown to be antigenic in humans. Correlation of antigen-specific immune responses in patients with the autoimmune disease vitiligo, therapy-induced hypopigmentation, and cutaneous melanoma has not been well studied. We examined antibody responses to a melanocyte autoantigen, tyrosinase-related protein-2 (TRP-2), as it is highly expressed in cutaneous melanoma and melanocytes. TRP-2 recombinant protein was synthesized for western blot and affinity anti-TRP-2 enzyme-linked immunosorbent assay. We demonstrated that patients with malignant melanoma, vitiligo, and active-specific immunotherapy-induced depigmentation had significant anti-TRP-2 IgG titers. The highest level of anti-TRP-2 IgG response was found in vitiligo patients. Induction and enhancement of anti-TRP-2 IgG responses were observed in melanoma patients treated with a polyvalent melanoma cell vaccine containing TRP-2. Active-specific immunotherapy could induce and/or augment the TRP-2 IgG antibody titers. Melanoma patients who developed hypopigmentation and had improved survival after polyvalent melanoma cell vaccine had significantly augmented anti-TRP-2 antibody responses compared with patients with poor prognosis. This study demonstrates that TRP-2 autoantigen is immunogenic in humans. TRP-2 antibody responses provide a linkage between autoimmune responses by vitiligo patients and melanoma patients responding to immunotherapy who have induced hypopigmentation.     

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 A resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.