Development of a semi-quantitative scoring protocol for gill lesion assessment in greenlip abalone Haliotis laevigata held at elevated water temperature.

Water temperatures that exceed thermal optimal ranges (~19 to 22°C for greenlip abalone Haliotis laevigata, depending on stock genetics) can be associated with abalone mortalities. We assessed histopathological changes in H. laevigata gills held in control (22°C) or elevated (25°C) water temperature conditions for 47 d by developing a new scoring protocol that incorporates histopathological descriptions and relative score summary. Lesions were allocated to 1 of 3 reaction patterns, (1) epithelial, (2) circulatory or (3) inflammatory, and scored based on their prevalence in gill leaflets. Indices for each reaction pattern were calculated and combined to provide an overall gill index. H. laevigata held in 25°C water temperature had significantly more epithelial lifting and hemolymph channel enlargement and significantly higher gill and circulatory reaction pattern indices than H. laevigata held in 22°C water temperature. One H. laevigata had a proliferation of unidentified cells in the v-shaped skeletal rod of a gill leaflet. The unidentified cells contained enlarged nuclei, a greater nucleus:cytoplasm ratio and, in some cases, mitotic figures. This cell population could represent a region of hematopoiesis in response to hemocyte loss or migration to a lesion. Without thorough diagnostic testing, the origin of these larger cells cannot be confirmed. The new scoring protocol developed will allow the standard quantification of gill lesions for H. laevigata, specifically for heat-related conditions, and could further be adapted for other Haliotis spp.

Recombinant Mycobacterium bovis BCG for immunotherapy in nonmuscle invasive bladder cancer.

In the past three decades, intravesical instillation of Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for treating bladder cancer and it still remains at the forefront of immunotherapy for cancer patients. Although BCG-based therapy is the most effective intravesical therapy for this kind of tumor and represents the only agent known to reduce progression into muscle invasive bladder cancer, BCG is ineffective in approximately 30-40 % of cases and disease recurs in up to 50 % of patients. Since that BCG is considered an effective vehicle for delivery of antigens due to its unique characteristics, the genetic manipulation of these mycobacteria has been appealing in the search for less toxic and more potent therapeutic agents for bladder cancer immunotherapy. Herein, we discuss current advances in recombinant BCG construction, research, concerns, and future directions to promote the development of this promising immunotherapeutic approach for bladder cancer.

Evaluation of a high-yield technique for pancreatic islet isolation from deceased canine donors.

Type 1 diabetes mellitus is one of the most frequently diagnosed endocrinopathies in dogs, and prevalence continues to increase. Pancreatic islet transplantation is a noninvasive and potentially curative treatment for type 1 diabetes mellitus. Institution of this treatment in dogs will require a readily available source of canine islets. We hypothesized that clinically acceptable islet yield and purity could be achieved by using deceased canine donors and standard centrifugation equipment. Pancreata were procured from dogs euthanized for reasons unrelated to this study. Initial anatomic studies were performed to evaluate efficacy of pancreatic perfusion. Infusion into the accessory pancreatic duct resulted in perfusion of approximately 75% of the pancreas. Additional cannulation of the distal right limb of the pancreas allowed complete perfusion. Collagenase digestion was performed with a Ricordi chamber and temperature-controlled perfusion circuit. Islets were separated from the exocrine tissue with the use of a discontinuous density gradient and a standard laboratory centrifuge. After isolation, islet yield was calculated and viability was assessed with dual fluorescent staining techniques. Islet isolation was completed in 6 dogs. Median (interquartile range) islet yield was 36,756 (28,527) islet equivalents per pancreas. A high degree of islet purity (percentage of endocrine tissue; 87.5% [10%]) and viability (87.4% [12.4%]) were achieved. The islet yield achieved with this technique would require approximately 1 pancreas per 5 kg body weight of the recipient dog. Purity and viability of the isolated islets were comparable with those achieved in human islet transplantation program. According to initial results, clinically relevant islet yield and quality can be obtained from deceased canine donors with the use of standard laboratory equipment.

Successful AV fistula creation does not lead to higher catheter use: the experience by the Northwest Renal Network 16 Vascular Access Quality Improvement Program. Four years follow-up.

BACKGROUND: In 2002, the Center for Medicare and Medicaid Services (CMS) required all 18 Renal Networks to participate in a Vascular Access Quality Improvement Program (QIP). The Northwest Renal Network (NWRN 16) chose to increase arteriovenous fistula (AVF) use. NWRN 16 hypothesized that strategies which targeted the improvement of AVF rate and the reduction of catheter use were the same. In December 2001, 44.2% of hemodialysis (HD) patients in the NWRN 16 received HD using an AVF which met the Dialysis Outcome Quality Initiative (K/DOQI) 40% AVF guideline for prevalent patients. However, 43% of HD facilities (2869 patients) had less than 40% of AVF and higher HD catheter rates than the average Network catheter rates (25.0 vs. 20.3%). To address the needs of underperforming facilities, NWRN 16 provided education and tools for their vascular access decision makers to promote AVF creation and catheter reduction. METHODS: In 2002, NWRN 16 sponsored four regional workshops targeted at nephrologists, vascular surgeons, HD nurses, and interventional radiologists. RESULTS: Percentage of AVFs in use in invited facilities increased from 31.3% pre-intervention to 56.2% at 4 yrs: 78% increase (99% confidence interval: 77.8% to 81.5%). Percentage of catheters increased from 25% to 25.8%: 3.2% change over 4 yrs (99% confidence interval: 2.5% to 4%). CONCLUSION: The success of Network 16's AVF interventions demonstrates the effectiveness of Network education promoting multidisciplinary teamwork, and innovative strategies to increase dramatically AVF use without substantial increase in catheter use.

Successful multidisciplinary interventions for arterio-venous fistula creation by the Pacific Northwest Renal Network 16 vascular access quality improvement program.

BACKGROUND: In December 2001, 44.2% of hemodialysis (HD) patients in the Northwest Renal Network (NWRN 16) received dialysis using an arteriovenous fistula (AVF). Substantial differences were noted in percentages of patients with AVF, ranging from 5% to 90% of the facility population, suggesting wide variation in physician practice patterns within the Network. To address the needs of facilities having < 40% AVF, NWRN 16 provided education and tools for their vascular access decision-makers to promote AVF creation. METHODS: In 2002, the Network sponsored 4 regional workshops targeted to nephrologists, vascular surgeons, dialysis nurses, and interventional radiologists. RESULTS: 46 facilities (43% of all Network facilities) had <40% AVF in use in December, 2001, dialyzing 2940 patients (Invited Units). Percent AVF in use in all the Invited Facilities increased from 31.3% pre-intervention to 39.8% at 1 year (p<0.001 vs pre) to 56.2% at four years: 79.8% increase in the prevalent AVF rate over a four-year period (95% confidence interval: 77.8% to 81.7%). CONCLUSION: Low prevalent AVF rates in many NWRN 16 facilities may have resulted from differences in physician practice patterns. The success of Network 16 AVF Intervention demonstrates the effectiveness of Network education promoting multidisciplinary teamwork, innovative strategies to increase AVF rates among dialysis patients.

Mobilization of iron from cells by hydroxyquinoline-based chelators.

With the aim of identifying an iron (Fe) chelator which is effective at mobilizing intracellular Fe, two novel ligands were synthesized and tested. Hydroxyquinoline is known to possess a high affinity for Fe and was thus chosen as the Fe binding motif for the hexadentate chelators, C1 (2,2'-[ethane-1,2-diylbis(iminomethylene)]diquinolin-8-ol) and C2 (2,2'-[cyclohexane-1,2-diylbis(iminomethylene)]diquinolin-8-ol). Both chelators are lipophilic, with Fe3+ complexes slightly more hydrophilic than the free ligands. C1 and C2 were equally toxic to K562 cells, and partial protection was afforded by supplementing the culture medium with human holotransferrin, suggesting that some of the toxicity of the ligands is due to cellular Fe depletion. Micromolar concentrations of both ligands effectively mobilized 59Fe from reticulocytes and K562 cells. In reticulocytes, 50 microM C1 caused the release of 60% of the cells' initial 59Fe uptake after a 4h incubation. Under the same conditions, C2 revealed a release of 50% of the 59Fe. Overall, both ligands merit in vivo study for oral activity. Their effectiveness at low concentrations makes them candidates for therapeutic use.

Automated impedance cardiography for detecting ischemic left ventricular dysfunction during exercise testing.

UNLABELLED: Automated impedance cardiography (ICG) is an attractive method for noninvasive hemodynamic evaluation. The objective of our study was to evaluate the feasibility and diagnostic value automated ICG in patients with suspected coronary artery disease (CAD). We measured stroke index (SI) and cardiac index (CI) in 65 patients with suspected CAD at rest and during bicycle exercise testing. All patients underwent subsequent cardiac catheterization including coronary angiography (CA). Depending on the results of CA, patients were divided into three groups, patients without significant CAD (group 0), single vessel disease (group 1) or multivessel disease (group 2-3). In a subset of 20 patients, automated ICG was compared to measurements of CI by the thermodilution (TD) method. RESULTS: There were no significant differences in SI and CI at baseline between the three groups. At 75- and 100-watt exercise, patients in group 2-3 showed significantly lower mean values of SI and CI as compared to patients of group 0 and group 1 (all p < 0.05), indicating exercise-induced ischaemic left ventricular (LV) dysfunction. Three patients had to be excluded because of inappropriate quality of the ICG signals during exercise. Comparison of automated ICG with TD measurements of CI showed good correlations between both methods at rest (r = 0.73) and during exercise (r = 0.89-0.91). CONCLUSIONS: We conclude that hemodynamic monitoring by automated ICG is both feasible and practical during exercise testing. Automated ICG can provide reliable and valuable additional diagnostic information on LV function during exercise which is helpful for selecting those patients for angiography who are likely to benefit from coronary interventions.

The role of oxidative stress in the toxicity of pyridoxal isonicotinoyl hydrazone (PIH) analogues.

Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Targeting proteins to membranes, using signal sequences for lipid modifications.

Changing an existing lipid or appending a lipid to a cytosolic protein has emerged as an important technique for targeting proteins to membranes and for constitutively activating the membrane-bound protein. The potential for more precise or regulated interactions of lipidated proteins in membrane subdomains suggests that this method for membrane targeting will be of increasing usefulness.

Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines.

The reactive cysteines in H-ras are subject to oxidative modifications that potentially alter the cellular function of this protein. In this study, purified H-ras was modified by thiol oxidants such as hydrogen peroxide (H(2)O(2)), S-nitrosoglutathione, diamide, glutathione disulphide (GSSG) and cystamine, producing as many as four charge-isomeric forms of the protein. These results suggest that all four reactive cysteines of H-ras are potential sites of regulatory modification reactions. S-nitrosylated and S-glutathiolated forms of H-ras were identified by protocols that depend on separation of alkylated proteins on electrofocusing gels. S-nitrosoglutathione could S-nitrosylate H-ras on four cysteine residues, while reduced glutathione (GSH) and H(2)O(2) mediate S-glutathiolation on at least one cysteine of H-ras. Either GSSG or diamide S-glutathiolated at least two cysteine residues of purified H-ras. Iodoacetic acid reacts with three cysteine residues. In intact NIH-3T3 cells, wild-type H-ras was S-glutathiolated by diamide. Similarly, cells expressing a C118S mutant or a C181S/C184S double mutant of H-ras were S-glutathiolated by diamide. These results suggest that H-ras can be S-glutathiolated on multiple thiols in vivo and that at least one of these thiols is normally lipid-modified. In cells treated with S-nitrosocysteine, evidence for both S-nitrosylated and S-glutathiolated H-ras was obtained and S-nitrosylation was the predominant modification. These results show that oxidative modification of H-ras can be extensive in vivo, that both S-nitrosylated and S-glutathiolated forms may be important, and that oxidation may occur on reactive cysteines that are normally targeted for lipid-modification reactions.

Analysis of function and regulation of proteins that mediate signal transduction by use of lipid-modified plasma membrane-targeting sequences.

It is now established that the function of many signaling molecules is controlled, in part, by regulation of subcellular localization. For example, the dynamic recruitment of normally cytosolic proteins to the plasma membrane, by activated Ras or activated receptor tyrosine kinases, facilitates their interaction with other membrane-associated components that participate in their full activation (e.g., Raf-1). Therefore, the creation of chimeric proteins that contain lipid-modified signaling sequences that direct membrane localization allows the generation of constitutively activated variants of such proteins. The amino-terminal myristoylation signal sequence of Src family proteins and the carboxy-terminal prenylation signal sequence of Ras proteins have been widely used to achieve this goal. Such membrane-targeted variants have proved to be valuable reagents in the study of the biochemical and biological properties of many signaling molecules.

Murine guanylate-binding protein: incomplete geranylgeranyl isoprenoid modification of an interferon-gamma-inducible guanosine triphosphate-binding protein.

Farnesylation of Ras proteins is necessary for transforming activity. Although farnesyl transferase inhibitors show promise as anticancer agents, prenylation of the most commonly mutated Ras isoform, K-Ras4B, is difficult to prevent because K-Ras4B can be alternatively modified with geranylgeranyl (C20). Little is known of the mechanisms that produce incomplete or inappropriate prenylation. Among non-Ras proteins with CaaX motifs, murine guanylate-binding protein (mGBP1) was conspicuous for its unusually low incorporation of [(3)H]mevalonate. Possible problems in cellular isoprenoid metabolism or prenyl transferase activity were investigated, but none that caused this defect was identified, implying that the poor labeling actually represented incomplete prenylation of mGBP1 itself. Mutagenesis indicated that the last 18 residues of mGBP1 severely limited C20 incorporation but, surprisingly, were compatible with farnesyl modification. Features leading to the expression of mutant GBPs with partial isoprenoid modification were identified. The results demonstrate that it is possible to alter a protein's prenylation state in a living cell so that graded effects of isoprenoid on function can be studied. The C20-selective impairment in prenylation also identifies mGBP1 as an important model for the study of substrate/geranylgeranyl transferase I interactions.

Mutation of Ha-Ras C terminus changes effector pathway utilization.

In PC12 cells, Ha-Ras modulates multiple effector proteins that induce neuronal differentiation. To regulate these pathways Ha-Ras must be located at the plasma membrane, a process normally requiring attachment of farnesyl and palmitate lipids to the C terminus. Ext61L, a constitutively activated and palmitoylated Ha-Ras that lacks a farnesyl group, induced neurites with more actin cytoskeletal changes and lamellipodia than were induced by farnesylated Ha-Ras61L. Ext61L-triggered neurite outgrowth was prevented easily by co-expressing inhibitory Rho, Cdc42, or p21-activated kinase but required increased amounts of inhibitory Rac. Compared with Ha-Ras61L, Ext61L caused 2-fold greater Rac GTP binding and phosphatidylinositol 3-kinase activity in membranes, a hyperactivation that explained the numerous lamellipodia and ineffectiveness of Rac(N17). In contrast, Ext61L activated B-Raf kinase and ERK phosphorylation more poorly than Ha-Ras61L. Thus, accentuated differentiation by Ext61L apparently results from heightened activation of one Ras effector (phosphatidylinositol 3-kinase) and suboptimal activation of another (B-Raf). This surprising unbalanced effector activation, without changes in the designated Ras effector domain, indicates the Ext61L C-terminal alternations are a new way to influence Ha-Ras-effector utilization and suggest a broader role of the lipidated C terminus in Ha-Ras biological functions.

S-Nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells.

Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.

Transient palmitoylation supports H-Ras membrane binding but only partial biological activity.

H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but <10% membrane-bound if only farnesyl is present, implying that palmitate provides major support for membrane interaction. However the direct contribution of palmitate to H-Ras membrane interaction or the extent of its cooperation with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover, a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal palmitoylation signal, derived from the GAP-43 protein. Although 40% of the GAP43:Ras(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, the chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorrect targeting or gross structural changes, because G43:Ras61L localized specifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:Ras61L both the N-terminal and the two remaining C-terminal cysteines of G43:Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys residues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L was only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especially transformation, while palmitates may provide potentially dynamic regulation of membrane binding.

Paramagnetic oligonucleotides: contrast agents for magnetic resonance imaging with proton relaxation enhancement effects.

An antisense paramagnetic oligonucleotide analogue targeted to a model macromolecular receptor (5S rRNA) was prepared. The paramagnetic agent's relaxivity (dependence of the relaxation rate on paramagnetic agent concentration) in the presence and absence of the macromolecular receptor was measured at 1.5 and 6.3 T. The relaxivity of the targeted agent increased specifically in the presence of the macromolecular receptor (16% at 6.3 T and 15% at 1.5 T). This effect was specific for a paramagnetic oligonucleotide targeted to the receptor and was larger than the relaxivity enhancement due simply to receptor-induced viscosity differences. Maximizing this relaxivity enhancement of tumor targeted paramagnetic oligonucleotides will aid in contrast agent development for magnetic resonance imaging.

A non-farnesylated Ha-Ras protein can be palmitoylated and trigger potent differentiation and transformation.

Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins.

Murine GBP-2: a new IFN-gamma-induced member of the GBP family of GTPases isolated from macrophages.

We have cloned a new member of the interferon (IFN)-induced guanylate-binding protein (GBP) family of GTPases, murine GBP-2 (mGBP-2), from bone marrow-derived macrophages. mGBP-2 is located on murine chromosome 3, where it is linked to mGBP-1. With the identification of mGBP-2 there are now two human and two murine GBPs. Like other GBPs, mGBP-2 RNA and protein are induced by IFN-gamma. In addition, mGBP-2 shares with the other GBPs important structural features that distinguish this family from other GTPases. First, mGBP-2 contains only two of the three consensus sequences for nucleotide binding found within the classic GTP binding regions of other GTPases. A second amino acid motif found in mGBP-2 is a potential C-terminal site for isoprenoid modification, called a CaaX sequence. mGBP-2 is prenylated, as detected by [3H]mevalonate incorporation, when expressed in COS cells and preferentially incorporates the C-20 isoprenoid geranylgeraniol. Surprisingly, despite having a functional CaaX sequence, mGBP-2 is primarily cytosolic. GBP proteins are very abundant in IFN-exposed cells, but little is known about their function. mGBP-2 is expressed by IFN-gamma-treated cells from C57Bl/6 mice, whereas mGBP-1 is not. Thus, the identification of mGBP-2 makes possible the study of GBP function in the absence of a second family member.

Analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis.

Two-dimensional (2-D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin alpha1, haptoglobin alpha2, haptoglobin beta and alpha1-anti-chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin alpha2, haptoglobin beta, alpha1-antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A-I, apolipoprotein A-IV, transthyretin, Gc-globulin, alpha2-HS glycoprotein, alpha2-macroglobulin and alpha1-B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of alpha1-antiprotease and SAA but raised the levels of transthyretin, Gc-globulin, alpha2-HS-glycoprotein and alpha2-macroglobulin in RA patients. C-reactive protein (CRP) could not be quantified on 2-D gels but, when measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute-phase proteins in RA patients could clearly be distinguished from that of piroxicam.