Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine.

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Avoiding intubation in the injured subglottis: the role of heliox therapy.

Intubation in the child presenting with severe viral tracheobronchitis or prior subglottic injury can be detrimental to the child and the subglottis. Intubation may lead to further mucosal ischemia, scar, subglottic stenosis, or failed extubation requiring a tracheotomy. Heliox is a combination of helium and oxygen that produces less-dense gas exchange. Its use leads to a decrease in turbulent airflow, which may obviate the need for intubation. Here we report our experience using heliox as initial therapy in 14 consecutive children presenting with severe airway distress and the need for intubation. (Five had viral tracheobronchitis, 5 had inflammatory exacerbation of subglottic stenosis, and 4 had acute iatrogenic subglottic injury.) In 10 of the 14 children, intubation, which can lead to mucosal injury and scarring, was avoided by the use of heliox therapy. Of the 4 children in whom heliox therapy failed, 3 had a prior history of subglottic stenosis. Heliox is a relatively safe and reliable alternative to intubation of children with severe subglottic edema or injury. Heliox should be considered before intubation for selected children with subglottic inflammation and severe airway distress.

Diagnostic laryngeal electromyography: The Wake Forest experience 1995-1999.

BACKGROUND: Laryngeal electromyography (LEMG) is a valuable diagnostic/prognostic test for patients with suspected laryngeal neuromuscular disorders. OBJECTIVE: To report our experience with diagnostic LEMG at the Center for Voice Disorders of Wake Forest University and to evaluate the impact of LEMG on clinical management. METHODS: Retrospective chart review of 415 patients who underwent diagnostic LEMG over a 5-year period (1995-1999). RESULTS: Of 415 studies, 83% (346 of 415) were abnormal, indicating a neuropathic process. LEMG results altered the diagnostic evaluation (eg, the type of radiographic imaging) in 11% (46 of 415) of the patients. Unexpected LEMG findings (eg, contralateral neuropathy) were found in 26% (107 of 415) of the patients, and LEMG results differentiated vocal fold paralysis from fixation in 12% (49 of 415). Finally, LEMG results altered the clinical management (eg, changed the timing and/or type of surgical procedure) in 40% (166 of 415) of the patients. CONCLUSIONS: LEMG is a valuable diagnostic test that aids the clinician in the diagnosis and management of laryngeal neuromuscular disorders.

Elective extracorporeal membrane oxygenation: an improved perioperative technique in the treatment of tracheal obstruction.

The surgical management of children with tracheal stenosis and obstruction is complicated by the perioperative needs of pressure ventilation and indwelling endotracheal tubes. These factors predispose to surgical failure and anastomotic breakdown, restenosis. and pneumomediastinum. The use of extracorporeal membrane oxygenation (ECMO) to manage ventilation during tracheal repair allows better visualization at the surgical site and obviates the need for indwelling endotracheal tubes and high-pressure ventilation. Six children were treated with elective ECMO at a tertiary care hospital. All 6 underwent successful surgical repair, and 4 of the 6 were ultimately extubated. There were no significant complications at the surgical site. There was 1 death from postoperative complications, and 2 patients required tracheotomy. One tracheotomy was performed for upper airway obstruction secondary to retrognathia, and this patient was subsequently decannulated. Medical complications were confined to 2 patients and included sepsis, hyperbilirubinemia, seizure disorder, renal failure, intracranial hemorrhage, and hydrocephalus. Elective ECMO provides a reliable perioperative technique for airway management of children with tracheal stenosis or obstruction. This technique offers the advantage of improved visibility at the operative site and eliminates the need for high-pressure ventilation, thereby likely reducing the risk of perioperative morbidity.

Major groove recognition by three-stranded beta-sheets: affinity determinants and conserved structural features.

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition.

NMR structure and functional studies of the Mu repressor DNA-binding domain.

The repressor protein of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. It interacts with several repeated DNA sequences within the early operator, preventing transcription from two divergent promoters. It also directly represses transposition by competing with the MuA transposase for an internal activation sequence (IAS) that is coincident with the operator and required for efficient transposition. The transposase and repressor proteins compete for the operator/IAS region using homologous DNA-binding domains located at their amino termini. Here we present the solution structure of the amino-terminal DNA-binding domain from the repressor protein determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The structure of the repressor DNA-binding domain provides insights into the molecular basis of several temperature sensitive mutations and, in combination with complementary experiments using flourescence anisotropy, surface plasmon resonance, and circular dichroism, defines the structural and biochemical differences between the transposase and repressor DNA-binding modules. We find that the repressor and enhancer domains possess similar three-dimensional structures, thermostabilities, and intrinsic affinities for DNA. This latter result suggests that the higher affinity of the full-length repressor relative to that of the MuA transposase protein originates from cooperative interactions between repressor protomers and not from intrinsic differences in their DNA-binding domains. In addition, we present the results of nucleotide and amino acid mutagenesis which delimits the minimal repressor DNA-binding module and coarsely defines the nucleotide dependence of repressor binding.

NMR structure of the Tn916 integrase-DNA complex.

The integrase protein catalyzes the excision and integration of the Tn916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel beta-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-Ppol-DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent approximately 35 degrees and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.

Site-specific DNA binding using a variation of the double stranded RNA binding motif.

The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.

A canine model for determination of the therapeutic index of cytokine inhibitors.

Using tumor necrosis factor (TNF) inhibition in dog blood as a measure of efficacy, and canine emesis as a measure of toxicity, we were able to assign a therapeutic index to rolipram, a prototypic anti-inflammatory compound. Because both assays were performed in the same species, the ambiguities associated with comparing the physiologic effects of drugs on various species was avoided. Rolipram, a standard phosphodiesterase type IV inhibitor, was a prototypic test compound characterized by a number of cardiovascular and central nervous system side effects, as well as its in vitro and in vivo inhibition of TNF. Initial experiments with canine whole blood incubated with lipopolysaccharide resulted in nanogram-per-milliliter concentrations of TNF that could be significantly reduced by in vitro addition of a 0.03 microM concentration of rolipram. Because rolipram inhibited canine TNF production in vitro, a protocol was devised in which TNF inhibitory activity was measured in a series of blood samples from dogs infused with increasingly high doses of rolipram. This yielded the efficacy half of the therapeutic index, whereas the emetogenic dose represented the side effect portion of the index. Rolipram was infused stepwise into conscious dogs at gradually increasing doses. The infusion was stopped when vomiting occurred, and the cumulative dose was reported as the emetic dose. Rolipram caused emesis in dogs at a cumulative dose of 0.1 mg/kg. At each dose of rolipram, blood was collected. The whole blood was incubated in vitro with lipopolysaccharide to induce TNF production, which in turn was quantified by the L929 bio-assay. Theoretically, if the rolipram infusion raised blood values high enough, the rolipram in whole blood would inhibit TNF production and be reflected by a lack of TNF activity in the L929 assay. In this assay system, rolipram's 50% effective dose in the TNF assay was always at least 33-fold lower than its emetic dose of 0.1 mg/kg. This gave rolipram a therapeutic index of at least 33:1 (0.003 versus 0.1 mg/kg) on the basis of its activity in a canine efficacy model (TNF inhibition) and a toxicity model (emesis induction). Experimental compounds were tested for their emetic dose as well as TNF 50% effective dose, with the goal of obtaining a therapeutic index better than that of rolipram. Thus the coupling of cytokine activity with overt toxicity was used to arrive at the therapeutic index of a compound. The therapeutic index was used to rank compounds as to their efficacy/toxicity profile. This ranking was used to eliminate several anti-inflammatory compounds that had a therapeutic index less than that of rolipram.

Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation.

Inhibitors of cyclic nucleotide phosphodiesterases are known to suppress lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production in vitro in human monocytes. The most potent of these have selectivity for type IV PDEs, suggesting that this class of PDE is the major type involved in the regulation of human TNF-alpha production. Using compounds of two distinct chemical structural classes, a quinazolinedione (CP-77059) and a 4 arylpyrrolidinone (rolipram), we show here that PDE-IV-specific inhibitors are also potent in suppressing LPS-induced TNF-alpha production in vitro in sodium periodate-elicited murine macrophages (IC50s of 1 and 33, respectively). We then report the in vivo anti-inflammatory effect of PDE-IV inhibition in five murine models of inflammation: (i) elevation of serum TNF-alpha induced by a sublethal LPS injection; (ii) LPS-induced endotoxic shock; (iii) LPS/galactosamine-induced endotoxic shock; (iv) carrageenan-induced paw oedema; and (v) adjuvant arthritis. Following a sublethal (5 micrograms/mouse) injection of LPS, serum TNF-alpha levels in mice peaked sharply, reaching concentrations of 3-12 ng/ml 90 min after injection. In this sublethal LPS assay, CP-77059 was about 30 times more potent than rolipram, with a minimum effective dose of 0.1 mg/kg versus 3 mg/kg for rolipram. This rank order is in keeping with the relative in vitro IC50s for CP-77059 and rolipram, as well as their relative Ki against the human PDE-IV enzyme (46 nM and 220 nM, respectively). In LPS-induced endotoxic shock, rolipram and CP-77059 at relatively high doses of 30 and 10 mg/kg, respectively, significantly reduced serum TNF-alpha levels, and also inhibited mortality 66%. In the LPS/galactosamine shock model, in which mice are rendered exquisitely sensitive to LPS by co-injection with galactosamine, only 0.1 microgram of LPS/mouse is necessary for serum TNF-alpha elevation and death. Both rolipram and the CP-77059 caused dose-dependent reduction of serum TNF-alpha and lethality. In the carrageenan-induced paw oedema model, in which there is a pronounced local TNF-alpha response (without a serum TNF-alpha elevation), rolipram significantly inhibited paw swelling as well as localized TNF-alpha levels in the paw. In the adjuvant arthritis model, a chronic model of inflammation also possessing localized TNF-alpha elevation in the inflamed paw, rolipram and CP-77059 suppressed ankle swelling and radiological evidence of joint damage. These data are consistent with a major role for PDE-IV in regulation of TNF-alpha production and inflammatory responses in murine systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-inflammatory activity of salmeterol: down-regulation of cytokine production.

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.

Evaluation of the significance of elevated levels of systemic and localized tumor necrosis factor in different animal models of inflammation.

Elevated tumor necrosis factor (TNF) levels have been reported in various models of acute and chronic inflammation and used by many investigators to determine the stage of disease and effectiveness of treatment. Because of the documented involvement of TNF in the mechanism of septic shock, experiments were done to determine whether serum TNF levels paralleled the pathology in endotoxic shock and other models of inflammation. When mice received an intraperitoneal injection of lipopolysaccharide, serum TNF levels increased dramatically, peaking 90 minutes after injection. In a dose-response experiment with lipopolysaccharide alone, we found no correlation between serum levels of TNF and survival rate of mice. All three lipopolysaccharide concentrations resulted in comparable elevations of serum TNF, yet only in the high-dose group did the animals die. In a second model of endotoxic shock, TNF-alpha levels in serum were again compared with the survival rate of mice receiving lipopolysaccharide plus galactosamine. As in the first model, we found no relationship between the level of TNF in mouse serum and mouse survival rate. The two lowest concentrations of lipopolysaccharide/galactosamine induced identically low levels of serum TNF, yet in one group all of the animals survived and in the other all died. Discrepancies between serum TNF level and mortality rate were also seen in drug treatment experiments. GI 147404X, a standard phosphodiesterase type IV inhibitor, inhibited lipopolysaccharide/galactosamine-induced elevation of serum TNF by 90% at doses of 1 and 10 mg/kg. However, the high dose resulted in 66% protection while the low dose afforded no protection.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor.

Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

Elevated levels of TNF in the joints of adjuvant arthritic rats.

The primary purpose of this study was to determine whether local levels of tumor necrosis factor (TNF) were elevated in chronically inflamed joints in rats with adjuvant-induced arthritis (AA). We also wished to develop methodology for the quantitative measurement of joint TNF, and to examine the effects of known anti-inflammatory agents on joint TNF levels. TNF levels were measured in joints from AA rats taken during the systemic phase (day 20) of arthritic disease. Using the L929 bioassay, joint extracts from AA rats had significantly greater TNF levels (1054 +/- 147 pg/g tissue) than joint extracts from normal rats (110 +/- 42 pg/g tissue). Administration of ibuprofen failed to significantly inhibit TNF levels in the joint at a time point when paw swelling was significantly reduced. The immunomodulating agents, methotrexate, cyclosporin A (CSA) and HWA486 profoundly inhibited both joint TNF levels and paw swelling. The specificity of this assay for TNF was supported by studies with a polyclonal rabbit anti-mouse TNF antibody which neutralized 78-87% of the TNF activity in the joint extracts. Our studies demonstrate a quantitative increase in local TNF expression in adjuvant arthritis and support a role for TNF in chronic inflammation.

Evaluation of proliferating cell nuclear antigen (PCNA) as an endogenous marker of cell proliferation in rat liver: a dual-stain comparison with 5-bromo-2'-deoxyuridine.

Proliferating cell nuclear antigen (PCNA) was evaluated as a marker of cell proliferation in formalin-fixed rat liver tissue through a comparative study with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). The comparison was conducted through the introduction of a dual immunohistochemical procedure that allows the simultaneous detection of the two antigens. The results of this study suggest that although statistically similar indexes for each can be achieved, what has been reported to be the "S-phase fraction" of PCNA-labeled nuclei is significantly different from the population of cells marked by BrdU. The data also suggest that the reason for this difference is that the "S-phase fraction" of PCNA-labeled nuclei is the population of cells in late G1- and early S-phases. BrdU, by comparison, is incorporated into cells only during DNA synthesis. Therefore, although BrdU and PCNA labeling techniques may both be effective for evaluating cell proliferation rates, it must be recognized that labeling indices derived from each are not entirely synonymous. The method presented here for the simultaneous labeling of PCNA and BrdU antigens may have utility in studies of cell cycle perturbations.

Differential effects of anti-arthritic agents on subnormal plasma iron levels in adjuvant arthritic rats.

Nonsteroidal anti-inflammatory drugs (NSAID's) and other antirheumatic compounds such as disease modifying antirheumatic drugs (DMARD's), immunosuppressives and glucocorticoids were tested to determine if daily medication for two weeks could elevate subnormal levels of plasma iron in adjuvant-arthritic (AA) rats. Aspirin, indomethacin, ibuprofen and phenylbutazone were chosen as representative carboxylic acids and pyrazole NSAID's. Although NSAID's at all doses significantly reduced noninjected paw swelling, no NSAID significantly enhanced subnormal plasma iron levels in AA rats. In contrast, the standard DMARD's auranofin and gold sodium thiomalate, as well as the glucocorticoid, dexamethasone and the immunosuppressives, methotrexate and cyclosporin-A all significantly restored plasma iron levels 28 to 100 percent. Plasma iron depression, a parameter of the acute phase response probably under regulation by pro-inflammatory cytokines, is not reversed by NSAID treatment. This appears to be a useful method for distinguishing NSAID's from other anti-arthritic compounds.

Elevation of plasma fibronectin and serum amyloid P in autoimmune NZB, B/W, and MRL/1pr mice.

The acute-phase proteins, fibronectin (Fn) and serum amyloid P (SAP), are opsonins which by virtue of their adhesive properties may be involved in the glomerular nephritis associated with splenic lupus erythematosus (SLE). Because of their possible involvement in the pathophysiology of lupus, plasma Fn and SAP levels from three strains of autoimmune mice were measured over time to determine if Fn and SAP rose as the mice sickened and renal function degenerated. Baseline levels of Fn and SAP were measured when the mice were between 1.5 and 3 months of age. The characteristic rapid onset of autoimmune disease in MRL/1pr mice was accompanied by a two- to threefold increase in plasma Fn and SAP by Day 100. The B/W mice, which develop autoimmune disease more slowly, did not have a significant increase in plasma Fn and SAP until Day 240. The NZB mice, with the most delayed onset of disease, exhibited a modest but significant elevation of plasma Fn and SAP by Day 360. Histologic examination of the kidneys of B/W and NZB mice indicated that pathological abnormality of the glomeruli and tubules coincided with the elevation of plasma Fn and SAP levels. In contrast, blood samples taken over time from normal BALB/c mice did not possess abnormal levels of Fn or SAP. It appears that elevation of plasma Fn and SAP in the MRL/1 pr, B/W, and NZB mice is related to the onset and severity of autoimmune disease and the subsequent loss of renal function.

Lymphocyte activating factor (LAF) and the acute-phase response in adjuvant arthritic rats.

The purpose of the study was to determine the temporal relationship between lymphocyte activating factor (LAF) activity and the acute-phase response, as measured by plasma fibronectin (Fn), C-reactive protein (CRP), and albumin levels in adjuvant arthritic rats. LAF activity as measured in the thymocyte costimulator assay, and plasma Fn, CRP, and albumin levels were measured during the acute (Day 3), intermediate (Day 10), and systemic (Day 17) phases of arthritic disease. On Day 3, supernatants from whole spleen cells of adjuvant-injected rats did not exhibit abnormal LAF activity. By Day 10, LAF activity in splenic supernatants from arthritics was significantly (P less than or equal to 0.05) higher than normal, although the increase was no greater than 60%. On Day 17 the LAF activity from arthritic rats had increased an average 300% compared to normals. In contrast to the time course of IL-1 activity, Fn and CRP levels in the arthritic rat were significantly higher than normal at all three time points, although there was a transient fall in Fn and CRP concentrations on Day 10. Plasma albumin levels in arthritic rats were subnormal (P less than or equal to 0.01) on Days 3, 10, and 17, although the concentration of plasma albumin on Day 10 was significantly higher than that measured on Day 3. The acute, intermediate, and systemic phases of adjuvant arthritic paw inflammation paralleled the abnormal profile of Fn, CRP, and albumin concentrations over time. However, LAF activity from arthritic rat spleen cells increased gradually and more closely coincided with the systemic appearance of the disease. Since the appearance of abnormal plasma protein levels in arthritic rats preceded the appearance of increased splenic LAF activity, it appears that there is no causal relationship between enhanced splenic LAF activity and early alteration of plasma Fn, CRP, and albumin levels.