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The Anti-inflammatory Effects of Water Extract from Cordyceps militaris in Murine Macrophage
Mycobiology
Abstract and Figures
The aim of this study was to determine the in vitro anti-inflammatory effect of hot water extract from Cordyceps militaris fruiting bodies (CMWE) on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release in RAW 264.7 cells. The treatment of macrophages with various concentrations of hot CMWE significantly reduced LPS-induced production as well as NO, TNF-α and IL-6 secretion in a concentration-dependent manner. These results suggest that CMWE have potent inhibitory effects on the production of these inflammatory mediators.
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... Moreover, recent studies revealed that the hot water extract obtained from the fruiting bodies of C. militaris exhibits inhibitory effects on nitric oxide production, TNF-α, and IL-6 secretion in LPS-stimulated murine macrophage cells [22]. The inflammatory response is regulated by nitric oxide produced from macrophages through inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) modulation. ...
Background
Pathogenic infections can significantly impact the health of livestock. Traditionally, antibiotic growth promoters (AGPs) have been used in feed to enhance growth performance and disease control. However, concerns regarding antibiotic resistance have led to the exploration of traditional herbal medicine as a natural alternative, guided by the principle of medicine-food homology. The Taguchi method was employed to optimize the culture formula for cordycepin production, an active component of Cordyceps militaris (C. militaris). The influences of C. militaris supplementing solid-state fermentation (CMSSF) in feed on the growth performance and immune responses of grower pigs were evaluated in the present study.
Results
The C. militaris ethanol extract (CME) displayed potent free radical scavenging activity against 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) after undergoing fermentation. Additionally, the antibacterial testing revealed that CME effectively inhibits the growth of common pig pathogens such as Glaesserella parasuis, Pasteurella multocida, Staphylococcus hyicus, and Streptococcus suis. In lipopolysaccharide (LPS)-treated intestinal porcine enterocyte cell line (IPEC-J2), CME significantly suppressed the production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, higher antioxidative activity was detected as indicated by elevated concentration of superoxide dismutase (SOD) in pig serum. The levels of immunoglobulin M (IgM), IgA, and IgG antibodies, as well as classical swine fever virus (CSFV) antibodies (S/P ratio) in serum were all increased. Growth performance of pigs fed with dietary CMSSF supplementation was improved in comparison with the control.
Conclusions
Results demonstrated that CMSSF has the potential to be used as a natural growth promoter to enhance immunity, antioxidation, as well as overall health and growth performance of grower pigs.
... Moreover, recent studies revealed that the hot water extract obtained from the fruiting bodies of C. militaris exhibits inhibitory effects on nitric oxide production, TNF-α, and IL-6 secretion in LPS-stimulated murine macrophage cells [12]. The in ammatory response is regulated by nitric oxide produced from macrophages through inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) modulation. ...
Background: Pathogenic infections can significantly impact the health of livestock. Traditionally, antibiotic growth promoters (AGPs) have been used in feed to enhance growth performance and disease control. However, concerns regarding antibiotic resistance have led to the exploration of traditional herbal medicine as a natural alternative, guided by the principle of medicine-food homology. The Taguchi method was employed to optimize the culture formula for cordycepin production, an active component of Cordyceps militaris (C. militaris). The influences of C. militaris supplementing solid-state fermentation (CMSSF)in feed on the growth performance and immune responses of grower pigs were evaluated in the present study.
Results: The C. militaris ethanol extract (CME) displayed potent free radical scavenging activity against 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) after undergoing fermentation. Additionally, the antibacterial testing revealed that CME effectively inhibits the growth of common pig pathogens such as Glaesserella parasuis, Pasteurella multocida, Staphylococcus hyicus, and Streptococcus suis. In lipopolysaccharide (LPS)-treated intestinal porcine enterocyte cell line (IPEC-J2), CME significantly suppressed the production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, higher antioxidative activity was detected as indicated by elevated concentration of superoxide dismutase (SOD) in pig serum. The levels of immunoglobulin M (IgM), IgA, and IgG antibodies, as well as classical swine fever virus (CSFV) antibodies (S/P ratio) in serum were all increased. Growth performance of pigs fed with dietary CMSSF supplementation was improved in comparison with the control.
Conclusions: Results demonstrated that CMSSF has the potential to be used as a natural growth promoter to enhance immunity, antioxidation, as well as overall health and growth performance of grower pigs.
... Cordyceps militaris is a medicinal fungus that has long been used traditionally in East Asia, including China, for a variety of medical purposes [42]. For example, it has been found that Cordyceps militaris extracts and its active ingredients inhibit the expression of inflammatory mediators (IL-1β, IL-6, IL-18, TNF-α, NO) which may be regulated via signaling pathways such as TLR2, NF-κB, MAPK, and others [21,43,44]. However, largescale cultivation of Cordyceps militaris is not only labor-intensive, but also leaves a large amount of discarded solid culture-medium residue of the body of Cordyceps militaris after the substrate is harvested. ...
The aim of this study was to investigate the inhibitory effects of Cordyceps militaris solid medium extract (CME) and cordycepin (COR) on LTA-induced inflammation in MH-S cells and their mechanisms of action. In this study, the establishment of an LTA-induced MH-S inflammation model was determined, the CCK-8 method was used to determine the safe concentration range for a drug for COR and CME, the optimal concentration of COR and CME to exert anti-inflammatory effects was further selected, and the expression of inflammatory factors of TNF-α, IL-1β, IL-18, and IL-6 was detected using ELISA. The relative expression of TNF-α, IL-1β, IL-18, IL-6, IL-10, TLR2 and MyD88 mRNA was detected using RT-PCR, and the IL-1β, IL-18, TLR2, MyD88, NF-κB p-p65, NLRP3, pro-caspase-1, Caspase-1 and ASC protein expression in the cells were detected using Western blot; immunofluorescence assay detected the expression of Caspase-1 in MH-S cells. The results revealed that both CME and COR inhibited the levels of IL-1β, IL-18, IL-6, and TNF-α in the supernatants of LTA-induced MH-S cells and the mRNA expression levels of IL-1β, IL-18, IL-6, TNF-α, TLR2 and MyD88, down-regulated the LTA-induced IL-1β, IL-18, TLR2 in MH-S cells, MyD88, NF-κB p-p65/p65, NLRP3, ASC, pro-caspase-1, and caspase-1 protein expression levels, and inhibited LTA-induced caspase-1 activation in MH-S cells. In conclusion, CME can play a therapeutic role in LTA-induced inflammation in MH-S cells via TLR2/NF-κB/NLRP3, and may serve as a potential drug for bacterial pneumonia caused by Gram-positive bacteria.
... Cordycepin, peptides, polysaccharides, and other active compounds produced from C. militaris will be a key factor in the development of green pharmacognosy and pharmacology [7]. These numerous bioactive compounds obtained from this fungus have been looked as responsible for their biological and therapeutic properties, with their antitumor, and immunomodulatory effects [8,9,10,11,12], anti-inflammatory activity [13], and antioxidant activity [14]. ...
... The IC 50 demonstrated in the NT fruits, both AAE and water extracts, are comparable to Eucalyptus eximia, E. acmenoides, and E. notabilis leaf ethanol extracts that demonstrated 50% NO inhibition in LPS-stimulated RAW 264.7 cells at 34.14 ± 7.1, 56.93 ± 11.8 and 53.84 ± 7.7 µg/mL, respectively [49]. The highest concentration of NT water extracts (500 µg/mL) inhibited ≥90% of NO production like F. suspense aqueous extracts (2000 µg/mL, >90%) [50], whereas the highest dose of WA water extracts (500 µg/mL) reduced NO production (61%) greater than that of C. militaris fruit water extracts (1250 µg/mL, 51%) [51]. Our results, presented in Figure 2, show that the NT AAE extract was the most potent inhibitor of NO in LPS-stimulated RAW264.7 cells (Table 2). ...
Growing location is known to affect the metabolite content and functionality of wild harvested fruits. Terminalia ferdinandiana, commonly known as Kakadu plum (KP), is among the most commercially important native Australian bush foods. Therefore, we evaluated the composition and in vitro bioactivity of aqueous acidified ethanol (AAE) and water extracts prepared from KP fruit wild harvested in the Northern Territory (NT) and Western Australia (WA). Compositional analysis included vitamin C, total ellagic acid (TEA), and total phenolic content (TPC), while in vitro bioactivity was assessed through anti-inflammatory (RAW 264.7 macrophages) activity and cell viability (Hep G2) assay. The IC50 of the extracts ranged from 33.3 to 166.3 µg/mL for NO inhibition and CC50 from 1676 to 7337 µg/mL for Hep G2 cell viability inhibition. The AAE KP fruit extracts from the NT exhibited potent anti-inflammatory activity and impacted Hep G2 cell viability more than other extracts, most likely due to TEA (3189 mg/100 g dry weight (DW)), vitamin C (180.5 mg/g DW) and TPC (196 mg GAE/g DW) being higher than in any other extract. Overall, the findings of the present study are promising for using KP fruit and derived products in functional foods, nutraceuticals, or dietary supplements.
In this study, we investigated the effects of lentinan (LNT) on hematological parameters, immune indices, and metabolite levels in dairy cows. We randomly assigned forty Holstein cows to four treatment groups. The treatments consisted of 0, 5, 10, and 15 g/d of LNT. Compared with the control group, the addition of 10 g/d of LNT decreased the content of ALT and IL-8 but simultaneously increased the content of IL-4 in the cows’ serum. Supplementation with 10 g/d of LNT decreased the levels of lymphocyte, RDW, ALT, AST, TC, IL-2, and IL-8, but, concurrently, in-creased the levels of granulocytes and IL-4 in their serum. In addition, supplementation with 15 g/d of LNT decreased the levels of RDW, TC, IL-2, and IL-8, but, at the same time, increased the levels of IL-4 and IgM in their serum. For the metabolomic analysis, cows fed with 0 and 10 g/d of LNT were selected. The results showed that 10 metabolites, including reduced nicotinamide riboside and trehalose, were upregulated in the 10 g/d group. These differential metabolites were enriched in tyrosine metabolism and trehalose degradation and altered two metabolic pathways of ubiquinone and other terpene quinone biosynthesis, as well as starch and sucrose metabolism. These findings provide evidence that LNT could be used to reduce the risk of inflammation in dairy cows.
Natural products have long garnered the interest of scientific communities as they have proven to be an effective therapeutic resource that paved the path for medicinal research and drug development. Among them, Cordyceps militaris is an entomopathogenic caterpillar fungus with a renowned history of being utilized as a medicinal remedy for centuries in Eastern civilizations. The number of pharmacological functions reported by this specific fungus resulted in continuous efforts to unravel new effective bioactive compounds and their corresponding mechanism of action. As time progresses, computational techniques become the forefront of genomic and proteomic analysis, besides acting as a platform for integrating various up-to-date multidisciplinary data sources. This review briefly introduces Cordyceps militaris alongside the latest known biologically active compounds and their respective therapeutic potential. The need to implement computational applications to cope with the continuous phytochemical evolution of Cordyceps militaris will be illustrated. Moreover, many databases, mathematical algorithms, or sourcing tools that could benefit data visualization, dissemination, and interpretation aligned to fungal-based research are enumerated, in addition to describing some of the broad discoveries relative to Cordyceps militaris in the past. In conclusion, using advanced computational technology may be the foundation to leverage natural product discovery about Cordyceps militaris and contribute to future mass production of this fungus for commercial purposes in the world pharmaceutical industry.
The content of differentially abundant metabolites in the fermentation broth of grapefruit peels fermented by Cordyceps militaris at different fermentation times was analyzed via LC‒MS/MS. Small molecule metabolites and differential metabolic pathways were analyzed via multivariate analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. A total of 423 metabolites were identified at 0, 2, 6, and 10 days after fermentation. Among them, 169 metabolites showed differential abundance, with significant differences observed between the fermentation liquids of every two experimental groups, and the metabolite composition in the fermentation liquid changed over the fermentation time. In summary, the upregulation and downregulation of metabolites in cancer metabolic pathways collectively promote the remodeling of cancer cell metabolism, facilitating increased glycolysis, alterations in TCA cycle flux, and enhanced biosynthesis of the macromolecules required for rapid proliferation and survival. This study provides new perspectives on the development of high-value-added agricultural and forestry byproducts and the development and research of functional foods.
This study presents the bioactive compounds and the antioxidant, anti-bacteria, and anti-inflammatory activities of C. militaris fruiting body (FB) and fruiting body with substrate (FBS). C. militaris FB and FBS were extracted using water, ethanol, and methanol solvents. The chemical composition analysis of C. militaris extract shows bioactive compounds including adenosine, cordycepin, carotenoids, phenolics and flavonoids correlated with antioxidant activity in 2.153–5.105 mg GAE/g extract. Moreover, the ethanolic and methanolic extracts of C. militaris , and the bioactive compounds cordycepin and carotenoids, exhibited the greatest bactericidal activity against all enteric pathogenic bacteria; Escherichia coli, E. coli O157:H7, Shigella dysenteriae, Salmonella Typhi, Vibrio cholerae and Bacillus cereus . In addition, C. militaris extracts and bioactive compounds are confirmed as new agents to prevent the adhesion and invasion of enteric pathogenic bacteria on Caco-2 colon cells. This finding demonstrates the anti-inflammatory activity found in the aqueous extract of C. militaris and bioactive compounds on the LPS-stimulated Caco-2 cell model, which had the efficacy to suppress inflammatory moderators including iNos, Cox-2, NF-κB, TNF-α, AP-1, TLR-4, IL-1ß and IL-6. C. militaris extract and its bioactive compounds cordycepin and carotenoids impede the adhesion and invasion of enteric pathogenic bacteria on colonic epithelial cells, and also promote anti-inflammation mechanisms. This study attests to C. militaris as an alternative therapeutic agent to prevent enteric pathogenic bacterial infection and inflammation due to its proven health benefits and high level of antioxidants.
Cordyceps militaris is a valuable mushroom with wide use in food and medicine. This fungal product usage in many countries, especially in Southeast Asia, has become widespread. There is a growing realization that C. militaris can be used as a succedaneum for Chinese cordyceps (Ophiocordyceps sinensis) due to their similar chemical characteristics and therapeutic properties. In nature, the complicated life cycle of C. militaris consists of teleomorph stage, anamorph stage, and the lifespan of the host insects. The fruiting bodies propagated by inoculation on cereal substrates and silkworm pupae have been successfully mass-produced. A battery of active components such as cordycepin, adenosine, N6-(2-hydroxyethyl)-adenosine, carotenoid, and polysaccharide have been extracted from fruiting body. Evidence shows that C. militaris has various bioactivities such as immunomodulatory, anti-inflammatory, antitumor, antimicrobial, insecticidal, anti-fibrotic, liver protection, kidney protection, and pneumonia protection. This fungus finds can be found in functional food, healthcare fields, as well as skin care products in East Asian countries represented by China, Japan, and Korea. Full elucidation of the production capacities of different metabolites and the quality control of the products are critically needed in the future. This review will be helpful for the future research and application of this fungus.Keywords
Cordyceps militaris
Life cycleFruiting body cultivationCordycepinPharmaceutical and therapeutic potentialIndustry and application
In order to find relationship within and between entomopathogenic species, analysis of protein band pattern in mycelia of 25 isolates was conducted by UPGMA. The results allowed differentiation of three groups on 85% similarity coefficient. Similarity coefficient within C. militaris was , C. kyushuensis was 0.958-1.000 and C. pruinosa was 0.993-1.000. C210 and C298 isolates which had somewhat immersed perithecia, comparable to other C. militaris isolates, had 91% similarity. C108, C225-1 and C228 isolates pathogenic on Lepidopterous larvae had 89% similarity. Closely related species to C. militaris were C. kyushuensis and C. pruinosa. And similarity between C. pruinosa and C. kyushuensis was 88%. Similarity between C. bifusispora formed conidia on media and Paecilomyces tenuipes was 89%. C. scarabaeicola pathogenic specifically on adult Scarabaeidae had 82% similarity with above two species. C118 identified as C. militaris showed different protein banding patterns.
Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2, which is further metabolized to prostanoids. Two isoforms of COX exist: a constitutive (COX-1) and an inducible (COX-2) enzyme. Nitric oxide is derived from L-arginine by isoforms of nitric-oxide synthase (NOS; EC 1.14.13.39): constitutive (cNOS; calcium-dependent) and inducible (iNOS; calcium-independent). Here we have investigated inducible isoforms of COX and NOS in the acute, chronic, and resolving stages of a murine air pouch model of granulomatous inflammation. COX and NOS activities were measured in skin samples in the acute phase, up to 24 h. Activities in granulomatous tissue were measured at 3, 5, 7, 14, and 21 days for the chronic and resolving stages of inflammation. COX-1 and COX-2 proteins were assessed by Western blot. COX activity in the skin increased over the first 24 h and continued to rise up to day 14. COX-2 protein rose progressively, also peaking at day 14. COX-1 protein remained unaltered throughout. The iNOS activity increased over the first 24 h in the skin, with a further major increase in the granulomatous tissue between days 3 and 7, followed by a decrease at day 14 and a further increase at day 21. The rise in COX and NOS activities in the skin during the acute phase reinforces the proinflammatory role for prostanoids and suggests one also for nitric oxide. However, in the chronic and resolving stages, a dissociation of COX and NOS activity occurred. Thus, there may be differential regulation of these enzymes, perhaps due to the changing pattern of cytokines during the inflammatory response.
Neutrophil infiltration into inflammatory sites is one of the hallmarks of acute inflammation. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to the infiltration at inflammatory sites. Interleukin-8 (IL-8), a novel leukocyte chemotactic activating cytokine (chemokine), is produced by various types of cells upon stimulation with inflammatory stimuli and exerts a variety of functions on leukocytes, particularly, neutrophils in vitro. However, no definitive evidence has been presented on its role in recruiting and activating neutrophils in the lesions of various types of inflammatory reactions. We administered a highly specific neutralizing antibody against IL-8 in several types of acute inflammatory reactions, including lipopolysaccharide (LPS)-induced dermatitis, LPS/IL-1-induced arthritis, lung reperfusion injury, and acute immune complex-type glomerulonephritis. Anti-IL-8 treatment prevented neutrophil-dependent tissue damage as well as neutrophil infiltration in these conditions. These results suggest that IL-8 plays a causative role in acute inflammation by recruiting and activating neutrophils.
Entomopathogenic fungus Cordyceps militaris is famous for its medicinal efficacies. It has been reported to have various pharmacological activities such as anti-tumour, insecticidal, antibacterial, immunomodulatory and antioxidant. In this study, we investigated the effect of the extract of C. militaris (MPUN8501), which was identified by the analysis of the nucleotide sequences of 5.8S ribosomal RNA, on the function of liver. C. militaris powder was extracted using hot water extracts method as time, volume and temperature and using method as differential polarity of organic solvent. Each fraction was tested for the improvement of hepatic enzyme alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activity. The BuOH extracts (CME) had highest activity which was used for the test of toxicity and efficacy of C. militaris. The enhancing effect of CME on the activity of ADH and ALDH was much more than medicine, drink, natural tea etc. Thus CME promoted the resolution of alcohol and acetaldehyde in rats, inducing recovery to normal condition rapidly. Furthermore, oral administration of CME effectively protected the carbon tetrachloride-induced acute hepatic injury as revealed by the hematological parameters (levels of sGOT and sGPT) and histological observation. CME was ascertained to be safe by regulatory toxicity studies of single dose toxicity and genotoxicity. These results suggest that CME would be useful for the maintaining normal hepatic activity as a functional health food.
It has recently become accepted that the activated immune system communicates to brain via release of pro-inflammatory cytokines. This review examines the possibility that pro-inflammatory cytokines (interleukins and/or tumor necrosis factor) mediate a variety of commonly studied hyperalgesic states. We will first briefly review basic immune responses and inflammation. We will then develop the concept of illness responses and provide evidence for their existence and for the dramatic changes in neural functioning that they cause. Lastly, we will examine the potential roles that both pro-inflammatory cytokines and the neural circuits that they activate may play in the hyperalgesic states produced by irritants, inflammatory agents, and nerve damage. The possibility is raised that apparently diverse hyperalgesic states may converge in the central nervous system and activate similar or identical neural circuitry.
The nucleoside analogue cordycepin (3'-deoxyadenosine, 3'-dA) is substantially more cytotoxic to terminal deoxynucleotidyl transferase positive (TdT+) leukemic cells than to TdT leukemic cells in vitro in the presence of an adenosine deaminase inhibitor, deoxycoformycin (dCF), and has been considered as a therapeutic agent for TdT+ leukemia. The intracellular metabolism of 3'-dA was examined with HPLC, and the mechanism of its anti-TdT+ leukemic activity was analyzed. In the presence of dCF (2.5 microM), TdT+ leukemic cells (N = 5) were sensitive to the cytotoxic effect of 3'-dA, whereas TdT (N = 6) cells were not. A high level of 3'-dA-5'-triphosphate (3'-dATP) formation was detected in TdT+ NALM-6 cells (67 pmol/10(6) cells) and TdT- K562 cells (49 pmol/10(6) cells) when cultured with 1 microM [3'-3H]-labeled 3'-dA. A substantial level of 3'-dATP was detected in TdT HUT-102 cells (27 pmol/10(6) cells), whereas the level of 3'-dATP in TdT+ MOLT-4 cells was low (0.3 pmol/10(6) cells). The mean IC50 values of 3'-dA against phytohemagglutinin (PHA)-activated and resting peripheral blood mononuclear cells (PBM) (N = 5) were 8 and 32 microM, respectively. There was a modest level of 3'-dATP (7 pmol/10(6) cells) in PHA-PBM, whereas a lower level of 3'-dATP was detected in resting PBM (2.5 pmol/10(6) cells). These data suggest that the presence of 3'-dATP is not sufficient for the antileukemic effect of 3'-dA, but that TdT positivity is essential, and that PBM are significantly less sensitive to the cytotoxicity of 3'-dA in vitro. Further development of 3'-dA as a potential antileukemic agent to treat patients with TdT+ leukemia is warranted.
Large amount of nitric oxide (NO) are produced at sites of inflammation through the action of inducible nitric oxide synthase (iNOS) present in both infiltrating leucocytes and activated, resident tissue cells. However, the role of NO in inflammation remains unclear. NO is a vasodilator, which inhibits the adhesion of neutrophils to the vascular endothelium; it reduces the production of IL-6 by Kupffer cells and chondrocytes, and the production of gamma-IFN and TNF-alpha by splenocytes. The literature provides contradictory information on the effect of NO on vascular leakiness, chemotaxis, prostaglandin production and tissue damage. Increasingly, data suggest that NO is immunosuppressive. Inhibitors of NOS have potent prophylactic activity in several but not all, animal models of inflammatory disease. However, in rat adjuvant arthritis, therapeutic activity is weak. Whether inhibitors of iNOS will be therapeutically useful in human inflammatory diseases cannot be predicted on the basis of present information.
Analysis of cytokine mRNA and protein in rheumatoid arthritis tissue revealed that many proinflammatory cytokines such as TNFα, IL-1, IL-6, GM-CSF, and chemokines such as IL-8 are abundant in all patients regardless of therapy. This is compensated to some degree by the increased production of anti-inflammatory cytokines such as IL-10 and TGFβ and cytokine inhibitors such as IL-1ra and soluble TNF-R. However, this upregulation in homeostatic regulatory mechanisms is not sufficient as these are unable to neutralize all the TNFα and IL-1 produced.
In rheumatoid joint cell cultures that spontaneously produce IL-1, TNFα was the major dominant regulator of IL-1. Subsequently, other proinflammatory cytokines were also inhibited if TNFα was neutralized, leading to the new concept that the proinflammatory cytokines were linked in a network with TNFα at its apex. This led to the hypothesis that TNFα was of major importance in rheumatoid arthritis and was a therapeutic target. This hypothesis has been successfully tested in animal models, of, for example, collagen-induced arthritis, and these studies have provided the rationale for clinical trials of anti-TNFα therapy in patients with long-standing rheumatoid arthritis. Several clinical trials using a chimeric anti-TNFα antibody have shown marked clinical benefit, verifying the hypothesis that TNFα is of major importance in rheumatoid arthritis. Retreatment studies have also shown benefit in repeated relapses, indicating that the disease remains TNFα dependent. Overall these studies demonstrate that analysis of cytokine expression and regulation may yield effective therapeutic targets in inflammatory disease.