Single pass sequencing and physical and genetic mapping of human brain cDNAs.

We have performed single pass sequencing of 1,024 human brain cDNAs, over 900 of which seem to represent new human genes. Library prescreening with total brain cDNA significantly reduced repeated sequencing of highly represented cDNAs. A subset of sequenced cDNAs were physically mapped to their chromosomal locations using gene-specific STS primers derived from 3' untranslated regions. We have also determined that human brain cDNAs represent a rich source of gene-associated polymorphic markers. Microsatellite-containing cDNAs can be physically mapped and converted to highly informative genetic markers, thus facilitating integration of the human physical, expression and genetic maps.

Leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific mitogen and permeability factor. Malignant human tumors have been shown to produce VEGF. Elevated levels of VEGF have been detected in sera of cancer patients, but its origin is unsettled. We analyzed VEGF concentrations in serum, plasma, whole blood, and peripheral blood mononuclear cells (PBMNCs) and platelets in 56 cancer patients and 52 healthy controls using ELISA. The VEGF concentrations in the lysed whole blood samples [blood VEGF (B-VEGF)] were higher in cancer patients than in healthy controls (median, 464 versus 298 pg/ml; P<0.0001). The highest B-VEGF values were found in disseminated cancer. In cancer patients, a high B-VEGF concentration was associated with a high peripheral blood leukocyte count (P = 0.0012) and platelet count (P = 0.019). In healthy individuals, a high B-VEGF was associated with a high leukocyte count (P = 0.0001) but not with the platelet count (P>0.1). The cancer patients regularly had higher B-VEGF concentrations than healthy individuals with comparable leukocyte or platelet counts. The VEGF content of isolated PBMNCs and platelets was severalfold higher in cancer patients than in healthy controls (median, 10.6 versus 0.9 pg per 10(6) PBMNCs, and median, 1.6 versus 0.5 pg per 10(6) platelets; P<0.0001 and P = 0.0008, respectively). Serum VEGF and B-VEGF correlated strongly (P<0.0001). Very little or no VEGF was found in the plasma. The results indicate that VEGF in the bloodstream is transported by blood cells, including leukocytes and platelets. The blood cells of cancer patients contain greatly elevated amounts of this major angiogenic growth factor, and this reservoir of VEGF may have a role in tumor angiogenesis and metastasis formation. VEGF in serum samples originates from blood cells, and the use of VEGF of whole blood or of isolated blood cells may improve the clinical value of VEGF measurements.

Serum vascular endothelial growth factor is often elevated in disseminated cancer.

In adults, marked angiogenesis takes place only during the female reproductive cycles, during wound healing, and accompanying some disease processes, such as tumor development. Vascular endothelial growth factor (VEGF) is a secreted, endothelial cell-specific growth factor, which is induced by tissue hypoxia and is angiogenic in vivo. We measured serum VEGF (S-VEGF) concentrations by ELISA in patients with a variety of types of cancer, as well as in healthy volunteers, and in patients with diabetes or rheumatoid arthritis. Elevated S-VEGF concentrations were found in patients with locoregional (n = 39; median, 158 pg/ml; range, 8-664 pg/ml) or disseminated (n = 58; median, 214 pg/ml; range, 17-1711 pg/ml) cancer in comparison to individuals without cancer (n = 113; median, 17 pg/ml; range, 1-177 pg/ml; P < 0.0001 for both comparisons). Values higher than 200 pg/ml were observed in 74% of patients with untreated metastatic cancer, and high serum levels were measured regardless of the histological type of cancer. S-VEGF levels were found to be higher in untreated patients with disseminated cancer than in those with local cancer (P = 0.006), and patients undergoing cancer therapy had lower values than those without cancer therapy (P = 0.03). The results indicate that both patients with locoregional cancer and patients with disseminated cancer may have elevated S-VEGF levels, regardless of the histological type of cancer, and that S-VEGF is often elevated in cancer with distant metastases.

Postmenopausal hormonal replacement decreases plasma levels of endothelin-1.

Hormone replacement therapy (HRT) protects against cardiovascular disorders, but the mechanisms of this action are poorly understood. We assessed the plasma levels of vasoconstrictive endothelin-1 (ET-1) in 26 healthy postmenopausal women before and during HRT. The women were randomized to receive either continuous transdermal (estradiol 50 ug/24 hrs) complemented with periodic 12 days' courses with medroxyprogesterone (10.0 mg/day)(n = 13) or continuous oral estradiol (2.0 mg/day) and continuous norethisterone acetate (1.0 mg/day)(n = 13). ET-1 was measured with specific radioimmunoassay after concentrating the sample with solid phase extraction. Pretreatment plasma ET-1 (1.28 +/- 0.36 pmol/ml, mean +/- SD) in the whole study group decreased (p < 0.01) to 1.05 +/- 0.26 pmol/ml at 6 months and to 1.10 +/- 0.32 pmol/ml at 12 months of treatment. A subgroup analysis between the two HRT regimens revealed no significant differences in the response of plasma ET-1 to HRT. These first data on HRT-induced reduction in plasma ET-1 may provide a new explanation for the cardiovascular protection by HRT.

17 beta-estradiol stimulates prostacyclin, but not endothelin-1, production in human vascular endothelial cells.

The exact mechanisms by which estrogens protect against occlusive vascular disorders are not known. One possibility could be an effect on vascular endothelial vasoactive compounds, such as vasodilatory prostacyclin (PGI2) and vasoconstrictory endothelin (ET-1). Here we report on the effect of 17 beta-estradiol on the synthesis of PGI2 and ET-1 in cultured human umbilical vein endothelial cells. These cells were incubated in the absence (control) and presence of 17 beta-estradiol (0.001-1 mumol/L) for 3-24 h with serum (10%) or without serum. The release of PGI2, as assessed by its metabolite 6-keto-prostaglandin F1 alpha, and that of ET-1, were assessed by RIA. 17 beta-Estradiol (0.01-0.1 mumol/L) predissolved in ethanol (final concentration, 0.01%) increased PGI2 production by 26-30% in endothelial cells incubated without serum. This increase in PGI2 production was enhanced up to 66% when 17 beta-estradiol (1 mumol/L) was encapsulated within beta-cyclodextrin. The stimulation of PGI2 production was detectable after 12 h of incubation. The 17 beta-estradiol-induced stimulation of PGI2 production was blocked in dose-dependent manner by antiestrogenic tamoxifen. 17 beta-Estradiol failed to affect the production of PGI2 if the endothelial cells were incubated with serum and had no effect on ET-1 production under any conditions. 17 beta-Estradiol-induced stimulation of vasodilatory and antiaggregatory PGI2 production without a concomitant change in vasoconstrictory ET-1 production may provide one explanation for the ability of estradiol to maintain vascular health and protect against vascular disorders.

Transhepatic neutrophil and monocyte activation during clinical liver transplantation.

BACKGROUND: During experimental liver transplantation, neutrophil sequestration results in increased oxygen free radical production and correlates inversely with graft viability. Neutrophil activation in clinical liver transplantation is poorly understood. METHODS: We assessed leukocyte sequestration and transhepatic differences of neutrophil and monocyte CD11b expression, neutrophil free radical production, and plasma concentrations of interleukin 6 and interleukin 8 in nine patients during liver transplantation. RESULTS: Significant hepatic neutrophil sequestration occurred during initial graft rewarming with portal blood, after inferior vena cava declamping, and after hepatic artery declamping (all P<0.05). A positive transhepatic difference (i.e., outcoming - ingoing) in CD11b expression of neutrophils was observed after portal vein declamping (51+/-32 relative fluorescence unit [RFU]) and in CD11b expression of monocytes during initial graft rewarming (67+/-86 RFU, both P<0.05). A transcoronary increase in both unstimulated (74+/-80 RFU) and N-formyl-methionyl-leucylphenylalanine-stimulated (112+/-168 RFU) neutrophil free radical production took place after hepatic artery declamping (both P<0.05). A negative transcoronary difference of interleukin 6 occurred during initial graft rewarming (-192+/-176 pg/ml) and a positive difference of interleukin 8 occurred after hepatic artery declamping (17+/-23 pg/ml, both P<0.05). CONCLUSIONS: Hepatic sequestration and transhepatic activation of neutrophils, and hepatic production of interleukin 8 occur during clinical liver transplantation. A splanchnic influx of interleukin 6 occurs to the graft, possibly modulating neutrophil-mediated graft reperfusion injury.

Quantitative detection of low-copy-number mRNAs differing at single nucleotide positions.

Accurate analysis of mRNA expression levels of SNPs, highly homologous genes, and splicing variants requires techniques capable of quantifying low-copy-number mRNAs differing at single nucleotide positions. We have used an RT-PCR-based technique based on co-amplification of closely related target mRNA transcripts and assessed the effect of the stochastic distribution of low-copy-number templates on sampling variation when quantifying rare mRNA transcripts. The technique was optimized for maximal sensitivity to enable the analysis of samples containing a subpopulation of target cells and small microdissected samples. We demonstrate that the input level of template molecules is a critical determinant of the achievable assay precision. A minimum of approximately 50 molecules of template is required to discriminate between 2-fold differences in the expression levels of two transcripts. At levels above 1000 molecules of input template, the stochastic effects on sampling variation become negligible.

Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.

VEGF-C is a recently characterised endothelial growth factor structurally related to vascular endothelial growth factor (VEGF). We studied the expression of VEGF-C and VEGF in the cells of peripheral blood and in the umbilical cord blood CD 34+ cells, representing haematopoietic progenitor cells. Expression of VEGF-C was detected in the CD34+ cells. In peripheral blood VEGF-C mRNA was restricted to platelets and T-cells. In contrast to the expression pattern of VEGF-C, VEGF mRNA was detected in all peripheral blood cell fractions studied, and also in CD34+ cells. VEGF-C mRNA was also detected in fresh bone marrow samples of acute leukaemia patients, but the expression did not show lineage specificity. VEGF-C and VEGF polypeptides were present in platelets and they were released from activated platelets together with the release of beta-thromboglobulin, suggesting that VEGF-C and VEGF reside in the alpha-granules of platelets. VEGF-C and VEGF, released from activated platelets, may have a role in angiogenesis during wound healing, and possibly also in other pathological conditions, such as atherosclerosis, tumour growth, and metastasis formation.

Prodynorphin and vasopressin mRNA levels are differentially affected by chronic ethanol ingestion in the mouse.

Opioid peptides derived from the precursor, prodynorphin, are co-localized with vasopressin in the hypothalamus and posterior pituitary, and vasopressin and prodynorphin synthesis are coordinately regulated during salt-loading. We had previously found that chronic ethanol ingestion resulted in decreased levels of hypothalamic and extrahypothalamic vasopressin mRNA, and the current study investigated the effect of ethanol ingestion on prodynorphin mRNA levels. A cRNA probe was constructed from a PCR product amplified from mouse genomic DNA. Cloning and sequencing of the PCR product revealed that the sequence of the mouse prodynorphin gene used to synthesize the probe is highly conserved, with high sequence similarity to corresponding regions of the gene in other mammalian species. In situ hybridization using the cRNA probe showed a widespread distribution of prodynorphin mRNA in mouse brain. In dehydrated mice, prodynorphin mRNA was significantly increased in the hypothalamus and nearly all other brain areas examined. In ethanol-fed mice, prodynorphin mRNA was also significantly increased in hypothalamus (50-60%) and in most brain areas. In the same mice, measurement of hypothalamic vasopressin mRNA confirmed a significant (approximately 60%) decrease. These results indicate that hypothalamic vasopressin and prodynorphin mRNA can be differentially regulated in certain situations.

Role of ethanol metabolism in the inhibition of testosterone biosynthesis in rats in vivo: importance of gonadotropin stimulation.

The mechanisms by which ethanol (EtOH, 1.5 g/kg) inhibits testicular testosterone synthesis were studied in nonstimulated and human chorionic gonadotropin (hCG, 50 IU/kg)-treated male rats. To dissociate the effects caused by ethanol metabolism, the alcohol dehydrogenase inhibitor 4-methylpyrazole (4MP, 10 mg/kg) was given to half of the rats 30 min before EtOH. The 4MP had little or no effect in the nonstimulated rats on the EtOH-induced decreases in the concentrations of serum testosterone and of the intratesticular steroids of the testosterone biosynthetic pathway measured, but reduced the EtOH-induced elevation in the intratesticular pregnenolone-to-progesterone ratio. In contrast, 4MP pretreatment markedly reversed the EtOH-induced decrease in serum and intratesticular testosterone and increase in intratesticular pregnenolone concentrations in the hCG-stimulated rats. Simultaneously, the EtOH-induced elevations in the intratesticular pregnenolone/progesterone and androstenedione/testosterone ratios were abolished. In the EtOH-treated rats whose EtOH metabolism was blocked by 4MP pretreatment, the intratesticular testosterone concentrations were negatively correlated with the elevated serum corticosterone levels. It is concluded that: (1) EtOH metabolism is involved in the inhibition of testicular steroidogenesis in vivo. This effect is pronounced during gonadotropin-stimulated conditions. Thus, previously reported "discrepancies" between the in vivo and in vitro results are clarified; (2) corticosterone seems also to be involved in the EtOH-induced inhibition of steroidogenesis. This effect is also pronounced during gonadotropin-stimulated conditions; and (3) without external gonadotropin stimulation other inhibitory mechanisms, such as decreased stimulation by luteinizing hormone, are prevalent.

Different effects of oral and transdermal hormonal replacement on prostacyclin and thromboxane A2.

OBJECTIVE: To elucidate the mechanism of cardiovascular protection of hormone replacement therapy (HRT) by comparing the effect of oral and transdermal HRTs on the production of antiaggregatory, vasodilatory prostacyclin, and its endogenous antagonist, thromboxane A2. METHODS: Oral estradiol (2.0 mg/d) plus norethisterone acetate (1.0 mg/d) (n = 13) or transdermal estradiol (50 micrograms/d) plus medroxyprogesterone acetate (10 mg/d) as 12-day courses at 4-week intervals (n = 13) were given to postmenopausal women. Urinary excretion of the metabolites of prostacyclin, ie, 6-ketoprostaglandinF1 alpha and 2,3-dinor-6-ketoprostaglandinF1 alpha, as well as those of thromboxane A2, ie, thromboxane B2 and 2,3-dinor-thromboxane B2, were measured by radioimmunoassays, after purification by extraction and high performance liquid chromatography, before and during the sixth and the 12th treatment cycles. RESULTS: Oral HRT stimulated excretion of thromboxane B2 from 3.4 +/- 0.7 ng/mmol creatinine to 4.5 +/- 1.5 (mean +/- standard deviation, P < .05) and that of 2,3-dinor-thromboxane B2 from 16.6 +/- 8.0 ng/mmol creatinine to 26.2 +/- 10.7 (P < .01), and thus led to the dominance of thromboxane A2. No changes in prostanoids occurred during transdermal HRT. CONCLUSIONS: The effects of various HRTs on prostanoids may significantly differ.

Hormone replacement therapy modifies the capacity of plasma and serum to regulate prostacyclin and endothelin-1 production in human vascular endothelial cells.

OBJECTIVE: To determine if hormone replacement therapy (HRT) modifies the ability of plasma or serum to regulate the synthesis of vasodilatory prostacyclin and that of vasoconstrictive endothelin-1 by cultured human umbilical vein endothelial cells. DESIGN: Plasma and serum collected before and during the sixth treatment cycle of HRT from 13 healthy postmenopausal women were added to cultured endothelial cells. SETTING: Helsinki University Central Hospital, Department of Obstetrics and Gynecology, Helsinki, Finland. PATIENTS: Thirteen postmenopausal women (> or = 1 year since their last menstruation, FSH level > 40 mIU/mL [conversion factor to SI unit, 1.00], clear vasomotor symptoms) that suffered from incapacitating menopausal symptoms necessitating the initiation of HRT were studied. INTERVENTIONS: A combined regimen consisting of 2 mg oral E2 for 12 days followed by 2.0 mg oral E2 + 1.0 mg norethisterone acetate for 10 days and 1.0 mg E2 for 6 days. MAIN OUTCOME MEASURES: The releases of prostacyclin, as assessed by its metabolite 6-keto-prostaglandin F1 alpha, and that of endothelin-1 by cultured human umbilical vein endothelial cells in the presence of 10% plasma or 10% serum collected from the study subjects. RESULTS: Hormone replacement therapy enhanced the ability of plasma to stimulate prostacyclin production by 21% +/- 6% (mean +/- SEM) during the E2 + norethisterone acetate phase and tended to do so also during the E2-only phase (11% +/- 10%) but caused no change in endothelin-1 release. In contrast, HRT decreased the ability of serum to stimulate prostacyclin production by 12% +/- 5% during the E2-only phase and increased that of endothelin-1 by 8% +/- 4% during the E2 + norethisterone acetate phase. CONCLUSION: Because plasma flushes endothelial cells in vivo, our data on the HRT-induced stimulation of the capacity of plasma to enhance the production of vasoprotective prostacyclin without a concomitant change in endothelin-1 release in cultured human umbilical vein endothelial cells may provide one new explanation for the cardiovascular protection of HRT.

Effect of physiological concentrations of estradiol on PGI2 and NO in endothelial cells.

OBJECTIVES: To elucidate the mechanisms by which estrogens protect against occlusive vascular disorders, we studied the effect of 17 beta-estradiol on the production of prostacyclin (PGI2) and nitric oxide (NO) in primary cultures of human umbilical vein endothelial cells (HUVECs). METHODS: To study the effect of 17 beta-estradiol on PGI2 production, HUVECs were incubated in the absence and presence of 17 beta-estradiol (0.01-10 nmol/l) encapsulated within beta-cyclodextrin for 12 h in serum-free medium. To study the effect of 17 beta-estradiol (100 nmol/l) on maximal calcium-dependent NO production, we used different approaches. First, HUVECs were incubated with 2 mumol/l calcium ionophore A23187 with or without 17 beta-estradiol (100 nmol/l) for 24 h in serum-free medium. Second, HUVECs were preincubated with or without 17 beta-estradiol (100 nmol/l) for 12 h in medium supplemented with 2% fetal calf serum, and thereafter incubated in serum-free medium with 2 mumol/l of A23187 and with 100 nmol/l of 17 beta-estradiol (cells which contained 17 beta-estradiol during the preincubation period as well as cells which did not) or without it (only cells which did not contain 17 beta-estradiol during the preincubation period) for 6 h or 24 h. RESULTS: 17 beta-Estradiol (0.1 nmol/l) increased the concentration of 6-keto-prostaglandin F1 alpha, a stable metabolite of PGI2 in the incubation medium, by 16%, and no further increase occurred with higher 17 beta-estradiol concentrations. The stimulation was prevented by tamoxifen. 17 beta-Estradiol did not affect NO production in any of our experiments measured as accumulation of nitrate and nitrite in the experimental medium. CONCLUSIONS: The stimulatory effect on PGI2 production of physiological concentrations of 17 beta-estradiol, shown now for the first time, may provide one explanation for the ability of 17 beta-estradiol to protect against occlusive vascular disorders.

Neutrophils of healthy subjects with a history of reactive arthritis show enhanced responsiveness, as defined by CD11b expression in adherent and non-adherent whole blood cultures.

OBJECTIVES: To study innate immune responsiveness of HLA-B27 positive subjects recovered from Yersinia-triggered reactive arthritis (B27 + ReA+). METHODS: Whole blood samples from 15 B27 + ReA+, 15 B27 + ReA- and 15 B27 - ReA- subjects were heparinized, aliquoted and (i) kept at 0 degree C to preserve constitutive cell surface marker status, or (ii) cultured with or without bacterial lipopolysaccharide (LPS) supplement, in adherent and non-adherent conditions at 37 degrees C for 4 h. Neutrophil surface expression of CD11b, CD14 and CD16 was quantified flow cytometrically, and compared between the subject groups using Jonckheere-Terpstra test. RESULTS: The B27 + ReA+ group showed significantly higher CD11b levels than the B27 - ReA- group on non-adherent neutrophils cultured with LPS as 100 pg/ml (P = 0.027), 10 ng/ml (P = 0.048) or 1 microg/ml (P = 0.024), or on adherent neutrophils without LPS supplement (P = 0.040). CD14 and CD16 expression on cultured neutrophils and constitutive expression of all three markers were comparable between the groups. CONCLUSIONS: Enhanced neutrophil reactivity observed may exacerbate innate immune inflammation in HLA-B27 positive ReA patients.

Aberrant TNF secretion by whole blood in healthy subjects with a history of reactive arthritis: time course in adherent and non-adherent cultures.

BACKGROUND: The pathogenesis of reactive arthritis (ReA) apparently involves aberrations in innate immune functions such as monocyte tumour necrosis factor (TNF) generation. OBJECTIVE: To investigate TNF production in healthy subjects with previous yersinia triggered reactive arthritis. METHODS: The study comprised HLA-B27 positive subjects with previous reactive arthritis (B27+ReA+), and B27+ReA- and B27-ReA- subjects (n = 15 each). Whole blood TNF production was induced by lipopolysaccharide (LPS), which binds to CD14/TLR4 on the monocyte surface, or by a combination of phorbol 12-myristate 13-acetate (PMA) and Ca(2+) ionophore A23187, which activates monocytes independently of cell surface receptors. To further evaluate the possible role of adhesion mediated signalling on TNF production, blood samples were incubated in adherent or non-adherent conditions. TNF levels in culture supernatants were measured using an automated immunoassay analyser. The CD14(-159)C/T genotype was determined by a cycle minisequencing method. RESULTS: B27+ReA+ supernatants had higher TNF levels than B27+ReA- supernatants in PMA/A23187 wells in two hour (p = 0.004) and four hour cultures (p = 0.001). Rapid initial TNF release took place in adherent but not in non-adherent conditions. This adhesion associated difference was greater in the B27+ReA+ group than in the B27+ReA- or B27-ReA- group in response to PMA/A23187 (p values <0.001), and greater in the B27+ReA+ group than in the B27-ReA- group in response to LPS (p = 0.021). CD14(-159)T was associated allele dose dependently with an increase in the LPS induced TNF secretion allele (p = 0.030). SUBJECTS: who have recovered from yersinia arthritis show enhanced TNF production, which may be regulated at the level of monocyte adhesion.

Genome-controlled reverse transcriptase-polymerase chain reaction for targeted gene-expression analysis.

OBJECTIVE: Although gene-expression profiling has an important part to play in the classification of tumours and premalignant conditions, reproducibility of the present polymerase chain reaction (PCR)-based quantitative techniques needs to be improved for diagnostic purposes and to enable analysis of gene expression in formalin-fixed paraffin-embedded (FFPE) tissue samples. We have developed reverse transcriptase-PCR-based technology for quantitative assessment of the relative content of multiple mRNA transcripts in small tissue or cell samples. MATERIAL AND METHODS: A multiplexed sequence modifying cDNA synthesis reaction is performed with this technique to create a 4-5 degrees increase in the melting temperature of subsequent short (56-64 bp) PCR amplicons. Each cDNA template is competitively co-amplified with genomic DNA, which serves as a universal internal standard. The relative amounts of cDNA and genomic DNA-derived amplicons are quantified in-tube by homogeneous melting curve analysis. RESULTS: The dynamic range of the assay was three orders of magnitude, while the detection limit was 100 cDNA molecules. A prototype assay, consisting of the analysis of eight genes, displayed good reproducibility (inter-assay CV 5-20 %) compared to the TaqMan assay (inter-assay CV 7-43 %). Gene-expression analysis could be performed in 20 of 20 (100 %) archival frozen samples, in 30 of 35 (86 %) archival FFPE samples and in 26 of 27 (96 %) endoscopic biopsies. CONCLUSIONS: We demonstrate that this new technique enables accurate analysis of mRNA expression in cultured cells and endoscopic tissue biopsies. Sensitive analysis FFPE tissue is also possible thanks to the short PCR amplicons.

Polymorphism at position +896 of the toll-like receptor 4 gene interferes with rapid response to treatment in rheumatoid arthritis.

The aim of this study was to determine whether the +896 A-->G substitution of the Toll-like receptor 4 (TLR4) gene, causing the Asp299-->Gly change in the extracellular domain of TLR4, influences treatment response in recent-onset rheumatoid arthritis. 169 patients with rheumatoid arthritis were genotyped from the Finnish Rheumatoid Arthritis Combination Therapy trial, in which they were treated either with only one disease-modifying antirheumatic drug (DMARD) with or without prednisolone (single group), or with three DMARDs and prednisolone (combination group). Patients homozygotic for the wild-type +896A allele were compared with those having the polymorphic G allele in terms of early clinical response (at 6 months) by the 28-joint Disease Activity Score (DAS28). 1 of 20 (5%; (95% (confidence interval (CI) 1 to 5)) patients of the single group with TLR4 +896AG or GG and 29 of 67 (43%; (95% CI 31 to 56)) patients with AA were in remission (p = 0.001). DAS28 of the single group with TLR4 +896AG or GG was higher than with AA (p = 0.019). In the combination group, remission rates and DAS28 values were comparable between the genotypes. The polymorphic TLR4 +896G allele may impair treatment response to single DMARD treatment in recent-onset rheumatoid arthritis.