NFKB1 and IKK Epsilon in Rheumatoid Arthritis
Condition: Rheumatoid Arthritis
MeSH terms: Arthritis; Arthritis, Rheumatoid
Study type: Observational

Summary: 
      Rheumatoid arthritis (RA) is a systemic and auto-immune disorder whose primary characteristic
      is the chronic inflammation of joints. The objective of this study was to evaluate whether
      there was an association between the NF-KB1/IKK epsilon genetic expression and the clinical
      activity in RA.

      60 RA patients were included in the study, 30 with clinical activity and 30 with clinical
      remission The NF-KB1/IKK epsilon genetic expression was performed by real time quantitative
      Polymerase chain reaction (qPCR) through the Pfaffl method of relative quantification with
      Taqman probes.
    

Detailed description: 
      Patients with a confirmed diagnosis of RA were allocated in two groups based on the Disease
      Activity Score 28 (DAS28): a) with clinical activity and b) with clinical remission. Key
      exclusion criteria were patients with any other inflammatory or autoimmune disease and
      patients with infections. The control group was represented by healthy patients Sample size

      The sample size calculation was carried out through the following formula:

      n= Nz2 pq /d2(N-1) +z2 pq With N=30, Z=2.46 (99% of confidence), p=0.9, q=0.1, d=0.05 (95% of
      accuracy). The sample size contained 20 patients with RA.

      Anthropometric measurements Weight (kg) and height (m) were calculated in a mechanical column
      scale (SECA). The patients were classified according to their Body mass index (BMI, weight
      (Kg)/ height (m)2) as a) normal weight (BMI< 24.9), b) overweight (24.9 kg/m2 <BMI<29.9
      kg/m2) and c) obese (BMI> 30 kg/m2).

      Lymphocyte extraction Lymphocytes from peripheral blood were extracted using the ACK Lysing
      Buffer® (Life technologies, Grand Island, NY) kit. Briefly, a venous blood sample in EDTA
      tube (BD Vacutainer®, Franklin Lakes, NJ), was centrifuged at 3500 rpm during 5-8 minutes.
      The resulting intermediate white colored phase was extracted and placed in an eppendorf tube;
      1 mL of ACK Lysing Buffer® was added and carefully re-suspended. Once again, the suspension
      was centrifuged at 3500 rpm during 5-8 minutes and the supernatant was discarded. This last
      step was repeated until the leukocyte package was completely white. Finally, it was added 100
      mcl of ACK Lysing Buffer® and frozen at -70ºC for later use.

      Genetic expression From the leukocyte package (approximately 10 to 15 mg), it was performed a
      messenger RNA (mRNA) extraction using the Magna Pure LC RNA isolation kit III (Roche) in the
      Magna Pure LC 2.0 Instrument. The A260/A280 nm absorbance ratio was >1.8 (quality) and total
      RNA concentration was calculated by determining absorbance at 260 nm established with the
      NanoPhotometer (Implen GmbH, Germany) and the extracts were adjusted to a concentration of 20
      µg of DNA for the PCR reaction.

      Subsequently, the cDNA was synthesized with the Transcriptor High Fidelity cDNA Synthesis Kit
      (Roche Applied Science). It was performed a real time polymerase chain reaction (qPCR), using
      the specific primers and probes for NF-KB1, IKK epsilon and 18s (as constitutive gene), using
      a 7500 Fast Real Time PCR System (Applied Biosystems, Applera UK, Cheshire, UK), mixing
      TaqMan® Universal PCR Master Mix and the specific probes for each gene NF-KB1
      (Hs00765730-m1), IKK epsilon (Hs01063858-m1) and 18S (Hs99999901-s1), (Applied Biosystems,
      Applera UK, Cheshire, UK).

      According to the Ct obtained from the two different groups it was calculated the relative
      units of expression through the Pfaffl relative quantification method, in which it was used
      as control a healthy patient.

      Statistical analysis The statistical analysis was performed through the Sigmaplot software
      version 12.0. Parametric and non-parametric test were used in function to the variable
      distribution: among the parametric test that were performed to compare the groups, the
      Student´s T-test was included; the non-parametric tests applied were: The Mann-Whitney U test
      and the Shapiro-Wilk test. Finally, a receiver operating characteristic (ROC) was used for
      the analysis of the genetic expression of NF-KB1/IKK epsilon with the SPSS software version
      22.0.
    

Intervention name(s): Genetic relative expression
Intervention type(s): Other
Intervention description(s): The genetic expression of NF-KB1 and IKK epsilon were quantified in both groups.
Observational model: Case-Only
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        The key inclusion criteria were patients with RA who met the criteria established by the
        American College of Rheumatology (ACR). Two groups were conformed based on the Disease
        Activity Score 28 (DAS28): a) with clinical activity (DAS28 > 3.6) and b) with clinical
        remission (DAS28 < 2.4). Key exclusion criteria were patients with any other inflammatory,
        autoimmune or neoplastic disease and patients with infections. The control group was
        represented by healthy patients
      
Keywords: Nuclear factor Kappa B; IKK epsilon