الفهرس 
TitleOnly 14 pages are availabe for public view
 |  | from | 174 |  |  |
| | | from | 174 | | |
Abstract
Rheumatoid arthritis, a chronic, systemic, autoimmune disease is one of the most common inflammatory joint diseases with a worldwide prevalence of about 1%. The disease primarily affects articular cartilage and bones and characterized by synovial hyperplasia and cartilage erosion accompanied by joint swelling and destruction.
Early treatment can delay or even prevent sever disability, although the lack of therapeutic efficiency in a significant number of patients remains problematic. To date, RA diagnosis requires a careful medical history, physical examination and selected laboratory tests to identify the biological features that are characteristic for RA. Because the disease always progresses with various manifestations due to its complex pathogenesis, it is frequently misdiagnosed and also prediction of remissions and response to treatment in established RA using the available markers is not satisfactory.
Therefore, there is an urgent need for additional biomarkers to early identify the disease onset, better assess disease severity, and evaluate the response to treatment and to monitor the efficiency of intervention in a personalized approach of the patients.
MicroRNAs are small non coding RNAs that act through base pairing to mRNA to mediate mRNA cleavage, destabilization or translational repression. MicroRNAs are involved in critical cellular processes and their deregulation has been discovered in different diseases. Increasing number of studies have reported alterations of several miRNAs expression in RA patients, availability of their detection in peripheral blood samples, raising the possibility of their use as non-invasive disease markers.
Among these miRNAs, miR-125b an evolutionary conserved miRNA, has been described to play a significant role in modulation of inflammation, B and T cells differentiation. Several researches have revealed that miR-125b is involved in RA and plays a crucial role in its pathogenesis.
Summary, C onclusion & Recommendations
124
FLCs are light chains that are not bound to intact immunoglobulin, can be detected as circulating FLCs under physiological conditions. The significance of FLCs in various disease conditions is increasingly recognized.
The interest in B cell activation markers has been rising over the past few years due to the pivotal role of B cell in RA development and progression and the proved efficacy of B cell targeted therapy in patients with RA. For RA patients, determination of disease activity and prediction of treatment response are usually made using a combination of clinical parameters, patient reported symptoms and non-specific markers of systemic inflammation. In order to make early diagnosis of RA, to monitor the disease progression and to ensure optimal treatment, a more accurate diagnostic tool is required.
The present study aimed to evaluate the expression levels of free light chains and PBMCs miR-125b and correlating them with other clinical and laboratory parameters of RA among Egyptian patients. Additionally, the diagnostic potential of FLCs & miR-125b and their ability to predict the clinical outcomes of treatment in Egyptian patients with RA were assessed.
The current study included fifty subjects, divided into three groups of which; group 1 involved twenty rheumatoid arthritis patients who did not receive treatment (naïve RA patients), group 2 involved twenty patients who have received treatment (RA patients on treatment). This group was further subdivided according to the treatment effect, where nine were controlled and eleven were uncontrolled. The third group involved ten healthy volunteers, matched in age and sex, as a control group.
Serum kappa and lambda FLC levels were measured in all RA patients and healthy controls using ELISA kit. While, quantification of PBMCs miR-125b was done by the TaqMan miRNA assay using two step RT-PCR in a StepOneTM real-time PCR system.
In the present study, we found that the expression of PBMCs miR-125b was down regulated in naïve rheumatoid arthritis patients compared with healthy controls. ROC curve showed that PBMCs miR-125b levels could distinguish RA from HC with (AUC) of 0.780 (95% (CI) 0.569-0.981) with 80% sensitivity, 90% specificity. It was suggested that this reduced expression could be related to increased inflammation in RA as miR-125b is considered as a negative regulator for several pro-inflammatory cytokines, such as TNFα, IFN-γ, chemokine CCL4 and matrix metalloproteinase (MMP)-13.
Summary, C onclusion & Recommendations
125
Significantly higher expression levels of miR-125b was observed in the controlled subgroup of RA patients on treatment than uncontrolled subgroup, the ROC curve analysis presented that miR-125b expression level in PBMCs could have a prognostic value in predicting effectiveness of treatment in RA patients on treatment with an AUC: 0.889, 95% CI: 0.684-1, sensitivity of 88.89% and specificity of 90.91%.
An inverse correlation was established between miR-125b expression levels and other disease activity parameters (DAS-28, ESR and CRP) in naïve RA and RA patients on treatment.
Interestingly, an inverse correlation was found between cellular miR-125b expression and (RF, ACPA, kappa and lambda FLCs). It could be explained by the effects of miR-125b on B cells as it was reported that miR-125b inhibits B cell differentiation into plasma cells and results in inhibition of Ig secretion through targeting BLIMP1 and IRF4, two transcription factors needed for post-GC plasma B cell differentiation.
Serum levels of kappa and lambda FLC in naïve RA and in RA patients on treatment were significantly higher in comparison to healthy controls. This increase can be attributed to polyclonal B cell activation and increased immunoglobulin production with relative increase of circulating λ and κ chains. Our study also reported a significant positive correlation between serum levels of kappa and lambda chains and other studied parameters (DAS-28 score, ESR, CRP, RF and ACPA) in both naïve and RA patients on treatment. This direct correlation may not only passively result from polyclonal B cell activation but also antigen-bound FLCs may activate B cells, through an autocrine activation loop, into plasma cells producing FLC and auto-reactive immunoglobulins.
To demonstrate κ and λ FLCs diagnostic power to discriminate RA patients from healthy subjects, ROC curve analysis was performed which revealed an area under curve (AUC) of 0.895, with 85% sensitivity with 90% specificity for kappa light chain. On the other hand lambda light chain yielded 80% sensitivity, 90% specificity with (AUC) of 0.890.
A significant decrease in serum concentrations of both kappa and lambda FLCs was observed in controlled patients compared to uncontrolled. The use of FLCs as predictors for treatment effectiveness have been analyzed by ROC analysis curve in which we found a sensitivity of 88.89% for both kappa and lambda free light chains and a specificity of 81.82% and 90.91% for kappa and lambda light chains respectively. Thus they can be utilized as novel biomarkers to predict treatment effectiveness.
Summary, C onclusion & Recommendations
126
6.2. Conclusion
from the present study, we can conclude that:
1) Cellular miR-125b expression was down-regulated in naïve RA patients and its expression was increased in controlled patients on treatment.
2) An inverse correlation was demonstrated between miR-125b expression levels and disease activity parameters.
3) Cellular miR-125b expression could serve as a feasible and promising diagnostic and prognostic biomarker.
4) Polyclonal free light chains, important inflammatory markers were elevated in serum and could be utilized as sensitive and specific non-invasive diagnostic and predictors of treatment effectiveness in Egyptian rheumatoid arthritis patients.
5) Serum kappa and lambda FLCs showed a significant correlation with disease activity parameters and may be used as indicators of disease activity in rheumatoid arthritis.Rheumatoid arthritis, a chronic, systemic, autoimmune disease is one of the most common inflammatory joint diseases with a worldwide prevalence of about 1%. The disease primarily affects articular cartilage and bones and characterized by synovial hyperplasia and cartilage erosion accompanied by joint swelling and destruction.
Early treatment can delay or even prevent sever disability, although the lack of therapeutic efficiency in a significant number of patients remains problematic. To date, RA diagnosis requires a careful medical history, physical examination and selected laboratory tests to identify the biological features that are characteristic for RA. Because the disease always progresses with various manifestations due to its complex pathogenesis, it is frequently misdiagnosed and also prediction of remissions and response to treatment in established RA using the available markers is not satisfactory.
Therefore, there is an urgent need for additional biomarkers to early identify the disease onset, better assess disease severity, and evaluate the response to treatment and to monitor the efficiency of intervention in a personalized approach of the patients.
MicroRNAs are small non coding RNAs that act through base pairing to mRNA to mediate mRNA cleavage, destabilization or translational repression. MicroRNAs are involved in critical cellular processes and their deregulation has been discovered in different diseases. Increasing number of studies have reported alterations of several miRNAs expression in RA patients, availability of their detection in peripheral blood samples, raising the possibility of their use as non-invasive disease markers.
Among these miRNAs, miR-125b an evolutionary conserved miRNA, has been described to play a significant role in modulation of inflammation, B and T cells differentiation. Several researches have revealed that miR-125b is involved in RA and plays a crucial role in its pathogenesis.
Summary, C onclusion & Recommendations
124
FLCs are light chains that are not bound to intact immunoglobulin, can be detected as circulating FLCs under physiological conditions. The significance of FLCs in various disease conditions is increasingly recognized.
The interest in B cell activation markers has been rising over the past few years due to the pivotal role of B cell in RA development and progression and the proved efficacy of B cell targeted therapy in patients with RA. For RA patients, determination of disease activity and prediction of treatment response are usually made using a combination of clinical parameters, patient reported symptoms and non-specific markers of systemic inflammation. In order to make early diagnosis of RA, to monitor the disease progression and to ensure optimal treatment, a more accurate diagnostic tool is required.
The present study aimed to evaluate the expression levels of free light chains and PBMCs miR-125b and correlating them with other clinical and laboratory parameters of RA among Egyptian patients. Additionally, the diagnostic potential of FLCs & miR-125b and their ability to predict the clinical outcomes of treatment in Egyptian patients with RA were assessed.
The current study included fifty subjects, divided into three groups of which; group 1 involved twenty rheumatoid arthritis patients who did not receive treatment (naïve RA patients), group 2 involved twenty patients who have received treatment (RA patients on treatment). This group was further subdivided according to the treatment effect, where nine were controlled and eleven were uncontrolled. The third group involved ten healthy volunteers, matched in age and sex, as a control group.
Serum kappa and lambda FLC levels were measured in all RA patients and healthy controls using ELISA kit. While, quantification of PBMCs miR-125b was done by the TaqMan miRNA assay using two step RT-PCR in a StepOneTM real-time PCR system.
In the present study, we found that the expression of PBMCs miR-125b was down regulated in naïve rheumatoid arthritis patients compared with healthy controls. ROC curve showed that PBMCs miR-125b levels could distinguish RA from HC with (AUC) of 0.780 (95% (CI) 0.569-0.981) with 80% sensitivity, 90% specificity. It was suggested that this reduced expression could be related to increased inflammation in RA as miR-125b is considered as a negative regulator for several pro-inflammatory cytokines, such as TNFα, IFN-γ, chemokine CCL4 and matrix metalloproteinase (MMP)-13.
Summary, C onclusion & Recommendations
125
Significantly higher expression levels of miR-125b was observed in the controlled subgroup of RA patients on treatment than uncontrolled subgroup, the ROC curve analysis presented that miR-125b expression level in PBMCs could have a prognostic value in predicting effectiveness of treatment in RA patients on treatment with an AUC: 0.889, 95% CI: 0.684-1, sensitivity of 88.89% and specificity of 90.91%.
An inverse correlation was established between miR-125b expression levels and other disease activity parameters (DAS-28, ESR and CRP) in naïve RA and RA patients on treatment.
Interestingly, an inverse correlation was found between cellular miR-125b expression and (RF, ACPA, kappa and lambda FLCs). It could be explained by the effects of miR-125b on B cells as it was reported that miR-125b inhibits B cell differentiation into plasma cells and results in inhibition of Ig secretion through targeting BLIMP1 and IRF4, two transcription factors needed for post-GC plasma B cell differentiation.
Serum levels of kappa and lambda FLC in naïve RA and in RA patients on treatment were significantly higher in comparison to healthy controls. This increase can be attributed to polyclonal B cell activation and increased immunoglobulin production with relative increase of circulating λ and κ chains. Our study also reported a significant positive correlation between serum levels of kappa and lambda chains and other studied parameters (DAS-28 score, ESR, CRP, RF and ACPA) in both naïve and RA patients on treatment. This direct correlation may not only passively result from polyclonal B cell activation but also antigen-bound FLCs may activate B cells, through an autocrine activation loop, into plasma cells producing FLC and auto-reactive immunoglobulins.
To demonstrate κ and λ FLCs diagnostic power to discriminate RA patients from healthy subjects, ROC curve analysis was performed which revealed an area under curve (AUC) of 0.895, with 85% sensitivity with 90% specificity for kappa light chain. On the other hand lambda light chain yielded 80% sensitivity, 90% specificity with (AUC) of 0.890.
A significant decrease in serum concentrations of both kappa and lambda FLCs was observed in controlled patients compared to uncontrolled. The use of FLCs as predictors for treatment effectiveness have been analyzed by ROC analysis curve in which we found a sensitivity of 88.89% for both kappa and lambda free light chains and a specificity of 81.82% and 90.91% for kappa and lambda light chains respectively. Thus they can be utilized as novel biomarkers to predict treatment effectiveness.
Summary, C onclusion & Recommendations
126
6.2. Conclusion
from the present study, we can conclude that:
1) Cellular miR-125b expression was down-regulated in naïve RA patients and its expression was increased in controlled patients on treatment.
2) An inverse correlation was demonstrated between miR-125b expression levels and disease activity parameters.
3) Cellular miR-125b expression could serve as a feasible and promising diagnostic and prognostic biomarker.
4) Polyclonal free light chains, important inflammatory markers were elevated in serum and could be utilized as sensitive and specific non-invasive diagnostic and predictors of treatment effectiveness in Egyptian rheumatoid arthritis patients.
5) Serum kappa and lambda FLCs showed a significant correlation with disease activity parameters and may be used as indicators of disease activity in rheumatoid arthritis.