It is estimated that 2.1 million people around the world have deformities due to leprosy. Reactions and nerve damage in leprosy require prompt and adequate treatment with steroids/anti-inflammatory drugs failing which permanent deformities result [18, 19]. Each of the markers mentioned earlier have been found to play a role in the pathogenesis of reactions and nerve damage in leprosy by other groups [10, 19, 20]. However each of these have not been studied individually and together in a single individual with values before, during and after a reaction.
We hypothesized that individuals respond differentially to steroids and that is reflected in increase or decrease of the levels of these serological markers and this variability is associated with reactions and nerve damage.
The follow up data of patients with leprosy reactions showed (Figure 1a to g) spikes at periodic intervals suggesting simmering inflammation in leprosy. The periodic increase and decrease in the markers could be due to the release of processed mycobacterial antigen into the immunological milieu and the concomitant rise of inflammatory markers. Similar phenomenon was observed in other progressive neurodegenerative diseases such as Multiple Sclerosis .
Of the individuals, 47% showed an increase in the level of markers when compared to their own individual existing level before the reaction. This is a unique analysis comparing the individual’s level of each molecule to their existing level prior and after reaction. The individual variability in the level of expression of markers were normalized and presented as percentage increase or decrease. This finding established that leprosy patients show a variable increase of different markers before reaction. Our analysis showed two or four serological markers when tested in combination showed about 70% sensitivity to detect reaction. The maximum fold increase was observed in Ceramide antibody followed by TNF-α, followed by S100 antibody and PGL-1 (Table 1 and Figure 2) suggesting the combination of these four serological markers could be a choice to understand reaction and their relationship to nerve damage. In addition, to the above mentioned four serological markers other inflammatory and autoimmune markers which have a fold increase such as Interferon-inducible protein-10 (IP-10)  and Myelin P0 [23–25] should be explored for understanding the nerve damage.
As shown in Table 1 nerve pain, tenderness, new NFI was considered at the time of reaction and were correlated with increase in any of the serological markers individually or in combination. When all seven markers showed an increase during reaction it was associated with new nerve function impairment. The markers were not significantly different a month after the reaction. The major limitation or constraint in leprosy studies is defining the onset of infection and the exact time point of reaction and nerve damage which creates a difficulty in the enrollment of homogenous patient group. Further studies would help in understanding the association between clinical symptoms (nerve pain, nerve tenderness and new NFI) and serological markers during progression of nerve damage.
We studied the steroid response after the reaction and our analysis showed a cumulative decrease in the levels of the markers about 65.4% (when four serological markers were tested at a time). The maximum decrease was observed in antibodies to S100, TNF-α and antibodies to Ceramide & LAM (Table 1). A study on steroids treatment of reaction and changes in the inflammatory cytokines showed that TNF-αand other cytokines continue to be produced for a considerable time during and after the reaction  suggesting that a sustained inhibition of the inflammatory process is warranted in the management of reactions. As clinical reactions are known to precede nerve damage, inhibition of S100 antibody and TNF-α in reaction could be a molecular mechanism by which reactions are controlled, thus facilitating a quick recovery of nerve function in leprosy.
Steroids have been the mainstay of treatment of reactions and prevention of nerve impairment. Even though the number of samples assayed by in vitro were small, PBMC when stimulated with PHA/Con A showed a significant increase in TNF-αproduction in patients as compared to healthy subjects (Table 3). Inhibition of TNF-α by steroid (DC) was 65.3% by in vitro as compared to 60% in vivo (Table 1) suggesting partial response in leprosy reactions. The present study has shown that steroids do not produce a consistent and/or sustained suppression of all the markers associated with reaction and nerve damage. There is a need for alternate drug combinations to manage reaction and prevent nerve damage. Azathioprine and thalidomide are now being used as substitutes for steroids [26–28]. Considering the serological & in vitro results this study recommends a combination of steroids and other drugs that can effectively prevent leprosy nerve damage.
In conclusion, this study helps in understanding the responses of a leprosy individual in reaction to steroids in vivo and in vitro. There is a heterogeneity in the immunological responses to steroids and suggest that the future therapies should be multi component and individually tailored. Inhibition of autoantibodies with steroids could be a significant mechanism in preventing nerve damage. An increase in the serological markers before a reaction had an association with clinical symptoms and signs of nerve pain, tenderness, and new NFI and thus needs to be considered in the management of nerve damage. Identification of steroid responders and non-responders by an in vitro test could benefit the physician in better management of reactions and nerve damage.
Future studies on protein microarray would provide a comprehensive evaluation of other inflammatory and regulatory markers that are involved in leprosy reaction and that are down regulated in response of prednisolone therapy. Furthermore it would be interesting to study whether similar immunological and molecular mechanisms occur in Type II reactions of leprosy and the role of steroids treatment in nerve damage.