**Acknowledgements**

*An Overview and Management of Multiple Chronic Conditions*

IL-6 secretion [70].

autoimmunity [67, 69]. IL-17A has been identified in the CNS following migration of Th17 cells in response to a pro-inflammatory event [74]. Clinical studies have reported that IL-17A may be responsible for breakdown of the blood-brain barrier facilitating the entrance of other inflammatory cytokines into the CNS [71]. IL-17A has been shown in preclinical studies to accelerate glial activation leading to neuroinflammation and neurodegeneration [68]. Thus, it would be expected that IL-17A levels may be lowest in RR-multiple sclerosis individuals who are in remission or most responsive to therapy [67]. Moreover, recent preclinical studies have reported that both IL-17A and IL-17F increase pro-inflammatory cytokine

In the present study, TNFα, a proinflammatory marker has been shown to increase in RR-multiple sclerosis [75, 76] and to be consistently elevated regardless of the status of disease type (i.e., clinically isolated syndrome, primary progressive, or relapsing-remitting), with serum levels 40–50% higher in multiple sclerosis subjects relative to controls. Preclinical studies have reported an association between TNFα and impaired memory in mice with EAE [77] and implicated astrocyte signaling as the downstream target of overexpression of the cytokine. Despite studies whereby TNFα knockdown mice had more severe EAE [69] and therapeutic administration of the cytokine protected against the disease [74], this cytokine is reported to be a major contributor to cognitive deficits related to late stages of multiple sclerosis. In the present study, age, gender, number of relapses, or length of disease had no effect of TNFα levels, but in general, the expression levels of this cytokine were elevated in RR-multiple sclerosis subjects relative to cohorts of RR-multiple sclerosis patients presumably in remission or non-multiple sclerosis. LDN is an off-label therapeutic used in substantially lower dosages than prescribed for drug overdose or alcohol use (3 vs. 50 mg). Its use is increasing worldwide [78, 79] and is consistently reported to be well-tolerated over extended periods of time [46]. Small clinical trials have reported few, if any, side effects [42, 43]. In this study, OGF serum levels appear to be correlated with IL-17A. Alternatively, whereas β-endorphin has been associated with the propensity to avoid alcohol or opioids, it was not related to RR-multiple sclerosis. The serum levels of cytokines IL-17A and TNFα did not appear to be discriminating biomarkers at least with the current population of RR-multiple sclerosis subjects. Perhaps with additional study, and possibly other DMTs, these cytokines will be able to discern progression of

In summary, the preclinical and clinical data illustrate that enkephalin levels (i.e., OGF) are decreased in animals with EAE and humans with relapsing remitting multiple sclerosis. LDN as a biotherapy is associated with elevating enkephalins and from all aspects, appears to reduce symptomatology of multiple sclerosis. LDN therapy to upregulate the body's own production of enkephalins has been shown by a number of clinical trials to be a safe adjuvant, or primary, treatment for RR-multiple sclerosis. LDN continues to be associated with stabilizing multiple sclerosis and does not appear to interfere with other disease-modifying therapies. Whether enkephalin levels directly or indirectly alter the therapeutic pathways is unclear at this time. Moreover, it is difficult to determine at this point whether specific disease-modifying therapies are more advantageous for manipulating enkephalin levels. However, it is evident that restored serum enkephalin expression is associated with reduced inflammatory cytokines and better patient outcome. Moving forward, studies will be conducted to determine the mechanistic role of

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disease.

**3. Conclusions**

This research was supported in part by a generous gift from The Paul K. and Anna E. Shockley Family Foundation, and private donations to the Penn State LDN Fund.

The authors acknowledge the support of Indira Purushothaman from the McLaughlin and Zagon lab as well as Syndi Reed and Trang Doan from the Institute of Personalized Medicine at Penn State College of Medicine.
