As it is summarized in Figure 1, after the approval of the ethical committee (RIF.CE: 4520) a 39-year-old with disease onset in 2005 with visual acuity disturbance and diagnosis of RRMS on July 2008, regularly followed in MS Center S. Andrea Hospital, University of Rome Sapienza. She started Natalizumab treatment, stopped after 30 infusions due to de-risking strategy in JCV positivity. After pregnancy on 2011, neurologist opted for treatment with Dimethyl fumarate, stopped after 8 months because persistence of disease activity, so patient was shifted to Fingolimod with partial response and worsening in EDSS score. Considering the young age, Alemtuzumab was started in 2016, with second cycle in February 2017. Unfortunately, patient showed an additional clinical progression, with a stable worsening of 1.5 point in the EDSS at 12 months despite the treatment, configuring a secondary-progressive form of disease. She not experienced relapses at least 1 month before the start of the protocol, the EDSS evaluation was 4.5 at baseline evaluation and she received the authorization to practice Adapted Physical Activity from Sport Medicine Physician at the City University; she was not engaged in physical activity exercise during the 6 months before the beginning of the protocol. All baseline characteristics are reported in Table 1.
The primary outcome of the study was to evaluate the effect of a combined training on expression levels of total adiponectin and its oligomerization state. Before (T0), after 4 months of concurrent training (see following) (T1) and 6 months after the end of the protocol (T2) the patient underwent to a functional and psychological (QoL) evaluation as well as a blood sample collection. The second aim was to identify the adiponectin expression and its oligomerization state in a healthy female group and in un untrained MS patient group. Baseline demographic and clinical characteristics of the subject are presented in Table 1. Ten sex- and age- matched healthy controls (all females, mean age 41 +- 2) were enrolled in the study. Ten sex- and age- matched secondary progressive MS patients (all females, mean age 40 +- 3) were recruited. The study was approved by local Ethical Committee, all patients gave their informed consent to participate in the study.
The assessment of body composition (fat mass percentage (FAT %), and free fat mass (FFM %) was obtained using the digital bioelectrical impedance device (Handy 3000; DS Medica, Milan, Italy), with a frequency of 50 and 100 kHz. Body mass index (BMI) was also determined. In order to make the measurement reproducible, the patient has to be fasting for 4 h, no physical activity for 12 h, no alcohol, and diuretics (unless prescribed) for 48 h, well hydrated (water only).
The 6-min walking test (6MWT) was used to assess endurance and functional capacity; the Handgrip test was performed to measure the muscular strength through the handgrip dynamometer (Jamar Plus ; Patterson Medical Ltd.) for each arm; and the Timed Up and Go Test (TUG) to evaluate the muscular function and mobility. The National Institute of Clinical Evidence (NICE) guidelines also advocate the use the TUG test for assessment of gait and balance in the prevention of falls.
The Multiple Sclerosis Quality of Life-54 (MSQOL-54), which investigate physical functions, limitations related to the physical and emotional sphere, perception about health, social, cognitive and sexual functions, and general QoL. The MSQOL-54 summary scores are the physical health composite summary and the mental health composite summary. The Fatigue Severity Scale (FSS) it is designed to differentiate fatigue from clinical depression in these patients and to explore severity of fatigue symptoms. The PHQ-9 Questionnaire used as a screening tool for major depressive disorder.
A total of 24 h before training and follow final training session, a venous blood sample (10 ml) was collected after a 10-h overnight fast between 7:30 a.m. and 9:00 a.m. Total serum adiponectin of healthy controls, MS patients, and the MS case undergone a physical activity training was measured by ELISA-test using house-produced polyclonal antibodies as previously described. Each serum sample was tested three times in triplicate.
Serum proteins were isolated from healthy controls, MS patients, and the MS case undergone a physical activity training as previously described by. For the immunoblot analysis, an equal amount of proteins (10 mug) was resolved in SDS-polyacrylamide (BIO-RAD) gels (10%) and transferred onto nitrocellulose membranes (Amersham). Thereafter, membranes were incubated with adiponectin antibodies (Novus Biologicals, Littleton, CO, United States) appropriately diluted in Tween Tris-buffered saline (TTBS). Proteins were revealed by the enhanced chemiluminescence (ECL) with Kodak BioMax Light film, (GE Healthcare Bio-Sciences Pittsburgh, PA, United States), digitalized with a scanner (1200 dpi) and analyzed by densitometry with ImageJ Software1. All samples were tested twice in duplicate.
After 6 months, during which the patient was not enrolled into a structured physical activity protocol, the same evaluations were performed to verify the long-term effect of the concurrent training, and the IPAQ questionnaire was administered in order to assess the total level of physical activity during the follow up.
Adapted concurrent training and 1-RM assessment: The patient was enrolled in a structured concurrent training, which last 4 months (34 sessions, twice a week, 50 min per session). The single session of this protocol was divided in four phases: warm-up, resistance training, endurance training, and cool down. The first phase of warm up consisted in 3 min of stationary bike without slope and resistance and 2 min of dynamic warm up for the upper body. In the second phase the patient was involved in strength exercises, where the loads of the upper and lower limbs were set at 50% of 1-RM of the patient, using six types of strength machines (Technogym equipment): Leg Press, Leg Extension, Leg Curl, Vertical Traction, Row Pull, and Abdominal Crunches. After a period of familiarization, the maximal strength was assessed by one repetition maximum (1-RM) test using Brzycki equation. The same equipment used for the 1-RM assessment were used for the resistance training that lasted 25 min and consist in eight repetitions for two sets, with 1 min of rest between the sets. The third phase consisted in aerobic training including 10 min of stationary bike at moderate intensity, 65% heart rate reserve (HRR) evaluated using the Karvonen formula: Exercise HR = % of target intensity (HRmax - HRrest) + HRrest. The last phase consisted in 10 min of cool down involved static stretching for all major muscle trained: shoulders and upper limbs, trunk and lower body, both in sit and in stand position.
As shown in the Elisa-test (see Figure 2A), we analyzed total serum adiponectin in healthy controls, MS patients, and baseline (T0), post physical activity intervention (T1) and at follow-up (T2) in the MS case patient, in which, we found a decrease of serum adiponectin levels [9.05 mug/ml (T0) vs. 7.78 mug/ml (T1)] before and after physical activity intervention. Interestingly, at the follow-up (T2), adiponectin levels remained stable (7.44 mug/ml) indicating that the effects of the training last for at least 6 months. As control, we confirmed that higher levels of serum adiponectin were found in untrained MS patient compared to healthy controls (9.25 vs. 8.13 mug/ml, respectively, p < 0.05).
Furthermore, as shown in the Western blots (Figures 2B,C), we analyzed the distribution of serum Acrp30 in healthy controls, MS patients, and baseline (T0), post physical activity intervention (T1) and at follow-up (T2) in the MS case patient. Three bands corresponding to HMW (>=250 kDa), MMW (180 kDa), and LMW (70 kDa) oligomers were evident in MS patient (Figure 2B). The densitometric evaluation of oligomeric distribution showed that the expression of HMW, MMW, and LMW oligomers are higher in MS patients compared to healthy controls (p < 0.05). When we analyzed adiponectin oligomeric profile in the MS case undergone physical activity, we found that all oligomers are lower in the MS patient after physical activity intervention compared to baseline. In particular, a more evident decrease in HMW oligomers, the most biologically active oligomers, was found (Figure 2C). The adiponectin oligomerization state at the follow-up (T2), do not substantially change for all oligomers (Figure 2).
As shown in Table 2, all functional parameters improved after 4 months of concurrent aerobic and resistance training. The patient reported a decreased BMI (-0.9%), more in deep the impedance analysis showed an increase of FFM (+0.8%) and a decrease of FAT (-2.6%) suggesting a positive modification of body composition. The tests related to walking ability and autonomy showed an improvement at T1 (10mwt = -4.3%; 6mwt = 6.7%), as well as the strength (Handgrip Sx = 8.9%; Handgrip Sx = +23%) and flexibility (TR Dx = +37%; TR Sx = 106.1%) analysis, confirming the functional improvements on these patients due to an adapted exercise. The TUG test performance reported a slight decrease after the protocol (+1.6%), instead the Berg Balance score did not reveal changes over the time, probably due to the good starting point of the patient. After 6 months, from the end of the protocol, most of the evaluated parameters decrease returning to the basal level. As shown in Table 2, BMI increase at T2 compare to T1 and T0 (T1/T2 = +2.8%; T0/T2 = 1.8%), the body composition analysis revealed that FFM decrease (T1/T2 = -2.0%; T0/T2 = -1.2%), and FAT increase (T1/T2 = +13.2%; T0/T2 = +9.4%). The tests related to walking ability and autonomy reported a worsening in these parameters after 6 months (T2), compared to T1 and T0 (TUG T0/T2 = +1%; 10mwt T1/T2 = +4.5%; 10mwt T0/T2 = 0%). An exception was reported by the TUG analysis between T1 and T2 (-0.6%) and the 6mwt that did not showed changes at T2, both comparing to the data collected at T0 and T1. The IPAQ at T2 revealed a LOW level meaning that the volunteer did not reach the criteria for either moderate or high levels of physical activity. The EDDS evaluation showed a decrease after training (EDDS T0/T1 = -11.1%) that remain the same after 6 months of follow up, suggesting the general positive effect of this protocol on the disease severity and on patient functionality.
The psychological parameters (Table 2) reported an increase in QoL, both in physical (45MSQOL P T0/T1 = +5.3%) and mental (45MSQOL M T0/T1 = +11.2%) Score after the concurrent training, the patient experienced a general well-being after the protocol, she also revealed a decrease in Fatigue perception (FFS T0/T1 = -5.4%) and Depressive status (PHQ T0/T1 = -40%) at T1. According to the results Fatigue and Depression have decreased further after 6 months (FFS T1/T2 = -5.7; PHQ = -16.7) and the scores did not return to the baseline level over the time (FFS T0/T2 = -12%; PHQ T0/T2 = -100%), suggesting that the effect of these training could be a long term effect. The QoL, instead, decreased after 6 months compare to T1 (45MSQOL P T1/T2 = -4.3%; 45MSQOL M T1/T2 = -12.5%) and almost return to the baseline level (45MSQOL P T0/T2 = +0.6%; 45MSQOL M T0/T2 = -2.8%).