s2fieldsofstudy: Biology Medicine

text:
Medial septal cholinergic mediation of hippocampal theta rhythm induced by vagal nerve stimulation

Background Electrical vagal nerve stimulation (VNS) has been used for years to treat patients with drug-resistant epilepsy. This technique also remains under investigation as a specific treatment of patients with Alzheimer’s disease. Recently we discovered that VNS induced hippocampal formation (HPC) type II theta rhythm, which is involved in memory consolidation. In the present study, we have extended our previous observation and addressed the neuronal substrate and pharmacological profile of HPC type II theta rhythm induced by VNS in anesthetized rats. Methods Male Wistar rats were implanted with a VNS cuff electrode around the left vagus nerve, a tungsten microelectrode for recording the HPC field activity, and a medial septal (MS) cannula for the injection of a local anesthetic, procaine, and muscarinic agents. A direct, brief effect of VNS on the HPC field potential was evaluated before and after medial-septal drug injection. Results Medial septal injection of local anesthetic, procaine, reversibly abolished VNS-induced HPC theta rhythm. With the use of cholinergic muscarinic agonist and antagonists, we demonstrated that medial septal M1 receptors are involved in the mediation of the VNS effect on HPC theta field potential. Conclusion The MS cholinergic M1 receptor mechanism integrates not only central inputs from the brainstem synchronizing pathway, which underlies the production of HPC type II theta rhythm, but also the input from the vagal afferents in the brain stem.

Subjects and surgical procedure
The data were obtained from 35 male Wistar rats (120-150 g) housed on a 12 h light/dark cycle with free access to water and food. The rats were initially anesthetized with isoflurane (Baxter, Belgium) while a jugular cannula was inserted. Isoflurane was then discontinued, and urethane (0.6 g/ml, Sigma Chemical Co., USA) was administered via the jugular cannula in order to maintain anesthesia throughout the experiment. Anesthesia levels were maintained such that theta field potentials and the transition from theta to large irregular activity (LIA) could occur spontaneously. Body temperature was maintained at 36.5 ± 0.5˚C by a heating pad, and heart rate was monitored constantly throughout the experiment.

Vagal electrode implantation
Since it is generally observed that left VNS minimizes potential cardiac effects, such as bradycardia or asystole [4], in the second stage of the procedure, the left cervical-vagal nerve was gently separated from the muscles and then isolated from the carotid artery using glass section sticks. Before implantation of the stimulating electrode, the vagal nerve was moisturized with glycerine. A custom-made platinum-iridium cuff electrode (SPM35 10HY, MicroProbe, USA) was gently placed around the vagal nerve ( Fig 1A). The platinum-iridium wire leads were tunnelled subcutaneously for about 2 cm and then connected with the input from a PSIU6 isolation unit (Grass-Astromed, West Warwick, USA).

Hippocampal electrode implantation
The rats were placed in a stereotaxic frame with the plane between bregma and lambda levelled to horizontal. An uninsulated tungsten wire placed in the cortex, 2 mm anterior to bregma, served as an indifferent electrode, and the stereotaxic frame was connected to the ground. A tungsten microelectrode (0.1-0.9 MΩ) for recording the hippocampal field activity was placed in the right dorsal HPC, in the stratum lacunosum-moleculare (3.7 mm posterior to bregma, 2.0-2.2 mm lateral from the midline and 2.6-2.9 mm ventral to the dural surface [53]; Fig 1B). An AC amplifier (P-511, Grass-Astromed, West Warwick, USA) was used for recording field potentials, with the low filter set at 1 Hz and the high filter set at 0.3 kHz. The mentioned band pass filter was applied to the data prior to any analysis. The field activity was displayed using a digital storage oscilloscope (Tektronix TDS 3014, USA) and a PC computer (Spike 2.7, Cambridge Electronic Design, GB). EEG signals were digitalized by interface (1410 plus, Cambridge Electronic Design, GB) and recorded onto a computer hard disk for subsequent offline analysis. After the optimal HPC field potential was obtained (theta amplitude of at least 400 μV), the electrode was fixed to the skull with dental cement.

Medial septal cannula implantation and injections
In preliminary experiments, we did not find evidence for the lateralization of the effects of intraseptal injection of drugs used on hippocampal theta (data not shown). Hence, in the initial experiments, microinjection (26 gauge, 5 ml Hamilton 701N microsyringe) of saline and all agents used were always performed into the right MS ( Fig 1B). The coordinates of Hamilton canulae were as follows: 0.6 mm superior from bregma, 0.3 mm lateral from the midline, and 6.5-7.0 mm ventral to the dural surface [53]. All 35 animals receiving the drug injection to the medial septum were divided into six experimental groups (5 or 6 animals each, Table 1): 1/ The animals of group I were administrated with 1 μl of 0.9% NaCl. 2/ The animals of group II were administrated with a local anesthetic, 1 μl procaine hydrochloride (20%). 3/ The animals of group III were injected with cholinergic receptor antagonist atropine sulphate (20 μg/1μl). 4/ The animals of group IV were administrated with cholinergic M1 receptor antagonist dicyclomine hydrochloride (4μg/1 μl). 5/ The animals of group V were administrated with cholinergic M2 receptor antagonist gallamine triethiodide (2 μl/1 μl). 6/ The animals of group VI were injected with selective M1 receptor agonist McN-A343 (0.5 μl/1μl). All drugs used in this study were obtained from the Sigma Chemical Corporation (St. Louis, USA) and injected into the region of the medial septum at the same rate of 1 μl/30 s. The threshold concentration of procaine hydrochloride and atropine sulphate were developed previously [54,55] and the threshold concentrations of dicyclomine hydrochloride, gallamine triethiodide and McN-A343 were developed in preliminary experiments (data not shown).

Vagal nerve stimulation
Based on our previous experiments [30], in the present study, the following square pulse parameters were applied: pulse duration (1 ms), train duration (10 s), frequency (10 Hz) and current intensity of 8 mA. These VNS parameters were previously found to induce a direct, brief effect on HPC field potential, i.e., theta rhythm appearing during vagal stimulation. VNS was delivered through a PSIU6 isolation unit (Grass-Astromed, West Warwick, USA) from an S48 square pulse stimulator (Grass-Astromed, West Warwick, USA). VNS was applied twice or three times, depending on the applied protocol (Fig 2). In each protocol, the medial-septal drug injection was pre-treated by 5 min HPC field recording while 10 s VNS was applied (control). The VNS was always applied at a moment when no spontaneous theta was present in the HPC field potentials.

Experimental protocols
Initially, the experiments were performed on 39 rats, but only 35 animals were taken into consideration (in four animals, the injection sites in the medial septum were incorrect). Thirty five rats were divided into six experimental groups in which different arrangements of VNS and HPC field potential recording were applied (Fig 2). As shown in Fig 2, in groups I and V, VNS was applied twice: in control, pre-injection time and after the injection, between the 15 and 20 min. In groups II, III, IV and VI, VNS was applied three times: in control, pre-injection time and twice post-injection: between the 15-20 min and then between the 60 and 65 min (group II and VI) or 180 and 185 min (groups III and IV). Recording procedure and data analysis HPC field activity was recorded continuously and analyzed in five-minute panels before drug injection and at strictly defined times after the medial-septal drug injection, (see Fig 2). Power spectra in the 0-50 Hz range for the 10 s HPC field potential recordings obtained during each VNS session were generated using the fast Fourier transform (FFT) algorithm implemented in the Spike 2.7 software package (Cambridge Electronic Design, GB) after a Hanning window was applied to the time series, which were obtained at a sample rate of 100 Hz. In this study, theta epochs were defined as rhythmic high amplitude sinusoidal waveforms in a strictly defined frequency band (3-6 Hz). These were identified by peaks in the power spectra within that frequency band, and confirmed by visual inspection of the raw EEG traces. Theta frequency for each 10s VNS epoch was then defined as the frequency with maximum power within the 3-6 Hz range, and theta power for each 10s VNS epoch as the peak power value within the same frequency range.

Statistics
Mean values and standard errors of the mean (± SEM) of two measured theta parameters (power and frequency) obtained during VNS performed before the medial septal drug injection and in successive time periods after the injection were computed and compared. The power and frequency of HPC theta rhythm induced during VNS, before and after the medialseptal drug injections, were subjected to the Shapiro-Wilk test to check the normal distribution of the data. Then, the Mann-Whitney U test was performed (StatSoft Poland).

Histological procedure
The recording electrode tip location was marked by passing 15 μA current for 14 min (7 min cathodal, 7 min anodal, S48 stimulator; Grass-Astromed, West Warwick, USA). Next, the rat was sacrificed by an overdose of urethane for histological examination. The brain was removed and stored in 10% formalin. Frozen brain sections (30 μm) were taken serially and mounted on glass slides for the reconstruction of the medial septal injection sites and the evaluation of HPC theta recording sites (data not shown).

Results
Only data obtained in experiments performed on rats with the correct location of the HPC recording sites and the correct medial septal injection sites were presented in this study (n = 35).

The effect of MS 0.9% NaCl injection on VNS-induced HPC theta rhythm
The effect of intra-septal injection of 0.9% NaCl on VNS-induced type II theta is shown in Fig  4. Fig 4A provides a representative example taken from one animal illustrating the effect of 0.9% NaCl micro-infusion into the MS on VNS-induced HPC type II theta in anesthetized rats. This figure also provides a corresponding power spectrum estimated from each data segment in pre-and post-injection time using the FFT (15 min). Before the MS microinjection of 0.9% NaCl, VNS-induced HPC theta rhythm in the power spectrum had a peak frequency 5.0 ± 0.1 Hz. At 15 min post-injection of 0.9% NaCl, VNS-elicited HPC theta (peak frequency 4.9 ± 0.2 Hz in power spectrum) had a mean power and frequency similar to VNS-induced theta observed in the pre-injection segment (p > 0.05 for power, p > 0.05 for frequency, Mann-Whitney U test; Fig 4B, Table 2).

The effect of MS procaine injection on VNS-induced HPC theta rhythm
The effect of intra-septal injection of procaine on VNS-induced type II theta is shown in Fig 5.  Fig 5A provides a representative example taken from one animal illustrating the effect of procaine microinjection into the MS on VNS-induced HPC type II theta rhythm in anesthetized rats. Fig 5A also provides a corresponding power spectrum estimated from each data segment in pre-and post-injection time using the FFT (at 15 and 60 min). Before the microinjection of procaine into the MS, VNS-induced HPC theta rhythm in the power spectrum had a peak frequency 4.6 ± 0.1 Hz. At 15 min post-injection, VNS no longer elicited HPC theta (absence of a peak in the power spectrum). At 60 min post-injection of procaine, VNS again elicited HPC theta with an power and frequency similar to the control, pre-injection conditions (p > 0.05 for power, p > 0.05 for frequency, Mann-Whitney U test; Fig 5B, Table 2). Pharmacological profile of VNS-induced theta rhythm

The effect of MS atropine injection on VNS-induced HPC theta rhythm
The effect of intra-septal injection of atropine on VNS-induced type II theta is shown in Fig 6. This figure provides a representative example taken from one animal illustrating the effect of atropine micro-infusion into the MS on VNS-induced HPC type II theta in anesthetized rats. Fig 6 also provides a corresponding power spectrum estimated from each data segment in preand post-injection time using the FFT (at 15 and 180 min). Before the microinjection of atropine into the MS control, VNS-induced HPC theta rhythm in the power spectrum had a peak frequency 4.9 ± 0.2 Hz. At 15 min post-injection, VNS no longer elicited HPC theta (absence

The effect of MS gallamine injection on VNS-induced HPC theta rhythm
The effect of intraseptal injection of M2 cholinergic antagonist gallamine on VNS-induced type II theta is shown in Fig 8. Fig 8A provides a representative example taken from one animal illustrating the effect of gallamine microinjection into the MS on VNS-induced HPC type II theta in anesthetized rats. This figure also provides a corresponding power spectrum estimated from each data segment in pre-and post-injection time using the FFT (at 15 min). Before the microinjection of gallamine into the MS, VNS-induced HPC theta rhythm in the power spectrum had a peak frequency 5.2 ± 0.1 Hz. At 15 min post-injection of gallamine, VNS-elicited HPC theta (peak frequency 5.0 ± 0.1 Hz in the power spectrum) had a mean power and frequency similar to VNS-induced theta observed in the pre-injection segment (p > 0.05 for power, p > 0.05 for frequency, Mann-Whitney U test; Fig 8B, Table 2).

The effect of MS injection of McN-A343 on VNS-induced HPC theta rhythm
The effect of intraseptal injection of cholinergic M1 agonist McN-A343 on VNS-induced type II theta is shown in Fig 9. Fig 9A provides a representative example taken from one animal Table 2 Table 2). In contrast to the frequency, the power of VNS-induced HPC theta increased, especially 60 min post injection (p < 0.01, Mann-Whitney U test; Fig 9B, Table 2).

Discussion
The basic experimental model used in the present study to evaluate the central effect of VNS was HPC type II theta rhythm recorded in urethanized rats. We have recently provided the first evidence that, using different protocols and current pulse parameters, VNS produced a very well synchronized HPC type II theta [30]. Pharmacological profile of VNS-induced theta rhythm The theta rhythm is considered to be a "fingerprint" of the limbic cortex [56]. Previous studies demonstrated that theta is involved in a number of physiological regulations, including LTP, learning and navigation, locomotor activity, and sensory-motor integration [57][58][59][60][61][62][63][64][65]. The studies also showed, both behaviorally and pharmacologically, that theta is not a homogenous field oscillation. In fact, two distinct types of theta rhythms have previously been distinguished: anesthetic-resistant and cholinergic-mediated type II theta at a frequency range of 3-6 Hz, and anesthetic-sensitive, probably serotonin-mediated type I theta (7)(8)(9)(10)(11)(12), which is related to motor behaviors [38,[66][67][68][69][70][71][72]. This type of theta depends on the non-cholinergic pathway that arises from the neocortex and cingulate cortex and reaches the HPC via the entorhinal cortex [69].
Recently, Larsen et al. [73,74] using a model of freely moving rats (i.e. type I theta), demonstrated that VNS slowed theta rhythm and decreased its power. In contrast to Larsen et al. [73,74], in the present study, we tested type II theta rhythm which typically is observed in anesthetized rats. The generation of the HPC type II theta is determined by structures of pontine region, hypothalamus and basal forebrain which form the ascending brainstem hippocampal Pharmacological profile of VNS-induced theta rhythm synchronizing pathway [39,43,48,75,76]. The medial septum is considered to be a nodal point of this pathway [44]. On the basis of the above-mentioned data, it seems that in addition to the different model used and different VNS parameters and protocols applied previously [73,74], the difference in neuronal substrate between type II and type I theta could additionally determine a completely different response of HPC rhythmic field potential to VNS in the previous experiments [73,74]. Larsen et al. [73,74] had decisive arguments to suggest that VNS induces a decrease in hippocampal excitation since, in addition to the decrease in amplitude and power of type I theta rhythm, these authors observed decreased efficacy in synaptic Pharmacological profile of VNS-induced theta rhythm transmission. Considering our data concerning type II theta, which appears when the animal is still or anesthetized, we would not go so far as to suggest that VNS induces inhibition of the hippocampal neuronal network. Quite the opposite. The presented data actually indicates the excitation of the hippocampal formation during VNS, as previously suggested [30]. This suggestion is also supported by earlier findings that VNS potentiates hippocampal LTP and enhances hippocampal synaptic transmission in freely moving and anesthetized rats [19,20,23].
The medial septum, is widely accepted to be the pivotal extrinsic regulator of theta rhythm occurring in the limbic system [43,48,77]. The main function of the MS/vDBB is the distribution of inputs to the cingulate cortex, entorhinal cortex, and hippocampal formation, i.e. limbic structures in which well synchronized, local theta field potentials are observed. Rhythmic outputs from the medial septum area act as a "pacemaker" for those structures, inducing theta rhythm [38,39,48,50,67,76,[78][79][80][81][82].
In the light of the above-presented data, new and important findings regarding the neuronal substrate underlying HPC type II theta rhythm emerged from the present study. The results of the second experiment, that temporal inactivation of the MS by the local anesthetic procaine reversibly abolishes VNS-induced HPC theta, clearly demonstrate that medial septum integrates not only central inputs from the brainstem synchronizing pathway which underlies the production of HPC type II theta rhythm, but also input from the vagal nerve.
The pharmacological profile of the MS involved in the VNS effect on HPC rhythmic field potentials was evaluated in the remaining experiments described. The experiments conducted in the group III and IV, with use of the MS injection of atropine, the nonselective muscarinic receptor antagonist and dicyclamine, the selective M1 antagonist, suggest that the M1 receptor subtype is involved in the medial-septal mediation of the VNS effect on hippocampal field potential since both agents irreversibly abolished VNS-elicited HPC theta. This suggestion was proved in the next experiments conducted with use of gallamine, the selective antagonist of M2 receptors. Specifically, the MS injection of this agent did not affect the HPC theta rhythm induced by VNS. The definitive confirmation of M1 receptor profile of VNS-induced type II theta was provided in the experiment with the use of McN-A343. This selective M1 receptor agonist was found to enhance the VNS-induced HPC theta power. The described cholinergic profile of VNS-induced type II theta rhythm supports earlier pharmacological findings concerning this type of theta [38,50,67,[83][84][85].
Although the involvement of the muscarinic M1 receptor subtype in the central pharmacological mechanism underlying VNS-induced HPC type II theta was proved in the present experiments, the possible participation of other noncholinergic receptors cannot be discounted. Previous electrophysiological and neurochemical data suggested that the modulatory effect of VNS on hippocampal LTP may involve the central noradrenergic systems. It was earlier pointed out that VNS potentiates noradrenaline (NE) release in the hippocampus [8, 17,18] and NE was also demonstrated to increase the discharge rate of HPC pyramidal and granular cells [86,87]. Interestingly, it was also shown that blockade of the dorsal ascending noradrenergic bundle abolished septal elicitation of HPC theta rhythm [88]. On the other hand, NE has never been demonstrated to affect hippocampal theta-related cells, which underlies this field potential [67]. Additionally, the bath perfusion of the hippocampal formation slices with NE did not elicit theta oscillation and did not even alter the production of cholinergic-induced theta rhythm in the HPC slice preparations [89].
Finally, one more issue should be addressed. The other possible explanation for the observed effect of VNS on HPC type II theta rhythm is that VNS may cause changes in peripherally released stress-related hormones, which, in turn, may affect hippocampal function through a mechanism not related to the medial septal area. For example, corticosterone was earlier demonstrated to induce the release of hippocampal acetylcholine [90] leading to the increase of the theta amplitude [91]. However, it should also be pointed out that the release of the corticosterone takes a relatively slow time and VNS-induced theta rhythm appeared in the present study immediately during stimulation.
In summary, the present data provide the first evidence for the role of the medial septum in the mechanism of the effect of VNS on hippocampal formation type II theta rhythm in rats. In addition, the present experiments, with the use of cholinergic muscarinic agonist and antagonists, demonstrated for the first time the involvement of muscarinic M1 receptor subtype in the medial septal mediation of VNS-induced HPC theta.


Dry immersion as a model of deafferentation: A neurophysiology study using somatosensory evoked potentials

Introduction Dry immersion is a ground-based experiment simulating the effects of weightlessness, and it is a model of acute symmetrical bilateral deafferentation. This exploratory study aimed to investigate the effects of three days of dry immersion (DI) on sensory thresholds and the functioning of lemniscal pathways, assessed by somatosensory evoked potentials (SEPs). Methods Twelve healthy male volunteers (32+/-4.8 years) participated in the study. Sensory thresholds and SEPs of the tibial nerve of both limbs were recorded before (D-1) and on the third day of dry immersion (D3). Results Sensory thresholds significantly decreased on D3 (-20.75 +/-21.7%; z = -2.54; p = 0.0109 on the right side and -22.18+/-17.28%; z = -3.059; p = 0.002 on the left side). The amplitude of P40 responses did not differ between D-1 and D3. Latencies of all central responses until P30 were shortened on D3 (N21 right:-0.57+/-0.31; z = -3.06; p = 0.002; N21 left -0.83+/-0.53; z = -2.94; p = 0.003; P30 right: -1.26+/-1.42; z = -3.059; p = 0.002; P30 left: -1.11+/-1.55; z = -2.27; p = 0.02) Conclusion Three days of dry immersion can induce hyperexcitability of lemniscal pathways. Significance This may be explained by a change in the expression of membrane channels and/or medullar plasticity and/or hypersensitization of peripheral sensory receptors induced by this acute deafferentation. Additional studies are needed to further elucidate the mechanisms.

Weightlessness and dry immersion
Studies of the effects of space environment on the human body have been carried out for many years: when the body is exposed to prolonged periods of weightlessness, the effects include significant loss of bone and muscle mass, strength, cardiovascular and sensory-motor deconditioning and hormonal and metabolic changes. Taking into account the limited number of flight opportunities and the difficulties encountered in performing in-flight experiments, ground-based experiments simulating the effects of weightlessness are used to get a better understanding of the mechanisms of physiological adaptation. They are also used to design and validate countermeasures (preventive methods developed to reduce or eliminate the deconditioning associated with prolonged weightlessness).
Dry immersion is a ground-based model used to simulate microgravity effects. The subject is immersed up to the neck in thermoneutral water. However since he is separated from the water with an elastic waterproof fabric (Fig 1),the immersed subject is freely suspended in the water mass, but remains dry.
Different studies have been conducted using this technique lasting from 10 hours up to 56 days [1,2]. The long-term effects of dry immersion are compared with those of bed rest and actual space flight.
For a relatively short period, as in bed rest, the model can faithfully reproduce most of the physiological effects of actual microgravity, including centralization of body fluids, support-unloading, and hypokinesia. Unlike bed rest, dry immersion provides a unique opportunity to study the physiological effects of the lack of a supporting structure for the body ('supportlessness') [3].
Consequently, dry immersion offers a unique opportunity to study the effects of the bilateral abolition of sensory afferents in humans. The central nervous system integrates sensory afferents (vision, vestibular system, cutaneous and musculo-tendinous afferents) to adjust posture and coordinate movements. During dry immersion, the usual mechanical constraints are no longer applied to the plantar soles and back. The loss of axial tone is one of the earliest effects of dry immersion [3]. It is triggered by supportlessness and the decrease of sensory afferents. Changes in motor control that are induced by dry immersion include spinal hyperreflexia, suppression of tonic motor neuron activities and alteration of the pattern of motor neuron recruitment, sensory and motor ataxia, and alterations in strategies for movement control [3]. Pathophysiological mechanisms underpinning these changes are not well understood, and the precise involvement of the central nervous system in sensory processing, sensorimotor integration and motor control as a result of these environmental modifications remains unclear.
Several imaging studies have reported a reorganization of the somatosensory cortex. People with arm or hand amputation present a shift of the mouth into the hand in the primary somatosensory cortex (S1) [1,2,4]. Short-term deprivation of sensory input by ischemic nerve block or by locoregional anesthesia are models of acute functional deafferentation [5][6][7]. Werhahn and colleagues found a focal increase in excitability in the hand motor representation contralateral to the deafferented cortex [6]. They also found a rapid improvement in tactile acuity and changes in cortical processing for the left hand during cutaneous anesthesia of the right hand [7]. Both could be influenced by transcallosal interactions and GABAergic transmission [7].
A study using regional anesthesia emphasized the occurrence of illusions similar to those observed in phantom limb pain and suggests that anesthesia could trigger similar cerebral plasticity mechanisms [5].
In healthy subjects, temporary functional deafferentation improves sensorimotor functions of the contralateral homonymous regions or neighboring ipsilateral regions. Similar results have been obtained following stroke. Changes in the interhemispheric balance, primary motor cortex plasticity and ipsilateral S1 cortical reorganization, could be involved [8,9].

Hypothesis
Dry immersion is a unique model of acute symmetrical bilateral deafferentation. This study aimed at elucidating the adaptation capacity of the peripheral and central lemniscal system following precociously acute and temporary bilateral deafferentation induced by 3 days of dry immersion in healthy subjects. We formulated the hypothesis that this acute deafferentation could induce early somatosensory cortex reorganization. The underlying mechanisms could involve changes in intracortical inhibition or facilitation levels. This functional rapid cerebral plasticity could be assessed by modifications of somatosensory evoked potentials (SEPs).

Methods
This was an open, single-center prospective study. Twelve healthy male subjects participated in the study. This study took place at the MEDES Space clinic located in the Toulouse hospital. It was supported by the French Space Agency (CNES) and coordinated by MEDES (Institute of Space Medicine and Physiology). The study was approved by the Ethics Committee (CPP Sud-Ouest Outre-Mer I) and the French Health Authorities.

Subjects
Inclusion criteria. The subjects were healthy male volunteers, aged from 20 to 45, neither over-nor underweight, with a BMI (weight Kg/height m2) between 20 and 26, height between 158 and 190 cm, with no personal or family history of chronic or acute disease or psychological disturbance which could affect the physiological data and/or create a risk for the subject during the experiment. They had fitness level assessments (if age < 35 years: 35 ml/min/kg < VO2max < 60ml/min/kg; if age > 35 years: 30 ml/min/kg < VO2max < 60ml/min/kg).
They were active and free from orthopedic, musculoskeletal and cardiovascular disorders, non-smokers, with no alcohol or drug dependence and no ongoing medical treatment. They all signed informed consent, and were free from all engagements during the study.
Exclusion criteria. Exclusion criteria were a history of any disease or risk factor for thrombosis, a subject already participating or in the exclusion period of a clinical research study, refusal to give permission to contact his general practitioner, incarcerated persons, a subject who, in the judgment of the investigator, was likely to be non-compliant during the study or unable to cooperate because of a language problem or poor mental development, or a subject under guardianship or trusteeship.

Methods
Dry immersion. The acute effects of dry immersion become apparent from the first 12 hours [3]. Changes in motor control that are induced by dry immersion are reproduced with three to seven days of dry immersion [10,11].
This three-days duration was determined by the French Space Agency. This three days experiment included several protocols in different domains (cardiovascular, muscular. . .). The choice of three days was adapted to the different experimental tasks. The length was chosen in order to be long enough to trigger the effects of supportlessness but not too long to improve tolerance of the experiment by volonteers.
Subjects were hospitalized at the MEDES Space clinic. The sensory threshold was assessed before the first day of dry immersion and at the end of the third day.
The dry immersion experiment was performed in a specially designed bath filled with tap water. A special high-elastic waterproof fabric was attached to a metal rim around the external border of the bath. The fabric area considerably exceeded the area of the water surface. The bath had a built-in lift for lowering and raising the subject. The subject, dressed in a comfortable athletic suit, was placed on the waterproof fabric after the fabric had first been covered with a cotton sheet for hygiene reasons. The subject was lowered slowly into the water on the lift and his body was gradually covered with the folds of the fabric together with the water they contained. The fabric was thin and of sufficient area to allow the subject to appear to be "freely suspended" in the water, under conditions that are similar to a complete lack of structural support (Fig 1). The subjects were permitted to put their hands out to work with a computer, eat, read and perform experimental tasks. The water temperature was regulated automatically. It was set at 32˚C to 34.5˚C (thermoneutral), and was adjusted for comfort within these limits at the subject's request. During the whole hospitalisation phases, diet was monitored.Subjects received three principal meals (breakfast, lunch and dinner) and one snack per day (afternoon). The meals was defined by the MEDES nutritionist, provided by Toulouse Hospital and delivered to the subjects by the health assistants under the supervision of the MEDES nutritionist. The main nutritional aspects are recalled hereafter: Coffee, tea, and alcohol were forbidden.
• Liquid intake:The liquid intake will be between 35 and 50 ml/Kg/day (total water taking into account beverages and food). Coffee, tea, and alcohol were forbidden.
• Caloric intake:On the first day of hospitalization, the basal metabolic rate (BMR) was calculated in kcal/day using the WHO equation: Age<30y: BMR = 15.3 x body weight (kg) + 679 Age>30y: BMR = 11.6 x body weight (kg) + 879 Volunteers ate their meal on a tray without getting out from their bath. Urination was done in a bedpan in the bath. For showering and defecation, once a day, the volunteer was lifted out of the bath to take a shower and to go to the toilet (lying down at-6˚, low head).
The air temperature was approximately 24˚C, in order to maintain the heat balance when the subject was raised from the bath. The subject remained under constant medical observation 24 hours a day. For physical examinations and visual inspections of the skin, the folds of fabric could be moved apart without substantially changing the experimental conditions.
Evaluation. Our tests were always done at the beginning of the afternoon. Measurements of SEPs were done just following dry immersion. Patients came out of their dry immersion ten minutes before at a maximum.Skin temperature was not checked at the time of our tests but the tympanic temperature of the patients was controlled twice a day and did not significantly vary. Tests were done in a room which was adjacent to the DI room. This room temperature was controlled and did not changed. Hydratation and liquid balance of the volunteers were controlled Sensory threshold. For sensory threshold assessment and SEP measurements we used a Dantec Natus device equipped with a headrest, with 3 channels linked to a laptop using Keypoint software.
We used the stimulator to assess the sensory threshold, applying it to the tibial nerve at the ankle and progressively increasing the intensity of the current until the subject felt it. This test was done twice in order to verify to reproducibility of the value. Then we noted this intensity in mA.
Somatosensory evoked potentials. SEPs consist of a series of waves that reflect sequential activation of neural structures along the somatosensory pathways. SEPs are elicited by electrical stimulation of peripheral nerves and are recorded by electrodes placed over the scalp (cup electrodes), spine, and peripheral nerves proximal to the stimulation site. The effect of dry immersion has been studied by the changes in latency and size of cortical responses.
We used a bipolar stimulator, and the intensity of the stimulation was fixed at twice the sensory threshold, and it was sufficient to be above the motor threshold triggering movements of the first toe. We stimulated the tibial nerve at the ankle with a 3Hz frequency and the length of the stimulus was 0.2 ms.
We used a bipolar recording with two cup electrodes (active-reference) placed at these sites: • popliteal recording: the active was placed over the route of the tibial nerve at the popliteal fossa, exactly 4 to 6 cm over the popliteal fold, at equal distance between tendons of the semimembranosus and semitendinosus muscles within, and the biceps femoris tendon outside, and the reference was glued over the head of the fibula.
• medullar recording: the active electrode was placed at the first lumbar level and the reference was placed just over the umbilicus. To ensure placement of electrodes at the same place, both before and after DI, a mark was drawn on the skin with a marker at L1.
• Scalp recording: The active was placed at C'z, two centimeters behind Cz (Cz was located per the 10-20 international system) and the reference was located on the contralateral lobule of the ear.
The impedance of each electrode was checked before beginning SEPs recording. For each set, 500 responses were averaged and 2 sets were performed on both sides. The bandwidth was between 3 and 3000 Hz. The analysis window was 100 ms (10 ms/division). Sampling rate was 48kHz. (Fig 2).
Study design. At baseline and after three days of DI immersion, we evaluated sensory thresholds on both sides and registered the SEPs following tibial nerve stimulation on both sides.
Statistical analysis. Based on previous studies, we focused on the Cz electrodes (references). The peak latencies at Cz were identified in the N21, N30, P40, N50, and P60 components, and peak amplitudes were measured for P40 (baseline to P40), N50 (P40-N50), and P60 (N50-P60). Data were separately submitted to a Wilcoxon rank test comparing the following data Correlations between sensory thresholds variation and the changes of the latencies before/ after DI were investigated using the Pearson test.

SEPs
Concerning the amplitude of P40, we found no significant difference between the baseline and DI data. We neither found any significant difference between the baseline and DI data pour N50-P60 amplitude. Popliteal response latencies were shortened on the left side (0.5+/-0.36; z = -2.86; p = 0.003), and tended to be shorter on the right side, but they were not statistically We did not find any difference between peripheral, medullar or central conduction time between baseline and after the third day of DI.
We found no correlation between the decrease in sensory thresholds and electrophysiological data.

Sensory thresholds
A statistically significant decrease in sensory thresholds was found after three days of DI. The decrease in sensory thresholds is in accordance with the study by Lowrey et al. Anecdotal observational studies suggest that the sole of the foot becomes hypersensitive following space flight. In their study, Lowrey et al. measured the skin sensitivity of eleven astronauts by vibration perception at the great toe, fifth metatarsal and heel. Data were collected pre-and postflight. They found a decrease in skin sensitivity on the landing day at 3 Hz and 25 Hz on the great toe. Also, six astronauts presented increased sensitivity at 250 Hz at the heel [12]. However, these results were not confirmed later, and Strzalkowski et al. found that short duration space flight increases the low frequency (3 Hz) perception threshold [13].

SEPs
An acceleration of the conduction at the initial part of the lemniscal pathway until the cervicobulbar response was found after three days of DI.
Regarding the decrease in SEP mean latencies, this was mainly explained by a decrease in the early latencies up to P 30, but did not significantly affect cortical responses; the P30 response is mainly generated by the activity of the bulbothalamic neuron of the lemniscal pathway. This may be explained by the fact that, in SEPs interpretation, a dysfunction at a precise level does not systematically induce a modification of the responses generated downstream from the lesion [14], especially when the volley is only dispersed by a demyelinating process. In this case a component can be abnormal in latency or amplitude, but may not impact later responses because of synaptic synchronization [15]. In our study, synaptic synchronization occurs at the bulbothalamic neuron level and can explain the fact that cortical responses are not modified. This could explain why we only found a decrease in the early responses induced by DI, but no impact on cortical responses. This hyperexcitability could be explained by a modification of the expression of membranous channels caused by deafferentation. Prolonged hind limb unloading leads to changes in electrophysiological properties of L5 dorsal root ganglion neurons in rats after 14 days [16]. The shedding induces a decrease in the rheobase and excitability thresholds of these neurons. This could be linked to variations of ionic currents, which could be due to an increase in the expression of membranous sodium channels or to a decrease in potassium conductance. However, in these studies a deceleration of the sensory conduction is usually noticed, but these modifications were observed following 14 days of deafferentation, when structural demyelination signs may occur secondary to neuronal hyperexcitability [16,17].
Medullar plasticity could also be involved. Some key points related to sensory processing by the spinal cord are as follows: (1) within the musculoskeletal and cutaneous tissues is an extensive network of mechanoreceptors and metaboreceptors that continuously update the spinal cord on the physiologic state of the peripheral tissues; (2) these receptors provide an ensemble of highly integrated and perceptually meaningful information to the spinal cord; (3) the spinal cord is smart, as demonstrated by its ability to interpret and appropriately respond to highly complex and meaningful sensoryensembles; and (4) the human spinal cord demonstrates this smartness and automaticity [18]. It is well accepted that there exists short-term task dependant modulation of spinal reflexes and this modulation does not immediately impose any structural or long lasting functional change in spinal circuits. The prevailing notion is that the synaptic strength is alterned in a task specific manner [19].In animals, cat studies using distal cutaneous denervation showed increased magnitude of the responses evoked by stimulation of the motor cortex in unaffected proximal muscles suggesting incresed corticospinal efficacy [20]. Diminishing afferent input by dorsal rootlet rhizotoy in adults rats led to a significant increase in corticospinal connections, including those on cholinergic interneurons [21]. Hence we think that altering sensory afferences input could modify spinal circuitry responses.
These phenomena have been well studied to explain phantom limb pain triggered by amputation. The lack of peripheral afferents affects neurons of the dorsal horn of the medulla and modifies the structural conformation of the synapses. This process is called central sensitization and includes several phenomena. The neurons of the dorsal horns become hyperexcitable and a reduction of inhibitory processes occurs with the release of glutamate and neurokinins. It is associated with an anatomical reorganization of the sensory inputs in the laminae. The loss of the afferent influx can also provoke disinhibition of the medulla, with a decrease in GABAergic activity and a down-regulation of the opioid receptors of the dorsal horns [1,22].
Some studies using DI also found medullar hyperreflexia [3]. It would be interesting in further studies to complete this neurophysiological evaluation by a measurement of the F waves and H reflexes to prove this hypothesis.
However we can not rule out an involvement of an alteration upstream to the popliteal site (the most peripherical site studied): skin, fiber endings, or very peripherical part of nerve fibers. We did not controlled skin temperature but the experimental conditions were really reproducible. We did not find any data in the litterature to support this hypothesis.

Inflight experiments
Inflight experiments have demonstrated that the absence of gravity modifies the stimuli associated with proprioception and suggestthat microgravity could impair the state of the proprioceptive sensory receptors; neuromuscular spindles, Golgi tendon organs, tactile receptors and joint receptors, as a result of atrophy of the antigravity muscles and fluid shifts, or the sudden release of a constant muscle tone. Daily sleep on Earth however does not impair those mechanoreceptors. The virtual absence of gravity modifies the stimuli associated with proprioception and impacts knowledge of limb position [23]. During the Spacelab-1 flight objective measurement of limb position awareness was carried out by asking crew members to point at target positions with their eyes closed, both inflight and immediately postflight. Results of this experiment indicated that pointing accuracy was degraded during and immediately after flight [24]. Many studies have dealt with the effect of microgravity on these basic mechanisms involving proprioceptive information processing. However, most of them have specifically focused on one of these mechanisms, using either a reflexological approach to analyze spinal excitability [18,[25][26][27] or vestibulospinal reactivity [28,29], or psychophysiological methods to study higher cognitive functions such as body or limb movement perception [30][31][32][33] or body representation in terms of systems of coordinates [34,35]. The "illusions" experiment carried out on five astronauts during two Franco-Russian flights (Antarès 1992 and Altair 1993) and in the Russian post-Antares mission aimed at investigating the adaptive changes in human proprioceptive functions occurring in weightlessness at both the sensorimotor and cognitive levels, focusing on whole body postural reflexes and whole body movement perception [36]. The nature of these changes suggests that new relationships between proprioceptive sensory afferents and motor command are set up in weightless environments, which involves a high degree of sensorimotor adaptability [36] The second point concerns the finding that proprioceptive feedback contributes less efficiently to postural regulation during the early post-flight period, as demonstrated by the gradual increase in the amplitude of the vibration-induced postural responses recorded during the first week [36]. A large depression of the cortical response to vibration in weightlessness compared to normogravity was observed [37].
In contrast to these studies, our work suggests an enhancement of sensitivity to proprioceptive inputs. However, dry immersion is a model of weightlessness with suppression of the proprioceptive afferents, but in this model the vestibular afferents were not affected, contrary to microgravity. To our knowledge SEPs during space flight have never been evaluated and do not allow direct comparison with our results. Our results could be explained by early compensation strategies in response to the lack of proprioceptive afferents.

Conclusion
Three days of dry immersion are sufficient to induce a significant decrease in sensory thresholds associated with a shortening of SEP latencies, which may reflect hyperexcitability of lemniscal pathways. Further studies are needed to confirm this hyperexcitability and to clarify whether it is transient or not.
Supporting information S1


extfieldsofstudy: Medicine Biology

text:
Chronic lifestyle diseases display seasonal sensitive comorbid trend in human population evidence from Google Trends

Seasonal and human physiological changes are important factors in the development of many diseases. But, the study of genuine seasonal impact on these diseases is difficult to measure due to many other environment and lifestyle factors which directly affect these diseases. However, several clinical studies have been conducted in different parts of the world, and it has clearly indicated that certain groups of population are highly subjected to seasonal changes, and their maladaptation can possibly lead to several disorders/diseases. Thus, it is crucial to study the significant seasonal sensitive diseases spread across the human population. To narrow down these disorders/diseases, the study hypothesized that high altitude (HA) associated diseases and disorders are of the strong variants of seasonal physiologic changes. It is because, HA is the only geographical condition for which humans can develop very efficient physiological adaptation mechanism called acclimatization. To study this hypothesis, PubMed was used to collect the HA associated symptoms and disorders. Disease Ontology based semantic similarity network (DSN) and disease-drug networks were constructed to narrow down the benchmark diseases and disorders of HA. The DSN which was further subjected to different community structure analysis uncovered the highly associated or possible comorbid diseases of HA. The predicted 12 lifestyle diseases were assumed to be “seasonal (sensitive) comorbid lifestyle diseases (SCLD)”. A time series analyses on Google Search data of the world from 2004–2016 was conducted to investigate whether the 12 lifestyle diseases have seasonal patterns. Because, the trends were sensitive to the term used as benchmark; the temporal relationships among the 12 disease search volumes and their temporal sequences similarity by dynamic time warping analyses was used to predict the comorbid diseases. Among the 12 lifestyle diseases, the study provides an indirect evidence in the existence of severe seasonal comorbidity among hypertension, obesity, asthma and fibrosis diseases, which is widespread in the world population. Thus, the present study has successfully addressed this issue by predicting the SCLD, and indirectly verified them among the world population using Google Search Trend. Furthermore, based on the SCLD seasonal trend, the study also classified them as severe, moderate, and mild. Interestingly, seasonal trends of the severe seasonal comorbid diseases displayed an inverse pattern between USA (Northern hemisphere) and New Zealand (Southern hemisphere). Further, knowledge in the so called “seasonal sensitive populations” physiological response to seasonal triggers such as winter, summer, spring, and autumn become crucial to modulate disease incidence, disease course, or clinical prevention.

Introduction . Furthermore, GT provides the total volumes for requested diseases terms, normalized in a way that countries of different size can be compared [31]. Importantly, GT was used to study the precise interaction of search terms effect using combined diseases terms (https://support.google.com/trends/answer/4359550?hl=en-GB&ref_topic=4365530). The present study utilized GT to estimate the seasonality in the diseases as well as to measure the comorbid associations among them.
The current study had the following research objectives: (1) to decipher the genuine seasonal diseases using the HA related diseases and disorders from the literature mining; (ii) to conduct disease ontology based semantic similarity network and community detection approach to find the most likely seasonal comorbid diseases; (iii) to evaluate seasonal comorbid diseases, the GT search terms volume of the respective diseases were subjected to rigorous time series analysis. The main outcome of the analysis includes: (a) reveals that a considerable number of human populations is severely subjected to annual seasonal variations; (b) highlights of observed high seasonal rhythm among hypertension, obesity, asthma, and fibrosis diseases are widely spread among the human population. To our knowledge, this is the first study of its kind to examine the possibility of existence of seasonal association in comorbid life style diseases in human population.

Data mining
The aim of this study is to contribute a novel association generation among high altitude related diseases/disorders, symptoms, drugs, and medicines. Fig 1 illustrates various steps involved in the data collection and analysis. The first step in the study was to retrieve all relevant papers related to the topic of interest, i.e., high altitude. Abstracts that contain the most important and concise diseases/disorders and symptoms linked to high altitude were then chosen to be examined. Although the full-text analysis is more informative, the abstract based disease terms analysis has added advantages. Mainly, the analysis is more informative and concise. Also, it is faster to compute with reduced noise level. Keeping in view, a PubMed medical subject headings (MeSH) terms query was used to collect all possible high-altitude related abstracts. PubMed was queried for four types of key terms related to high altitude including: "High altitude disease", "High altitude disorder", "High altitude medicine" and "High altitude drug". PubMed abstracts related to human studies were extracted by using the search filter option "Human". The total number of Pubmed Identification numbers (PMIDs) retrieved from the four key terms was 6159 as on Jan 2017. The redundant PMIDs were reduced resulting in 3513 unique PMIDs. The abstracts of these unique PMIDs were fetched using "PubMed-Wordcloud" of R console [36]. Overall, 2976 abstracts were ready for knowledge discovery.

Entity selection and extraction
The next step is to extract the entities such as diseases/disorders, symptoms, drugs and medicines associated with high altitude. The association of the entities with high altitude means any loose relation that covers biological, biomedical or health related interest. To study the association, an online application programming interface (API) called BioMedical Concept Annotation System (BeCAS) was adopted [37]. This API is widely used to identify biomedical concepts in text [38][39][40]. BeCAS employs a standard pipeline that consists of sentence boundary detection, tokenization, lemmatization, part of speech tagging, and chunking and abbreviation disambiguation. 2976 abstracts were supplied as input to BeCAS. Among the 11 default entities of BeCAS, the term fetching for diseases and disorders entities was carried out. GDep, a dependency parser with the option of domain adaptation using unlabeled data of the target domain, was used to achieve most of the pre-procession steps in BeCAS. For entity recognition, BeCAS uses UMLS extended with a series of more specialized dictionaries such as the Joint chemical dictionary (Jochem).

Benchmark high altitude diseases/disorders
First, the high altitude related chemicals and their target diseases/disorders were used to generate a bipartite network. The known associations between chemicals (or equivalently, drugs) and disorders or its descendants were collected from Comparative Toxicogenomics Database (CTD) in Feb 2017 [41]. In this study, the researchers mainly extracted curated associations of chemical-diseases from CTD to ensure the strong association between chemicals (or equivalently, drugs) and disorders or its descendants. The size of the nodes is based on the frequency of the diseases that were occurring in the resulting dataset composed of the 2976 abstracts. The Gephi open source graph visualization software tool is used to develop a graphic representation Step 1: PubMed Central was queried for high altitude related abstracts.
Step 2: All high altitude abstracts were collected and redundancies were removed. Step 3: The Biomedical concept annotation system (BeCAS) was used to extract the high altitude related diseases/disorders and chemicals from the collected abstracts.
Step 4: The relations among diseases and chemicals from Comparative Toxicogenomics Database (CTD) were used to construct the Disorder-Chemical Network.
Step 5: Disease ontology based semantic similarity-based score was used to construct the Disease similarity network (DSN). of the extracted interaction network [42]. Analysis of the generated network was carried out by Cytoscape network analyser [43]. Diseases that missed their links with drugs/chemicals were left out from their disease module. Drug-disease network property and node size (frequency in literature) were used to narrow down the significant high altitude diseases/disorders and chemicals/drugs.

Clustering disease-disease networks
The disease-disease association among the benchmark high altitude diseases/disorders was depicted in the form of network. The association was established using the DOSE package for R based on computing semantic similarities among Disease Ontology (DO) terms associated with every disease pair [44]. The DO score in correlation matrix ranged between 0-1. Various types of cut-off techniques such as clustering based, score based, percentage based, etc., were employed. In the execution, the threshold value as topmost three edge score (even after transposition of matrix) arising from a single node was used. The clustered disease modules were constructed using fast greedy, edge betweenness, spin glass and walk trap based clustering methods [45][46][47][48]. These different disease network modules were superimposed with each other to identify the overlapped cluster disease modules. The DO score-based disease mapping was carried out to detect disease outliers. GT is used to study the temporal trends in web search using monthly and weekly Relative Search Volume (RSV). In the query, "all categories" and "all types of web search" were used, which are the default setting of GT. We searched "worldwide", using the default settings, for the time period of Jan 2004 to Dec 2016. The rationale behind the selection of default option was to include wide variety of web resources such as image, you tube, news and Google shopping in the GT web search. The "worldwide" search option allowed us to collect RSV from almost 250 political regions, including sovereign as well as dominion states with most of them (162 countries) in the northern hemisphere (NH), covering almost 90% of our human population. RSV is calculated based on how often search terms entered in Google relative to the total search volume in a specific region. These RSV were also collected with and without a reference disease such as obesity in our case. The rationale behind the selection of obesity as the reference disease will be explained in the discussion section under the sub heading "Widespread seasonal comorbid rhythm in the severe SCLD". The search volume of the term was normalized by dividing an unrelated common web search query. This normalization process of GT was compensated for population sizes, increased sensitivity in detection, and allowed the direct comparison of the search volume from countries/ cities and different diseases. The RSV scaled between 0 and 100, with 100 being the highest search proportion per week. GT uses internet protocol (IP) addresses from server logs to assign the origin of web search queries. The monthly and weekly RSV for 12 diseases was downloaded in � .csv format with and without reference diseases for the period of 2004 to 2016 across the world. It should be noted that the relative medical terminologies, similar medical conditions and related search strings excluded in the GT search. In our opinion, such a comprehensive analysis is beyond the scope of the current study. Our GT search terms were limited to English speaking population, because this population is widely spread in world. Furthermore, two English speaking countries, United States of America (USA) and New Zealand (NZL) were geographically selected above 23.5˚N and below 23.5˚S to represent Northern and Southern hemispheres (SH) seasonal changes respectively. In both the countries, RSV of the four severe seasonal comorbid diseases namely Asthma, Hypertension, Obesity and Fibrosis were collected with obesity as the reference for the period from January 2004 to December 2016.

High altitude disease-drug network
We developed a systems approach to infer benchmark diseases/disorders of high altitude to investigate their seasonality. As mentioned earlier, we have collected comprehensive list of high altitude related disorders/disease and drugs from PubMed. After excluding duplicate medical terms, they were annotated using the most commonly used Medical Subject Headings (MeSH). From 2976 high altitude related abstracts, 1710 diseases and 865 drugs were extracted using the text analytics method BeCAS. In search of closely connected high altitude diseases, the clinically reported drug-disease pairs associations were obtained from CTD [41]. After excluding duplicate drug-disease pairs and unlinked pairs, a high altitude related drug-disease bipartite network with six drugs/chemicals and 73 diseases connected by 95 edges was built ( Fig 3A). The node size of each disease term in the network corresponds to its frequency of occurrence in the high altitude related abstracts. The network showed large node size for edema, hypertension, asthma, fatigue, apnea, and obesity diseases/disorders. The outlier plot further showed these six diseases have the high term frequency than the rest of the 67 diseases. Interestingly, edema, hypertension, asthma, fatigue, apnea, and obesity have the high term frequencies (>90 percentile) and node sizes and were termed as key bottleneck diseases of high altitude ( Fig 3B). The pipeline for prediction of comorbid seasonal diseases from DSN. The pipeline starts from the DSN corresponds to step 5 in Fig  1. Step1: the DSN was clustered using four community clustering algorithms of igraph program. Step 2: These community clusters were overlapped to predict the 12 diseases (core-DSN).
Step 3: The monthly Google RSV were collected for the 12 diseases with obesity as the benchmark disease.
Step 4: The low RSV diseases were removed, and the remaining diseases were subjected to seasonal Mann-Kendall and Dynamic time wrapping methods.
Step 5: Based on their seasonal trend p-values and shape distance matrix clustering the diseases were divided into two groups severe (high) and moderate (low).
Step 6: Their seasonal trends were further verified using seasonal decomposition analysis by TABTS and LOESS. The periodicity was analyzed using Autocorrelation and Fourier series analysis.

High altitude associated diseases community structure analysis
To generate a high altitude disease network in which diseases that have similar signs and symptoms were clustered together. Firstly, we generated the disease similarity network (DSN) based on semantic similarities scores among DO terms associated with every disease pair. We computed the pairwise disease-disease semantic similarity matrix from the 73 high altitude related diseases. Among 73 diseases, only 41 diseases (including the six key bottleneck diseases) have disease pair similarity scores with other diseases. The weighted average disease similarity scores were used to generate the DSN. The DSN consisted of 41 nodes and 820 edges (after removing duplicates and self-loop edges) as shown in Fig 4. The DSN network was further subjected to most widely used community detection algorithms, namely-fast greedy (FG), edge betweenness (EB), spin glass (SG) and walk trap (WT) available in the "igraph" package [56]. Each algorithm clustered the DSN into four major disease communities or sub networks ( Fig 5). The sub networks identified from the four algorithms were overlapped to uncover the highly associated or possible comorbid diseases. The bottleneck disease associated community clusters were chosen for detailed analysis. Interestingly, in these community clusters, hypertension formed intra-community interactions with hyperglycemia, hyperhomocysteinemia, hypercholesterolemia, and acidosis, whereas obesity formed intra-community interactions with fibrosis and esophagitis. Overall, these six bottleneck diseases and their six community disease pairs formed a core DSN (Fig 6). The core DSN (12 diseases) possessed almost 60% of the similarity score of DSN (41 diseases).

Monthly google seasonal trend analysis
Using obesity as the benchmark keyword, the month wise varying RSV for the 12 diseases (including obesity) were analyzed for seasonal trend in the 2004-2016 period. All the six bottleneck diseases showed high RSV. Except fibrosis, the remaining five community diseases with low RSV were excluded from the trend analysis (Fig 7). The seasonal Mann-Kendall and Mann-Kendall showed positive seasonal trend for asthma, hypertension, obesity, fibrosis, apnea, and fatigue whereas edema showed no seasonal trend ( Table 1). The seasonal Mann-Kendall and Mann-Kendall of the month wise RSV of the seven diseases without benchmark keyword also showed similar trends (Table 2). Based on their trend P-values, the seven diseases were clustered into two groups (1 and 2) ( Fig 8A, Table 1). The strong seasonal trend in the group 1 diseases was exhibited by TBATS and LOESS analysis (Figs 9 and 10). The auto correlation analysis also revealed the presence of six months periodicity in the group 1 diseases (Fig  Table 1). In this direction, we further calculated the optimal shape alignments among the seven diseases RSV using dynamic time warping method. Importantly, the clustering of the Here, we clearly see that among the six bottleneck diseases (square shape edges) only hypertension community (green colour) and obesity community (magenta colour) are tightly maintained by the four community detection algorithms (encircled). Please note that in all the community detection algorithms, the hypertension community associated diseases (acidosis, hypocholesterolemia, hyperhomocysteinemia, and hyperglycemia) formed a major community cluster. Similarly, the obesity community diseases (esophagitis and fibrosis) formed a short community cluster. Whereas, other bottleneck diseases not able to maintain a separate community cluster. Moreover, the overall inter community cluster interactions (red colour edges) are more than intra community cluster interactions (gray colour edges). Visualization of the network was done using cytoscape (Shannon et al., 2003).
Using obesity as the benchmark keyword, the month wise varying RSV from USA and NZL for group 1 diseases (including obesity) were analyzed for seasonal trends in the 2004-2016 period. The seasonal Mann-Kendall and auto correlation analysis of month wise RSV from both the countries revealed positive seasonal trends and 6 months periodicity respectively in most of the group 1 diseases ( Table 3). The BMA of the group 1 diseases from the same periods were combined for USA and NZL separately. The BMA scores revealed that at any point of time USA and NZL have completely opposite search trends for group 1 diseases (Fig 12).

Weekly google seasonal trend analysis
The RSV seasonal trends of the query diseases may vary depending upon the selection time interval. To avoid this, weekly RSV of the group 1 diseases were collected for the 2004-2016 period without any benchmark diseases. Furthermore, the group 1 diseases were subjected to Mann-Kendall and seasonal Mann-Kendall analysis. Both the analysis The monthly widespread google trend relative search volume (RSV) collected for the "highly comorbid diseases of high altitude (core DSN)" with obesity (dark blue colour) as the benchmark disease. The average RSV of the core-DSN was diseases more than 20 for the entire period were boxed. These seven diseases were named as "severe seasonal comorbid lifestyle diseases (SCLD)".
https://doi.org/10.1371/journal.pone.0207359.g007 Table 1. Widespread monthly RSV of GT time series seasonal analysis of the core DSN diseases (Jan 2004 to Dec 2016) with obesity as the reference term. (Complete raw data is given in S1 Table).  showed strong seasonal trends in the group 1 diseases, also supported by LOESS seasonal decomposition analysis (Table 4). Further, seasonal decomposition analysis by TBATS revealed high seasonal trend in obesity even in the noisy weekly RSV of the four diseases. The optimal shape alignments were also achieved among the group 1 diseases weekly RSV (S4 Table). All these results again strongly suggested that group 1 diseases maintain widespread seasonal comorbid trend.

Discussion
A fundamental question in biology and medicine is to what degree the seasonality is related to the manifestation of human disorders, a hypothesis that we aimed to test in the present work. We find: (i) HA diseases and disorders with similar signs and symptoms reduced into the core DSN (12 diseases); (ii) RSV time series analysis revealed that most of the core DSN diseases  The hierarchical clustering clearly divided them into diseases with (red box) and without (blue box) seasonal pattern matching. Please note that the four diseases (obesity, asthma, hypertension, and fibrosis) in the red boxes have similar seasonal trends and seasonal patterns named as "Group1" diseases (S1 Table). https://doi.org/10.1371/journal.pone.0207359.g008 Seasonal sensitive comorbid life style diseases (except edema) have significant seasonal trends; (iii) Among the core DSN diseases, the public interest in asthma, hypertension, obesity, and fibrosis diseases exhibit strong global seasonal comorbid trend in human population. The merits of each findings, limitations, and implications are discussed below.

Seasonal (sensitive) comorbid lifestyle diseases (SCLD) network
The DSN is derived from many clinical and systemic studies of major HA disorders and diseases. The core DSN renders a dense network of highly similar signs and symptoms reveal their strong comorbid association. Several clinical evidences support the strong comorbid  Seasonal sensitive comorbid life style diseases association of the core DSN (Fig 13). The core DSN is exploited further to study their shared underlying disease etiologies. There are several clinical evidences that support that the major common etiology of core DSN is seasons along with lifestyle factors such as biological clock, inadequate exercise, and bad diet, which further contribute to their disease progression and activation (Table 5). Based on these findings, we named the "core DSN" as "seasonal (sensitive) comorbid lifestyle diseases (SCLD) network". One of the key objectives in the identification of comorbid diseases is to find a common pathological process of significant clinical importance [57]. Interestingly, the major common pathological process of SCLD flares turned out to be the onset of hypoxic state during their patho physiological process.  68] create endogenous hypoxic state in lungs, which leads to the disruption of energy supply functions and plastic processes. This lung hypoxic environment increases pulmonary hypertension [69][70][71] in turn leads to fatigue [72][73][74], respiratory acidosis [75] and lethal esophagitis [76] in certain maladapted population. Unlikely, to our knowledge, none of the clinical studies addressed the onset of hypoxic state in hypercholesterolemia  Table).  [77][78][79], hyperhomocysteinemia [80][81][82][83], and hyperglycemia [84][85][86] patients disease progression. Surprisingly, all the three disorders impair endothelium-dependent vasodilatation by reducing the key hypoxia responsive molecule nitric oxide (NO) production [87]. Overall, the onset of hypoxic state during the disease progression appears to be shared by SCLD. This predicted common pathological process is of clinical importance in the sense that inhaled NO is proposed as a long-term therapy or used as a rescue therapy to some of the SCLD patients [88][89][90][91]. Furthermore, individual genetic makeup is one of the major factors that determines an individual's susceptibility to seasonal changes by invoking complex patho-physiological pathways [92,93].

Classification of SCLD with benchmark
Several studies have successfully correlated the Google RSV with disease prevalence and their seasonal trend with diseases progression [94,95]. For the first time, a benchmark disease such as obesity based RSV is used to classify the SCLD into severe, moderate, and mild categories. First, based on high RSV, the present study classifies hypertension, obesity, asthma, and fibrosis diseases as the most severe widespread seasonally associated SCLD. In other words, their high RSV indicates that a majority of the human population is more severely subject to  seasonal changes in these diseases. Their RSV pattern shape alignments further indicates their strong widespread high bi-seasonal comorbid in the human population. These disease's high seasonal severity and comorbidity is becoming a major health issue in most of the countries around the world [96]. Second, based on the moderate RSV, the present study classifies edema, apnea, acidosis and fatigue as the moderate SCLD diseases. Several clinical studies indicate the presence of bi and quarterly seasonal variations in them [97][98][99]. Even though they have the moderate average RSV widespread, in certain countries, the average RSV of the disease terms edema (South America, Italy, Spain, Indonesia, and Lithuania), and apnea (Venezuela, Italy, Spain, and Turkey) is more than the severe SCLD. This may be due to the influence of the language bias in these disease search term RSV. Finally, the present study classifies hypercholesterolemia, hyperglycemia, hyperhomocysteinemia, and esophagitis as mild SCLD diseases/disorders with bi and quarterly seasonal variations. The results, also supported by several clinical studies, demonstrate the seasonal variation of lipid and glycemic levels in blood and serum from the hypercholesterolemia and hyperglycemia patients respectively [100][101][102].  Table. https://doi.org/10.1371/journal.pone.0207359.g013

Seasonal sensitive comorbid life style diseases
Although we classify hyperhomocysteinemia as mild SCLD, the evidence of its seasonal variation of blood homocysteine level remains controversial [103]. In tune, the weekly RSV analysis of hyperhomocysteinemia shows no seasonal trend. In USA, a mild but consistent seasonal variation in the diagnosis of esophagitis was observed, which corroborates with the present study [104]. Interestingly, the bi-annual peaks (six months periodicity) in most of the diseases occur in the late winters (October through December) and falls (March through April) (Fig 7). This observation is also of significant clinical importance, especially for the chronic disease asthma. The annual exacerbations rate of asthma follows these two peaks, one in October through December, and the other from March through April and is a well reported global phenomenon [105][106][107][108]. Several potentially important SCLD disease risk factors such as seasonal variations in the serum level of insulin, cholesterol, and glucose which also tend to follow the same trend [109].

Widespread seasonal comorbid rhythm in the severe SCLD
Apart from RSV and high significant seasonal trend, even from the noisy weekly data, we consider obesity as our benchmark disease to predict the comorbidity based on multiple factors. Worldwide obesity (higher BMI values) has nearly doubled since 1980, and current estimates indicate that >1.4 billion adults are overweight or obese [110]. Whilst, there is considerable evidence that obesity is strongly associated with seasonality [111]. In correlation, our study finds the highly significant widespread seasonal trend in the public interest on obesity for the period 2004-2016. However, in modern society, especially those who access internet, are mostly used to living in artificial lighting, heating and air-conditioning systems that considerably reduce the exposure of individuals to seasonal (day and light exposure) and environmental changes [112]. Probably, studies also claimed that the extensive use of these artificial aids may develop mismatch between the season and the body clock that may promote obesity [113,114]. Importantly, being overweight or obese leads to higher prevalence of risk association with chronic diseases such as systemic and pulmonary hypertension, chronic kidney diseases, stroke, obstructive sleep apnea, gastroesophageal reflux disease, type 2 diabetes, osteoarthritis etc. [115]. The risk association of these diseases at any given level of obesity varies with ethnicity. For example, Asians have been shown to have a higher absolute risk of  Seasonal sensitive comorbid life style diseases diabetes and hypertension and African Americans to have a lower risk of cardiovascular disease than other groups [116]. Similarly, the prevalence of obesity risk associated diseases varies according to geographical location of the country. For example, the relative risk of death associated with diabetes in Mexico far exceeds that in the United States and Europe [117]. Significantly, first time, our study has shown that public interest in major chronic lifestyle diseases such as obesity, asthma, hypertension, and fibrosis follow a similar strong seasonal pattern or seasonal rhythm which is independent of ethnicity and most likely dependent on seasons. For example, October is commonly associated with the season of autumn in USA and with spring in NZL. In October, our study showed the SCLD search patterns of USA and NZL follow totally opposite seasonal trends and supported by clinical studies [118][119][120][121][122]. In this electronic search study, trends of internet user interest on these SCLD determined for 250 regions from seven continents, suggesting that this is a global phenomenon. This predicted pattern of increase in the prevalence of seasonal comorbid association among asthma, hypertension and obesity is highly supported by clinical evidences [123][124][125]. Even though fibrotic diseases strongly associate with season, their seasonal comorbid association with obesity, hypertension, and asthma is poorly evaluated [10,126,127]. Further studies in this direction could help healthcare providers to design season based strategies for the better management and prevention or efficacy of treatment start at different months of the year to control the seasonal flare.

Limitations
This study has several limitations in the text mining as well as electronic search (GT) that need to be considered while interpreting the results. In text mining, the coverage of diseases and symptoms are limited to the abstracts of "PUBMED", the search terms "high altitude disease", "high altitude disorder", "high altitude medicine", and "High altitude drug" and the search period (Jan 2004-Jan 2017) [128]. In addition, the DO semantic similarity between DSN diseases should be available in the DOSE package [44]. These limits variety of other diseases, and their interactions could be left out in the DSN probably of decisive importance for the generality of results. In the electronic search, our search study revealed the seasonal trends of public interest in the 12 DSN diseases, but not the seasonal trend of the 12 diseases itself. Furthermore, the individual performing the search is not necessarily suffering from the diseases. To validate our predictions, they should be correlated with clinical data. Meanwhile, the demographic characteristics were not available for the users who were performing the search. Besides the GT ability to cover a large geographical area (250 regions from seven continents), 90% of human population living in the northern hemisphere dominate the worldwide RSV seasonal patterns. Moreover, efforts were not made in our study to give special attention to the remaining 10% of human population living in southern hemisphere seasonal changes. In addition, the seasonal patterns were not studied using any language other than English and with a search engine other than Google. Finally, important covariates other than 12 diseases terms affect the development of these 12 diseases or search behaviors could not be assessed.

Conclusions
Despite several limitations, there are several strengths in this study. Majority of the human population adapts well to these seasonal changes. But significant world human population maladaptive to seasonal changes, and render their body highly susceptible to one or other kind of disorders [129,130]. Less progress is made to classify this highly seasonal sensitive population from the normal population. This is mainly due to the challenge in prioritizing seasonal sensitive diseases from the environmental sensitive diseases and lifestyle diseases. In this direction, the present study has successfully addressed this issue by predicting the SCLD, and indirectly verified them in the world population using Google Search Trends. Furthermore, based on the SCLD seasonal public interest trend, the study also classified them as severe, moderate and mild. To our knowledge, for first time, these results provide a basis to predict and classify seasonal sensitive population. The study also necessitates the need to study these categories of seasonal sensitive population separately, because they are genetically susceptible host for the SCLD flares. The dense semantic similar diseases network of SCLD further reflects the most possible comorbid seasonal sensitive diseases. Further, knowledge in the so called "seasonal sensitive populations" physiological and molecular response to seasonal triggers such as winter, summer, spring, and autumn become crucial to modulate disease incidence, disease course, or clinical prevention.
Supporting information S1


text:
Adjunct Therapy of rIFN-γ and rIL-17A along with Sub- optimal Dose of Amphotericin-B effectively Control the Leishmania donovani Parasitic Growth in Infected Mice

Containment/dissemination of Leishmaniasis depends on the dominance of Th1/ Th2 immunity. IFN-γ and IL-17A are well known for protection from leishmaniasis. Synergistic effects of these two cytokines are also known in various autoimmune diseases. However, the therapeutic, as well as adjunct therapeutic use of rIL-17A and rIFN-γ in combination with sub-optimal dose of amphotericin-B (AmpB) is still not validated in visceral leishmaniasis. In the present study, we have evaluated the adjunct therapy in a mouse model of VL. After twenty-one days of post infection, in the therapeutic group, mice were intra-peritoneally injected with two doses of recombinant cytokines at one week interval. In adjunct therapeutic groups of mice, immune components were primed for three days with recombinant cytokine(s) followed by injection of sub-optimal dose of AmpB. Body weight, parasitic load in visceral organs and fold change in cytokines’ gene expression was evaluated. We observed significant gain in body weight, inhibition of parasitic load in visceral organs {(liver; 71.7% 95%), (spleen; 70%-88.7%) (Bone marrow; 46.6 -87.1%)}; significant up regulation in fold change of proinflammatory cytokine(s) gene expression (TNF-γ,iNOS, IL-2 and IL-12) as well as marginal increase of anti-inflammatory cytokine(s) gene expression (IL-4, IL-10&TGF-γ} in adjunct therapeutic groups of mice. Our results suggest that though the therapeutic use of recombinant cytokine(s) is not the best option; however, use of recombinant cytokine(s) along with suboptimal dose of amphoterocin-B to reduce drug toxicity could have a way for better treatment options.

INTRODUCTION
Visceral leishmaniasis (VL), also known as kala-azar (black fever) is a disease of reticuloendothelial system caused by Leishmaniadonovani and Leishmania infantum 1,2 . The clinical manifestations caused by these organisms are usually not distinguishable. However, the decisions for treatment usually do not require species identification. The infection spreads from skin (site of infected insect bite) to visceral organs, especially spleen, liver and bone marrow which additionally progress in spleenomegaly and hepatomegaly that ultimately leads to death, if not treated 3 . In experimental Leishmania donovani infection mice model susceptibility (BALB/c) and resistant (C57BL/6J) in mice acquire via a macrophageactivating, Th1 cell-dependent mechanism governed by different interdigitating cytokines 4 . Use of recombinant cytokine-based therapies, over the past few decades, boosts the clinical relevance, especially in the anticancer immune response [5][6][7] . Recombinant cytokines milieu virtually regulates the innate and adaptive immunity, including the imitation, execution and extinction of pathogen through the induced immune response.
Cytokines are known for their pleiotropic functions so that they function locally or at a distance to suppress or enhance the immunity 8,9 . Cytokine/cytokine-combinations that selectively induce the dominance of Th1 response might be useful for VL treatment option. Strong effective immunity against VL parasite is characterised by the emergence of strong parasite-specific Th1 response. Interferon (IFN)-γ and its major inducer IL-12 dictate the blood circulating T cells and monocytes to assemble at the site of infection and engulf the infected macrophages 10,11 . Typically Th1 activating cytokines accompanied with tumor necrosis factor induces profound inflammation and activate inducible nitric oxide synthase (iNOS) that leads to parasite killing in infected macrophages 12,13 . However, the dominance of IL-4 and IL-10 alone or in additive are responsible for dissemination of parasite into visceral organs 14,15 . Murray et.al., first of all, paved the pathway for the use of cytokines as therapy in visceral leishmaniasis. They reported the killing of parasite in the liver of infected mice with the use of anti-IL-10 receptor antibody (anti-IL-10R mAb) through an inducible nitric oxide synthase-dependent mechanism 16 .
The available options for the treatment of visceral leishmaniasis are associated with severe problems like adverse effect, low efficacy, long treatment period and the development of resistance 17,18 . Hence there is an urgent need for an alternative treatment option for visceral leishmaniasis. In this context, we designed and executed this study in a mice model; in which we have studied three different aspects of the use of recombinant cytokine(s) for an alternative treatment options. i) Prophylactic use of recombinant cytokine(s) for protection from leishmaniasis ii) Therapeutic use of recombinant cytokine(s) once the disease establishes and iii) use of sub-optimal dose of amphotericin-Balong with recombinant cytokine(s) to reduce the drug induced toxicity.
We did not observe better response of recombinant cytokine(s) as a prophylactic or as a therapeutic agent. However, in adjunct therapy, we observed significant results (in terms of parasite clearance in visceral organs, increased proinflammatory cytokines response and decreased/ marginal increase of anti-inflammatory cytokine response). These findings may help in redefining the treatment module in the treatment of visceral leishmaniasis and decreasing the drug induced toxicity (especially amphotericin-Binduced toxicity). Furthermore, this study can also be done in the drug unresponsive cases.

MATERIALS AND METHODS Animal and parasites
Mice from Animal House Facility of CDRI were used as experimental hosts. This study was compliance with Ethical standard. Clinical strains of Leishmania donovani parasite were taken from patients those who were admitted for diagnosis and treatment of kala-azar in Balaji Utthan Sansthan (BUS), Patna. The clinical isolate of parasite were further cultured aseptically in our laboratory under the standard in vitro conditions as described elsewhere 19 . Further, grown parasite was injected into mice to establish and maintained in mice model with specific infectivity. Serial passages were done to maintain the amastigote to amastigote.

Mice and Infection
A total of 60 mice were utilized in this study. A 0.1ml of inoculum containing 10 6 amastigotes/ml was injected intravenously through tail vein to 52 naive Balb/c mice (25-30 g in body weight) {instead of 45 infected mice required for this study} with a sterile 26 gauge needle while rest of the 8 animals were kept as an uninfected control {instead of 5 uninfected mice required for this study} (Table 1 & 2). The magnitude of infection in infected animals was assessed 21 days later by necropsy of two mice from infected group. Treatment Ten groups containing five mice each were used for the study. These were treated with different recombinant cytokines alone and in various combinations with suboptimal dose of AmpB. The mice of uninfected controls (M1) were given PBS only (negative Control) and the rest groups of mice were infected with 1x10 6 amastigote parasites through the tail vein. No. 485-MI-100 and Group M8= rIL-17A+ rIFN-γ) was administered via intra peritoneal in respective group of mice. After seven days of the first dose, the second dose of the same amount of recombinant cytokines was again injected. After 10 days of second dose (i.e. day 45) mice were sacrificed and evaluated for study parameters (Table 1).

Adjunct therapeutic groups
From day 21 of post challenge, adjunct therapy was initiated in respective groups. In this, groups of mice where the only conventional drug, AmpB was used for therapy: two doses of optimal (5µg/mice) and sub-optimal (1µg/mice) AmpB (on day 21 and 28 post challenge respectively) was administered in M10 and M11 group of mice respectively. In groups of mice where sub-optimal dose of AmpB along with recombinant cytokine(s) used for therapy: Three days prior to sub-optimal dose of AmpB administration, mice were sensitized with recombinant cytokine(s) (GroupM12= rIL-17A; Group M13=rIFN-γ and Group M14= rIL-17A+ rIFN-γ). After 15 days of the second dose of drug (i.e. day 45) mice were sacrificed and evaluated for study parameters (Table 2).

Parasitic Burden Post Treatment
Necropsy of all the mice was done after 10 days post-treatment to assess the parasitic burden in spleen, liver and bone marrow. Therapeutic efficacy was assessed in terms of the parasitic load in each organ. Calculations for parasitic density were done as the number of amastigotes/1000 cell nuclei in each organ as compared to untreated controls (M2). Percentage inhibition was assessed in comparison to the infected control by following formula 20

Immunological assay Cytokine gene expression by Real time-PCR
To assess the quantitative analysis of mRNA expression of various cytokines and inducible NO synthase (iNOS) in spleen cells of various experimental groups of mice, Real-time PCR was performed. Spleen tissues were taken for RNA isolation. RNA isolation was carried out using Tri-reagent (Sigma-Aldrich: Cat No. 93289-100ML) after day 10 post treatment and quantified by using Gene-quant (Bio-Rad). cDNA was synthesized from 1µg of total isolated RNA using a first-strand cDNA synthesis kit (Fermentas: Cat No. K1622). Real-time quantitative polymerase chain reaction (RT-PCR) was conducted as per the protocol described earlier 20 . Housekeeping gene GAPDH was used for normalization in all quantifications. To avoid contamination or non-specific reactions, a no-template cDNA was included. Cycle threshold (CT) values obtained indicated the number of PCR cycles which are required for the fluorescence signal to exceed the detection threshold value (background noise). Comparative CT method was used to calculate the differences in gene expression 21 . The fold change in the expression of genes in recombinant cytokine treated groups as compared to that of the infected group was determined by the formula fold change in expression 2-∆∆CT 21 .

Therapeutic use of recombinant cytokines (IL-17A and IFN-γ) after infection has no effect on change in body and organ weight of mice
Analysis for change/gain in body and organ weight during the course of treatment is an important indicator for evaluation of efficacy of any intervention 22

Therapeutic use of recombinant cytokine(s) inhibit parasitic growth in visceral organs of Leishmania donovani infected mice
After infection, leishmania parasites from the local site (tissue) migrate through the lymph nodes to the internal organs (especially, liver, spleen and bone marrow). Hence, we compared the parasitic load in visceral organs in treated group of mice (M6, M7 and M8) and compared with non-treated group of mice (M2). In liver: significant decrease (49% to 56.6%) in parasitic load was observed in cytokine(s) treated groups;   2.6 ± 0.35)} compared with infected non-treated control group (Group M2) (Figure 2A III). Gene expression of IL-12: In treated groups of mice relative fold expression of IL-12 was up-regulated (1.3 to 2.6 times); {(Group M6; mean ± SD: 1.3 ± 1.0), (Group M7; mean ± SD: 1.8 ± 0.6) and (Group M8; mean ± SD: 1.6 ± 1.1)} compared with infected non-treated control group (GroupM2) (Figure 2A  IV). The up-regulation of relative gene expression was not significant probably due to small sample size.

Change in anti-inflammatory gene expression in infected mice upon therapeutic use of recombinant cytokine(s)
Similar to pro-inflammatory cytokine(s) gene expression, relative fold expression of antiinflammatory cytokines gene expression was also evaluated in infected after cytokine treated groups. Gene expression of IL-4: In treated groups of mice relative fold expression of IL-4 was almost

Adjunct therapy of recombinant cytokines (IL-17A and IFN-γ) along with sub-optimal dose of AmpB, influence significant improvement in body weight but not change in organ weight
Change/gain in body and organ weight was evaluated in groups of mice used for conventional drug therapy after infection: optimal (Group M10:AmpB; 5µg/mice) and suboptimal (Group M11: 1µg/mice) dose of drug, alone, and sub-optimal dose of drug along with recombinant cytokines (rIL-17A and rIFN-γ) alone or in combination (Adjunct therapy; M12, M13 & M14). After twenty-one (21) days of infection, therapy was started. Two doses of drugs and recombinant cytokines were given after a oneweek interval. In order to prime the immune components to the fullest function of drug, recombinant cytokine(s) was given three (3) days prior to drug infusion in adjunct therapy groups of mice. After forty-five (45) days of infection body weight of mice was taken before sacrifice and organ weight was taken after sacrifice. Significant change in body weight was observed in all the group of mice (M10 to M14) when compared with body weight at zero-day ( Figure 3A I). When  Further, we evaluated the parasitic load in visceral organs (especially, liver, spleen and bone marrow) of the study groups of mice (M1, M2, and M10 to M14) to see the effectiveness of drugs (optimal and suboptimal) alone or in combination with recombinant cytokine (suboptimal dose of drug). The parasitic load was compared with infected non-treated control group of mice (M2). In liver: significant decrease in parasitic load in optimal drug treated group (Group M10; mean± SD; 569 ±328.3; 90.53%) andin sub-optimal drug treated group (Group M11; mean± SD; 2675 ±124.3; 55.4%). Moreover, in sub-optimal drug along with recombinant cytokine(s) treated groups showed significant reduction in parasitic load:

DISCUSSION
The anti-leishmania drugs are the only treatment option for visceral Leishmaniasis till date 24,25 . This treatment option is always associated with adverse effect on the host like cost, toxicity, longer treatment duration, drug resistance etc 24,25 . Even though several research advances has been made to overcome the associated problems; drug resistance among VL patients is emerging as a major challenge. Researchers are continuously searching for alternative therapy and developing short duration combination therapy along with reducing drug dose in order to overcome the drug resistance 24 .

Journal of Pure and Applied Microbiology
In the present study, author(s) interested in evaluating a new prophylactic and therapeutic strategy with recombinant cytokine(s) for better treatment of visceral leishmaniasis. Change in body weight during the course of treatment has been recognised as a relevant determinant for clinical outcome of the infection with leishmaniasis 22,26 . Galindo et al. in 2013, showed the gain in body weight of hamsters with progression of disease 26 . However, we observed no significant change in body weight in both prophylactic and therapeutic groups of mice when compared with control group with the course of treatment. This shows that the recombinant cytokine(s) have some role in amelioration of disease. In our findings, we did not observe any change in the weight of visceral organs among the study groups compared with controls. Additionally, significant gain in spleen weight was observed in untreated group of mice. This is an indicative of profound parasitemia in leishmaniasis in untreated group of mice. This further substantiates our finding of the role of recombinant cytokine(s) in disease management. Sudharshan et al. in 2011 27 has shown that a significant correlation between parasitic load and clinical outcome in patients undergone treatment with L-AmpB. Mary et al. in 2006 proposed the persistence of more than one parasite/ml of blood after treatment is associated with high risk of relapse 16 . Few more studies were also shown an increase of more than 10 parasite/ml of blood after treatment is a strong indicative of relapse 16,27,28 . In our study, we observed significant inhibition of parasitic load in visceral organs in both therapeutic and adjunct therapeutic group of mice with the use of recombinant cytokine(s). When we evaluated the sub-optimal dose of AmpB with cytokine(s) primed group of mice, we observed more than 95% of parasitic clearance.
Furthermore, balance and dynamic change of pro-inflammatory and anti-inflammatory cytokines may help in predicting the clinical outcome of disease 14,29 . On one hand increased proinflammatory response amplifies the inflammatory reactions that trigger the immune response to overcome the disease; on the other hand this heightened pro-inflammatory response may cause collateral tissue damage. Simultaneously the antiinflammatory cytokines limit the pro-inflammatory cytokine response to prevent the collateral damage of tissue 30 . However, an excessive down regulation of pro-inflammatory cytokines may favour disease progression. Several reports show that the Th1 type of pro-inflammatory cytokine response (especially interferon-γ, tumor necrosis factor-±, interleukin-2, interleukin-12 and iNOS) are the crucial factors in the initiation of protective immunity against visceral leishmaniasis. In our study, we did not find any clear cut cytokine dominance in therapeutic group of mice. Moreover, in adjunct therapeutic group of mice, we observed a clear shift toward Th1 type of cytokine. Furthermore, we also observed increased expression of anti-inflammatory cytokine genes (especially IL-4, IL-10 and TGF-β). Probably this over expression of anti-inflammatory cytokine(s) was the activation of compensatory mechanism to limit the over expressed pro-inflammatory response. This was further confirmed with significant parasitic inhibition in visceral organs of this group.
Based on the above findings we propose that use of recombinant IFN-γ as well as IL-17A may curtain the parasitic load in visceral leishmaniasis. This is possibly due to Th1 induced macrophage activation of IFN-γ/IL-12 axis, which is further facilitated by IL-17A. Therefore we suggest that both therapeutic, as well as adjunct therapeutic use of recombinant cytokine(s) is/are beneficial in redefining the disease treatment. However, the adjunct therapeutic use of recombinant cytokine(s) shows much better outcome as it reduces the parasitic load in visceral organs and skews the cytokine milieu dominantly towards Th1.