Toxicological Study Employing Repeated Doses of Garcinielliptone FC, a Polyisoprenylated-Benzophenone Isolated from Seed of Platonia Insignis Mart Toxicological Study Employing Repeated Doses of Garcinielliptone FC, a Polyisoprenylated-Benzophenone Isolated from Seed of Platonia Insignis Mart Toxicological Employing Toxicological Study Employing Repeated Doses of Garcinielliptone FC, a Polyisoprenylated-Benzophenone Isolated from Seed of Platonia Insignis Mart

: The major constituent from the hexane extract of the seeds of P. insignis is GFC (garcinielliptone FC). Doses of 25, 50 and 75 mg/kg of GFC were aseptically suspended in 0.05% Tween 80 dissolved in 0.9% saline (vehicle) and orally administered for 30, 90 and 120 consecutive days to adult Swiss mice. In this work, the repeated oral administration, in animals of both sexes, demonstrates that this compound is not able to induce mortality and/or behavioral changes in adult mice. In addition, body weight gain, feed intake and disposal of excreta were not altered by the administration of this compound with repeated doses. Furthermore, no differences in weight and macroscopic structure of the brain, liver, kidney, lung, heart and spleen between groups of male and female adult mice were observed after treatment. During the periods of treatment, GFC produced no significant changes on haematological and biochemical parameters in male and female mice treated with all doses used. The aim of this study was to investigate the toxicological potential of GFC through behavioral, hematological, biochemical and morphological parameters in animals in order to ensure the safe use of Platonia insignis in folk medicine.

Introduction 
Platonia insignis Mart is a monotype specie that belongs to the Clusioideae family, popularly known as  [2,3].
Previous studies dealing with the chemical composition of the plant family Clusiaceae show that these species are rich in polyisoprenylated xanthones and benzophenones [1,[4][5][6]. Among these studies, from the hexane extract of the P. insignis seeds, it was isolated a pair of polycyclic polyisoprenylated tautomers, named Garcinielliptone FC (Fig. 1). This compound is unprecedent, in the genus Platonia.
These types of compounds and other benzophenone polyisoprenylated structures, such as 2,4,6-trihydroxy-benzophenone and alkyl aryl ketones have also been isolated from species of the genus Garcinia, Clusia and Rheedia (clusioideae subfamily).
They are not only found in the resin, but also in various organs of the plants, which reinforces the need for developing this study since these compounds may exhibit pharmacological activity of interest in pharmaceutical, cosmetic and/or food industry/field [7][8][9].
These compounds have shown various pharmacological activities, which can be highlighted the antidepressant [10]; antioxidant [1]; antifungal [11], cytotoxic [12], antiretroviral [11], antibacterial [13] and anticonvulsant [1] effects, suggesting once again that knowledge of its toxicity profile can expand its safer use. Because, as described in the literature, any substance can be considered as a toxic agent [14].
In this context, the aim of this study was to conduct an experimental protocol with repeated doses of GFC in mice to investigate its toxicological potential through behavioral, hematological, biochemical and morphological parameters in animals treated with this compound. The studies were made in order to ensure the safe use of Platonia insignis in folk medicine.

Plant Material, Extraction and Isolation
The seeds were obtained from fruits of specimens collected in the city of Barras, Piaui, Brazil, in March 2009, a voucher specimen (ICN TEPB27164) was deposited in the Herbarium Graziella Barroso, Federal University of Piaui. At first, 848.2 g of the seeds were dried at 55 °C and submitted to extraction (soxhlet) with hexane (63%). Then, the hexane extract was subjected to silica gel eluted with hexane containing increasing amounts of ethyl acetate and washed with methanol to end the process. One of the fractions was purified by TLC (thin layer chromatography) eluted with CHCl 3 -CH 3 OH (9:1), obtaining 22 mg of GFC which was identified by means of spectroscopic methods [1].

Animals
All animals were maintained in a controlled temperature (26 ± 1 °C) and a cycle of 12 h light/dark. The animals had free access to food and water. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, Department of Health and Human Services, Washington DC, 1985. The project was approved by the Ethics Committee on Experimental Animals of the Federal University of Piaui (ECEA/UFPI # 078/2012).

Behavioral Screening (Hippocratic Screening)
The method of Hippocratic Screening for evaluation of toxicity has been a very useful and commonly used method for the preliminary screening of plants and/or to detect chemical compounds with pharmacological and toxicological activities [15,16] as well as the study of physiologic daily habits.
Thirty mice between males and females were divided into three groups of 10 animals weighing 25-30 g for each dose (n = 10). The animals were treated orally for 30, 90 and 120 consecutive days with repeated doses of 25, 50 e 75 mg/kg of GFC. During the treatment period, systematic behavioral observations were conducted to evaluate the Hippocratic Screening, which provides a general estimate of the toxicity of the substance on the state of consciousness and general provision, activity and coordination of the motor system, reflexes and activities on the central and autonomic nervous system [17,18].
The parameters: general activity, vocal fremitus, irritability, touch response, contortion, righting reflex, muscle tone, strength to grasp, ataxia, atrial reflex, corneal reflex, tremors, convulsions, straub tail on, hypnosis, anesthesia, lacrimation, ptosis, urination, defecation, piloerection, hypothermia, breathing, cyanosis, hyperemia, death; were evaluated every two days during the period of 30, 90 and 120 days. The administration of the GFC was evaluated daily until the last day of administration. Signs of toxicity, time of onset, intensity, duration and progression were recorded for later analysis.
2.4.2 Repeated Doses Body weight and nutritional status of the animals were quantified and analyzed. The mice were placed individually in cages and acclimated for a period of 15 days. Groups of animals (n = 10) were treated daily at the same time, orally (gavage; o.r.) at doses of 25, 50 and 75 mg/kg of GFC or vehicle (0.1 mL/100 g, o.r.) over a period of 30, 90 and 120 days. The following measures were recorded daily for each animal: the volume of water consumed (mL), the weight of feed consumed (g), the volume of urine excreted (mL) and weight of stools produced (g) besides behavioral changes throughout the study of Cunha et al. [19]. At time intervals of seven days, the animals were weighed for determination of weight gain during the experimental period of 120 days.

Evaluation of Hematological and Biochemical Parameters
Forty mice divided into four groups of 10 animals (n = 10) were treated daily orally for up to 120 days, with vehicle (Tween 80 0.05% dissolved in saline 0.9%, control group) and the doses of 25, 50 e 75 mg/kg of GFC, respectively GFC 25 , GFC 50 and GFC 75 groups.
After each treatment period, the animals were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and blood collection was performed by puncture of the orbital venous plexus using a needle and syringe microhematocrit tubes. The blood was placed into two kinds of tube: one with anticoagulant hemoglobin (Laborlab ® ) for determination of hematologic parameters and the other without anticoagulant to The values for erythrocytes, leukocytes, platelets, hemoglobin, hematocrit and RBC indices MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin) and MCHC (mean corpuscular hemoglobin concentration) were determined immediately after collection using an automated analyzer hematologic cell 120/Hematology Siemens Advia. The differential leukocyte count was performed in extensions stained with May-Grünwald-Giemsa. In each experiment, 100 cells were analyzed and counted.
For biochemical analysis, the material was centrifuged at 3500 rpm for 10 minutes and then determined the glucose, urea, creatinine, AST (aspartate aminotransferase), ALT (alanine aminotransferase), total cholesterol, triglycerides, total and direct bilirubin uric acid. Assays were performed in automatic machine LabMax 240 (Labtest) with commercial systems Labtest ®.

Gross Morphological Analysis
After the treatment period of 120 days with repeated doses of GFC, animals received 40 mg/kg sodium pentobarbital. According to the protocol of Cornell University/Cornell Center for Animal Resources and Education as described by Flecknell [20] and Kohn et al. [21], mice were euthanized by cervical dislocation to perform the macroscopic evaluation. Therefore, the gross morphological analysis of the naked bodies view was held. In addition, there must had weighted the liver, lung, heart, kidney, brain and spleen to determine the relative weights to check whether there was gross morphological changes in the major organs evaluated.

Statistical Analysis
Results of hematological and biochemical parameters were expressed as mean ± S.E.M. (Standard Error of Mean). Data were evaluated using variance analysis (ANOVA) followed by t-Student Newman Keuls test as post hoc test (P < 0.05). An estimate of variance was applied for comparison between treated and control groups for each gender group.

Study of Toxicity at 30, 90 and 120 Days, Hippocratic Screening
One of the methodologies used to assess the toxic potential of the GFC administered in repeated doses was the analysis of the animals according to the Hippocratic Screening. This technique checks the effects produced by a substance as the state of awareness and provision, coordination, muscle tone, reflexes (ear, corneal), activity of the central nervous system and autonomic nervous system activity of the animal [18].
The Hippocratic Screening of animals treated with repeated doses of GFC in 30, 90 120 days are shown in Table 1. Physiological signals were kept steadily in all three doses, with a slight increase after treatments, not significant as clinical signs of toxicity.
During the study, it was observed that among the signs and symptoms that animals presented, there were an attendance of straub, piloerection, lack of response in the grip of the tail and corneal reflection, characteristic manifestations of toxicity but when they occur intensely and associated with other suggestive parameters of toxic processes. In this study, these events were a response to the organic compound studied and are not characteristic of toxicity, since these events were mild and did not cause mortality in animals.
During the Hippocratic Screening, the increased urination and defecation was unobtrusive and may have occurred only by simple manipulation of the animals after administration of the daily doses, since the action of the observer and the change of the eating habits can cause these changes [19]. Nonetheless, this is considered irrelevant for toxicity studies. Absence of signs and symptoms of intoxication. 120 Absence of signs and symptoms of intoxication. GFC 25 30 Increased urination and defecation. However, even with this understanding, the weights of the animals, the consumption of water and food and excreta presented during the same periods of the Hippocratic Screening assessment were measured to check possible changes in these parameters with an indication of toxicity.

Body Weights and Food Consumption of Animals
During the treatment period, intakes of food (Fig. 2) and water (Fig. 3) of animals were analyzed. The results showed that after the administration, in any of the three doses tested of GFC, food and water consumption were not significantly altered.
Regarding the production of excreta, animals treated with GFC during the three doses did not achieve significant change compared with the control group (Fig. 4), showing weak oscillations according to time and dose.
Body weight of the animals was evaluated, considering ingestion, elimination and weigh. The results showed no significant changes in these parameters even after chronic treatment period or dose increasing. There were slight increases for these two aspects (Fig. 5).

Analysis of Hematological and Biochemical Parameters
Hematological and biochemical parameters are useful for the investigation of systemic toxicity in animals treated for long periods of time with potentially harmful substances [19]. In this study, the results of hematology parameters of the animals treated with GFC are shown in Tables 2 and 3. The data demonstrated that there were no significant changes in the groups treated with GFC, when it was compared to the control group.
During the 30 days treatment with GFC there was a reduction of 5 and 7% in the MCHC index in male mice treated with doses of 50 and 75 mg/kg compared with the control group (P < 0.05), respectively. As for the females, it was observed a 7% reduction in this parameter only after the treatment with the dose of 75 mg/kg for the same period of observation (P < 0.05). Statistical analysis was performed using ANOVA followed by t-Student-Newman Keuls-test as a post hoc test (GFC-Garcinielliptone FC: 25 mg/kg, 50 mg/kg and 75 mg/kg).

Fig. 3 Effect of oral administration of GFC in repeated doses on the water consumption of animals.
Statistical analysis was performed using ANOVA followed by t-Student-Newman-Keuls test as a post hoc test (GFC-Garcinielliptone FC: 25 mg/kg, 50 mg/kg and 75 mg/kg). Statistical analysis was performed using ANOVA followed by t-Student-Newman-Keuls test post hoc test (GFC: 25 mg/kg, 50 mg/kg and 75 mg/kg). 75 mg/kg compared with the control group (P < 0.05), respectively.
Furthermore, during treatment with GFC for 90 days, there was a reduction of 6% on the MCHC index in male mice after treatment with a dose of 75 mg/kg as compared with the control group (P < 0.05), and decreases of 5, 7 and 7% in females on the MCHC index after treatment with doses of 25, 50 e 75 mg/kg when compared with the group control (P < 0.05) respectively.
Finally, similarly to these results, the administration of GFC for 120 days reduced by 3 and 4% in male mice and by 7 and 12% in females the MCHC index with doses of 50 and 75 mg/kg as compared with the control group (P < 0.05), respectively. Moreover, we detected a decrease of 25% in the number of leukocytes only in female mice after treatment with a dose of 50 and 75 mg/kg as compared with the control group (P < 0.05).
The biochemical parameters for animals treated with GFC are presented in Tables 4 and 5 and 120 days (MM = 12.5% (25 mg / kg), FM: 16.5% (25 mg/kg)) as compared with the control group (P < 0.05). Given these effects it can be suggested a beneficial role of this polyisoprenylated benzophenone serum in reducing triglycerides, that needs to be better evaluated.   The values represent the mean + S.E.M. (n = 10) animals. "%" represents the relative weight calculated based on the absolute organ weight (g) the value of live animal weight (g) x 100. P < 0.05 compared to the control group (one-way ANOVA).

Gross Morphological Analysis
In toxicity studies of repeated doses the main parameters evaluated are the general toxic signs, dietary changes, laboratory aspects and pathologic evaluation and behavioral [22].
In macroscopic anatomical review of the main dissected organs of male and female mice which underwent the treatment with repeated doses of GFC (animals Table 4), did not show significant changes compared to the control group (P < 0.05).
Additionally, there was no significant difference in the weights of the major organs (brain, spleen, heart, liver, lung and kidney) of male and female animals treated orally with doses of 25, 50 e 75 mg/kg of GFC compared to the control group. The dose also did not have a deleterious effect on the organs analyzed after treatment with repeated doses.

Discussion
The acute or chronic evaluation of the toxicity of bioactive compounds is of great relevance and interest for the pharmaceutical, cosmetic and/or food industry. Because they may interfere with several biological mechanisms, including the production of blood cells or damage to major organs that reveal important role in vital functions of the organism with biotransformation and/or excretion [23]. These data suggest that the toxicological safety profile of medicinal plants should be drawn, since its use in folk medicine is increasingly carried out by the Brazilian population, particularly the inhabitants of the state of Piaui, which has easy access to P. insignis and products derived from parts of the plant of this species. Furthermore, the safety assessment on the use of herbal medicines has recently been questioned due to reports of deaths and diseases, which reinforces the need for the present study [24,25].
In the present study, complementing tests on the acute toxicity of a single dose of GFC were made [26] by oral administration repeated doses of GFC. This study was carried out with the aim of evaluating the qualitative and quantitative effects for a long period of exposure, providing latency for the installation of toxic effects and the consequent appearance after the accumulation of the substance in the body during preclinical trials.
Literature has demonstrated numerous toxicological studies with plant extracts or isolated compounds from plants with therapeutic effect, which makes it increasingly necessary to establish parameters for toxicity to other species used in Brazilian folk medicine [24,27]. The species P. insignis cannot be considered an herbal yet, but the GFC isolated from the hexane extract of its seeds has been used in various experimental protocols and has demonstrated several biological properties such as anti-inflammatory [28], antioxidant [1,29], antileishmanial, cytotoxic [30] and vasorelaxing properties in rat mesenteric artery [31]. These data and the present study are useful to provide more information to strengthen the rational therapy and safety margins of pharmaceutical, cosmetic and/or food products originated from this species, such as, folk juices, ice creams, jellies, among others by the Brazilian Northeast population.
In this context, acute toxicity with repeated doses makes reference to the cumulative adverse effects of substances administration, preferably orally, in a limited period of time (15-30 days) [32]. In our study, administration of the substance GFC for 30 days in both sexes, demonstrated that this compound is not able to induce mortality and/or changes in behavior in adult mice. Moreover, body weight gain, feed intake and excreta disposal were not altered by acute administration with repeated doses of GFC. Our results confirm data found for the species of the genus Garcinia which belongs to the same family of P. insignis.
The studies with repeated doses for 30, 90 and 120 consecutive days are differentiated by the period of exposure of the animals to the GFC, aiming to verify toxicological effects for a long exposure on biochemical, hematological and physiological parameters of male and female adult mice [33]. Regarding the consumption of food and water, there were no significant differences on these parameters over the days of treatment as well as the volume of excreta was proportional to ingestion, suggesting the absence of interference of GFC on both parameters and on the main bodies' responses for metabolism and excretion.
The changes in body mass in studies of long-term toxicity are important indicators of adverse effects of a substance in animal's laboratory [34,35]. By means of this parameter it was possible to verify that, after repeated dosing, GFC increased the body weight of the animals, indicating a minimal or no degree of toxicity of this compound. As a general rule, most investigators consider the gain or loss of body weight is of toxicological significance if the reduction is at least by 10% compared to the average of the control group [36,37]. We can stress that this compound can be considered slightly toxic and therefore safe to be used in industrial process for obtaining technological products for human and/or animal medical practice In our study through the Hippocratic Screening, a previous screening test useful in the evaluation of pharmacological and toxicological activities [15,19,38], no changes were detected in general activity, awareness and sensitivity of male and female mice treated with doses of GFC in 25, 50 e 75 mg/kg. In addition, there were subtle changes on the autonomic nervous system and the absence of feces in individual during the treatment, suggesting a possible presence of mild constipation. However, it is notable that despite this observation, the animals in groups treated with GFC and the control group were uniformly healthy at the end of the administration period and in each of the experimental protocols, suggesting a mild toxicity for this compound isolated from P. insignis. In addition, to evaluate the toxicity parameters, Hippocratic Screening is important to consider the effects of GFC on the hematopoietic system, which is one of the most sensitive target for toxic compounds and is an important index for the physiological and pathological state of rodents. Moreover, changes in blood profile usually provide vital information about the body's response to tissue injury [39]. Daily administration of GFC for 120 days produced no significant changes in hematological parameters of male and female mice treated at all doses, and the changes found may be considered toxicologically irrelevant as they were within the limits of references, demonstrating the slight toxicity of this compound [40]. These data also suggest that this compound cannot exhibit toxic effects on the hematopoietic system, ensuring safe use of this substance in animals and generating promising future expectations for clinical studies.
Although, a general model of the biochemical profile of the animals were within the reference values [26], there were exceptions for urea, uric acid, triglycerides, cholesterol, AST and ALT. The reduction of urea and uric acid in plasma levels indicate an improvement on renal function, suggesting GFC can be evaluated in experimental models that detect kidney failure, to ensure that it can be used during a treatment of acute renal failure besides GFC can also be applied in order to produce a reduction in protein catabolism when its needed in some pathological conditions [41]. On the other hand, the increase in plasma levels of urea and creatinine provides evidence of renal overload, acute renal failure and increased protein catabolism [42,43]. In this study, a decrease in the serum levels of urea and uric acid were observed in the groups treated with GFC for 30, 90 and 120 days. Thus, it can be suggested that its use can be made safely, since no renal overload was observed in the mice.
In addition to these results, with regard to the biochemical parameters, cholesterol and total triglyceride levels were reduced in male and female mice treated with GFC for 30, 90 and 120 days. However, a pharmacological research on the possible hypolipidemic effect is required, since the doses were used to identify toxic parameters [44]. The assessment of the effects of the GFC on the biochemical parameters, the analysis of serum aminotransferase enzymes (ALT and AST) are important indicators of lesions in liver cells [26]. GFC does not cause any damage to the liver without interfering with the normal activity of these enzymes [44,45]. The present study suggests that the use of GFC for 30, 90 and 120 cannot induce hepatotoxicity, since a significant reduction of AST and ALT were observed, suggesting that the compound may induce a hepatoprotective effect, which needs to be better investigated [43].
Complementing the toxicological evaluation, it is important to perform a macroscopic morphological study, which is an important index to diagnose whether a body has been exposed to an injury, and can be done by calculating the ratio between the organ weights versus body weight of the animal [46]. In our study, no differences in weight and macroscopic structure of the brain, liver, kidney, lung, heart and spleen between groups of adult male and female mice treated with doses of GFC at 25, 50 e 75 mg/kg for 30, 90 and 120 days were observed when compared to the control group [27].
Our observations indicate that the polyisoprenylated benzophenones in GFC have a wide margin of safety in experimental animals commonly used in preclinical studies with emphasis on evaluation of toxicity. However, despite the knowledge of mild toxicity of GFC established in this study, it may be suggested that further studies should be conducted as in-depth investigation of hyper cholesterolemia in animals in order to prove the findings and advance the assessment of the safety use of GFC for the design of pharmaceutical, cosmetic and/or food formulations, since the species which GFC was isolated from is widely used in cooking and in popular medicine in Northeast Brazil.

Conclusions
These results indicate that treatment with repeated doses of GFC showed no haematological and biochemical toxicity, particularly in hepatic and renal parameters. In addition, GFC reduced triglyceride levels, which need to be investigated in animal models of hypercholesterolemia. Thus, in our study, the oral administration of GFC at 25, 50 and 75 mg/kg for 30, 90 and 120 days, in both sexes, demonstrates that the polyisoprenylated benzophenones in GFC have a good margin of safety in experimental animals commonly used in preclinical studies with emphasis on evaluation of toxicity.


extfieldsofstudy: Biology Medicine

text:
Modulation of the PI3K/Akt Pathway and Bcl-2 Family Proteins Involved in Chicken’s Tubular Apoptosis Induced by Nickel Chloride (NiCl2)

Exposure of people and animals to environments highly polluted with nickel (Ni) can cause pathologic effects. Ni compounds can induce apoptosis, but the mechanism and the pathway of Ni compounds-induced apoptosis are unclear. We evaluated the alterations of apoptosis, mitochondrial membrane potential (MMP), phosphoinositide-3-kinase (PI3K)/serine-threonine kinase (Akt) pathway, and Bcl-2 family proteins induced by nickel chloride (NiCl2) in the kidneys of broiler chickens, using flow cytometry, terminal deoxynucleotidyl transferase 2ʹ-deoxyuridine 5ʹ-triphosphate dUTP nick end-labeling (TUNEL), immunohistochemstry and quantitative real-time polymerase chain reaction (qRT-PCR). We found that dietary NiCl2 in excess of 300 mg/kg resulted in a significant increase in apoptosis, which was associated with decrease in MMP, and increase in apoptosis inducing factor (AIF) and endonuclease G (EndoG) protein and mRNA expression. Concurrently, NiCl2 inhibited the PI3K/Akt pathway, which was characterized by decreasing PI3K, Akt1 and Akt2 mRNA expression levels. NiCl2 also reduced the protein and mRNA expression of anti-apoptotic Bcl-2 and Bcl-xL and increased the protein and mRNA expression of pro-apoptotic Bax and Bak. These results show that NiCl2 causes mitochondrial-mediated apoptosis by disruption of MMP and increased expression of AIF and EndoG mRNA and protein, and that the underlying mechanism of MMP loss involves the Bcl-2 family proteins modulation and PI3K/Akt pathway inhibition.

Introduction
Nickel (Ni) is one of the essential elements found in abundance in the earth's crust occurring at an average concentration of about 75 μg/g [1]. Ni and Ni compounds have many industrial and commercial uses, and the progress of industrialization has led to their increased release into ecosystems [2,3]. Ni is considered an essential element in animals, microorganisms and plants, and is a constituent of enzyme proteins and nucleic acid [1,4]. However, symptoms of toxicity can occur when too much Ni is taken up [3]. Ni is potentially hazardous to living organisms due to its genotoxicity, immunotoxicity, mutagenicity and cancinogenicity [5][6][7]. Exposure of workers to Ni compounds can produce adverse effects on their health, such as Ni allergy, contact dermatitis, lung fibrosis, cardiovascular diseases, kidney diseases, and cancer of the respiratory tract [8]. Many forms of Ni may induce carcinoma in human beings and animals [9][10][11]. The findings of Zheng et al. [12] show that oxidative stress and the mitochondrial pathway play important roles in nickel sulfate (NiSO4)-induced apoptosis in Carassius auratus liver. Ni compounds can promote the generation of reactive oxygen species (ROS), interact directly or indirectly with nucleic acids and cause DNA damage [13]. It has been also suggested that nickel chloride (NiCl2) can induce DNA damage indirectly through the formation of ROS [14][15][16]. Efremenko et al. [17] have reported that nickel sulfide (Ni3S2)-caused inflammation and proliferation in the lungs of rats. Our previous studies have shown that dietary NiCl2 in excess of 300 mg/kg can cause immunotoxicity, oxidative damage and apoptosis in the kidneys, spleens, small intestines, and cecal tonsils of broiler chickens [18][19][20][21][22][23][24][25][26][27][28].
The phosphoinositide-3-kinase (PI3K)/serine-threonine kinase (Akt) signaling pathway plays a crucial role in cell growth and cell survival, and the pathway can be activated by many types of cellular stimuli or toxins [29]. Serine/threonine kinase Akt/PKB is the primary mediator of PI3K-initiated signaling. Akt, activated by PI3K, regulates cell survival through phosphorylation of a variety of downstream targets such as pro-apoptotic protein, transcription factors and another protein kinase [30,31]. The PI3K/Akt pathway can mediate cell-survival signals through the Bcl-2 family [4,[32][33][34]. Among the Bcl-2 family proteins, Bcl-2 and Bcl-xL promote cell survival, while Bad, Bak, Bid, and Bax can induce cell death [35,36]. The Bcl-2 family of proteins, which is located on the mitochondrial membrane, can alter mitochondrial membrane permeability and trigger apoptosis [35,37,38], and Ni-induced apoptosis is reportedly associated with the PI3K/Akt pathway [4,33,34]. Wang et al. [39] suggest that nickel acetate induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway is involved in the positive regulation of nickel acetate-induced renal cytotoxicity.
Although studies on apoptosis induced by Ni and Ni compounds have been reported, the mechanisms of Ni and Ni compounds-induced apoptosis are unclear. Therefore, the objective of this study was to determine potential mechanisms of NiCl2-induced mitochondria-mediated apoptosis in kidneys of broiler chickens and the alteration of the PI3K/Akt pathway and Bcl-2 family proteins. We monitored apoptosis, the change of mitochondrial membrane potential (MMP), and the mRNA expression of apoptosis inducing factor (AIF) and of endonuclease G (EndoG). We also measured the PI3K/Akt pathway (mRNA expression levels of PI3K, Akt1, Akt2) and the Bcl-2 family of proteins (the protein and mRNA expression of anti-apoptotic Bcl-2 and Bcl-xL and pro-apoptotic Bax and Bak).

Histopathological Changes in the Kidney
In Figures 1-4, NiCl2 resulted in dose-and time-dependent histopathological changes in the kidney, including tubular granular degeneration, vacuolar degeneration, necrosis and apoptosis. In the granular and vacuolar degenerated tubular cells, tiny particles and small or large vacuoles appeared in the cytoplasm. Karyorrhexis, karyolysis and hypochromatosis appeared in the necrotic cells. In the apoptotic cells, cytoplasm was intensely eosinophilic, and nuclei were shrunken, dense, ring-shaped and crescentic. Apoptotic bodies were also observed.

Effects of NiCl2 on Apoptosis in the Kidney
The effects of dietary NiCl2 on the apoptosis in the kidney were studied with TUNEL assay. The results presented in Figure 5, showed that the number of apoptotic cells was significantly greater (p < 0.05 or p < 0.01) in the 600 and 900 mg/kg groups at 14 days of age than in the control group. Apoptotic cells were significantly increased (p < 0.05 or p < 0.01) also in the three NiCl2-treated groups at 28 to 42 days of age. Data are presented with the mean ± standard deviation (n = 5 × 5); * p < 0.05, compared with the control group; ** p < 0.01, compared with the control group.

Effects of NiCl2 on MMP, and AIF and EndoG Protein and mRNA Expression in the Kidney
To further confirm the role of mitochondria in NiCl2-induced apoptosis, the changes of the MMP and release of AIF and EndoG protein from the mitochondria to the nucleus were examined.
As illustrated in Figure 6a,b, the results of the flow cytometry assay showed that NiCl2 caused a significant loss (p < 0.05 or p < 0.01) in the MMP of the three NiCl2-treated groups from 28 to 42 days of age and in the 600 and 900 mg/kg groups at 14 days of age as, compared with that in the control group. In Figure 7, AIF and EndoG protein expression was significantly higher (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 600 and 900 mg/kg group at 14 days of age, than in the control group.
AIF mRNA expression was significantly higher (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 600 and 900 mg/kg group at 14 days of age, than in the control group. EndoG mRNA expression was significantly higher (p < 0.05 or p < 0.01) in the 600 and 900 mg/kg group from 14 to 42 days of age and in the 300 mg/kg group at 42 days of age than in the control group, as shown in Figure 8.

Effects of NiCl2 on Phosphoinositide-3-Kinase (PI3K)/Serine-Threonine Kinase (Akt) Pathway in the Kidney
We investigated whether PI3K/Akt was involved in NiCl2-mediated apoptosis. As shown in Figure 9, PI3K mRNA expression was significantly decreased (p < 0.05 or p < 0.01) in the 600 and 900 mg/kg groups from 14 to 42 days of age and in the 300 mg/kg group at 42 days of age. The mRNA expression of Akt1 and Akt2 was significantly lower (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 600 and 900 mg/kg groups at 14 days of age than in the control group.  . The mRNA expression levels of PI3K, Akt1 and Akt2 in the kidney. Data are presented with the mean ± standard deviation (n = 5); * p < 0.05, compared with the control group; ** p < 0.01, compared with the control group.

Expression of NiCl2 on Bcl-2 Family Protein and mRNA Expression in the Kidney
It has been suggested that Bcl-2 can be a crucial mediator downstream of PI3K/Akt signaling. And Bcl-2 famly proteins have been shown to regulate the MMP. Therefore, we examined the effect of NiCl2 treatment on Bcl-2 family proteins in the kidney.
In Figure 10, Bcl-2 protein expression was significantly decreased (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 900 mg/kg group at 14 days of age when compared with those in the control group. Bcl-xL protein expression was significantly decreased (p < 0.05 or p < 0.01) in the 900 mg/kg group at 14 days of age, in the 600 and 900 mg/kg groups at 48 days of age and in the three NiCl2-treated groups at 42 days of age. The protein expression of Bax and Bak was significantly increased (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age. And, Bax protein expression was significantly increased (p < 0.05 or p < 0.01) in the 600 and 900 mg/kg groups at 14 days of age when compared with those in the control group.
The protein expression of Bax/Bcl-2 ratio was significantly higher (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 900 mg/kg groups from 14 days of age than in the control group, as shown in Figure 11.
In Figure 12, Bcl-2 mRNA expression was significantly decreased (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 900 mg/kg group at 14 days of age. Bcl-xL mRNA expression was significantly lower (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 42 days of age and in the 900 mg/kg group from 14 to 28 days of age than that in the control group. The mRNA expression of Bax and Bak was significantly increased (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 600 and 900 mg/kg groups at 14 days of age.
The Bax/Bcl-2 ratio was significantly higher (p < 0.05 or p < 0.01) in the three NiCl2-treated groups from 28 to 42 days of age and in the 600 and 900 mg/kg groups from 14 days of age than in the control group, as shown in Figure 13.    Data are presented with the mean ± standard deviation (n = 5); * p < 0.05, compared with the control group; ** p < 0.01, compared with the control group. Figure 13. The ratio of Bax/Bcl-2 mRNA expression in the kidney. Data are presented with the mean ± standard deviation (n = 5); * p < 0.05, compared with the control group; ** p < 0.01, compared with the control group.

Discussion
This study explores the molecular control pathways of dietary NiCl2-induced apoptosis in the kidney of young chickens. We found consistent evidence that dietary NiCl2 in excess of 300 mg/kg had adverse effects on the kidney cells. The results showed that NiCl2 significantly increased the TUNEL-postive cells, which were regarded as the apoptotic cells. The histopathological changes also showed that NiCl2 increased apoptosis in the kidney. Our previous studies have proved that NiCl2 induced apoptosis in the thymus, cecal tonsil and spleen [18,21,40]. Also, our findings are in agreement with the the results of Ma et al. and Hossain et al. [41,42] who demonstrate that nickel nanowires (Ni NWs) induce apoptosis in Hela cells and human pancreatic adenocarcinoma cells. Also, Gathwan et al. [43] have reported that NiCl2 can cause apoptotis in liver of male mice.
Mitochondria play an important role in the regulation of cell apoptosis [35]. Changes in the MMP are considered an early event in apoptosis and many proapoptotic proteins can be released from the mitochondria into the cytoplasm when the MMP is damaged [44]. We investigated whether mitochondria were involved in NiCl2-induced apoptosis in broilers. As shown by flow cytometry analysis, NiCl2 reduced the MMP in the kidney. Moreover, AIF and EndoG protein and mRNA expression were increased after disruption of the MMP. AIF and EndoG are thought to be mitochondrial cysteine proteases, whose release can be blocked by Bcl-2. After the MMP is damaged, AIF and EndoG can translocate from mitochondria to the nucleus, and there cause DNA fragmentation and cleavage of genomic DNA without being activated by caspases [45,46]. The data here shows that NiCl2 induces apoptosis through a mitochondria-mediated pathway in the kidney. Previous studies show that nickel acetate can leads to apoptosis via a mitochondrial-mediated pathway [39]. Also, Zhao et al. [47] have reported that nickel nanoparticles (Ni NPs) and nickel fine particles induce changes in the MMP, and up-regulate and mobilize AIF from mitochondria to cytoplasm in JB6 cells.
Recently, it has been reported that regulation of mitochondrial-mediated apoptosis requires the involvement of the Bcl-2 family proteins [48]. The anti-apoptotic members of the Bcl-2 family proteins tend to stabilize the barrier function of mitochondrial membranes, whereas pro-apoptotic members of Bcl-2 family proteins destabilize this function [36]. Loss of MMP is a prerequisite for mitochondrial-mediated apoptosis as it is associated with the reshuffling of Bcl-2 family proteins [49]. The ratio between anti-and pro-apoptotic proteins is a determinant of tissue homeostasis because it influences the sensitivity of cells to apoptosis [35,50]. In this study, NiCl2 increased pro-apoptotic Bax and Bak protein and mRNA expression, and concomitantly decreased anti-apoptotic Bcl-2, Bcl-xL protein and mRNA expression, which caused a significant increase in the Bax/Bcl-2 ratio, and then promoted apoptosis. Our previous studies have also shown that NiCl2 increases the mRNA expression levels of Bax and decreases the mRNA expression levels of Bcl-2 in the thymus, cecal tonsil and spleen [18,21,40].
The PI3K/Akt pathway also plays a role in the regulation of Bcl-2 family proteins, which are believed to be important targets for anti-apoptosis [29,51]. The activation of PI3K/Akt pathway leads to increase in Bcl-2, Bcl-xL expression, and decrease in Bad, Bax expression [30,[52][53][54]. We have investigated whether the PI3K/Akt pathway is involved in NiCl2-induced mitochondria-mediated apoptosis. The results showed that dietary NiCl2 reduced the expression of PI3K and Akt mRNA, implying that inhibition of the PI3K/Akt pathway is one mechanism of NiCl2-induced apoptosis. This implication is consistent with the report of Liu et al. [55] that NiCl2 induces apoptosis and decreases PI3K, p-Akt and Bcl-2 protein expression and increases Bax protein expression in the liver of Kunming mice. Our results are also in agreement with the results of Wang et al. [39] in which nickel acetate-induced apoptosis is characterized by decreasing the protein expression of Bcl-2 and Bcl-xL and increasing the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6 in human proximal tubule cells. Pan et al. [56] suggest that Ni3S2 can down-regulate several anti-apoptotic proteins, including Bcl-2 and Bcl-xL in human bronchial epithelial (BEAS-2B) cells. Contrary to our results and the above-mentioned references, Shi [54] suggests that NiCl2 up-regulates expression of Akt, Bcl-2, and Bcl-XL in BEAS-2B cells.
Based on the results of our study and the discussion above, the mechanism of NiCl2-caused mitochondria-mediated apoptosis in the kidney is summarized in Figure 14. NiCl2 modulates the Bcl-2 family proteins and inhibits the PI3K/Akt pathway. This action is followed by MMP disruption, which increases AIF and EndoG protein and mRNA expression. The highly expressed AIF and EndoG translocate from mitochondria to the nucleus and there causes DNA damage, which finally leads to renal-cell apoptosis.

Animals and Treatment
Two hundred and eighty one-day-old healthy broilers (Chia Tai Group, Wenjiang, Sichuan, China) were divided into four groups. There were seventy broilers in each group. Broilers were housed in cages with electrical heaters, and provided with water as well as under-mentioned experimental diets ad libitum for 42 days. The growth cycle of commercial broilers is about 42 days, after which they are used for consumption. In this rapid growth period food consumption is high, and broilers will easily be affected by diet containing metal pollutants (such as Ni). The aim of our study is to evaluate the effect of dietary NiCl2 on the broilers in this period of rapid growth.
To observe the time-dependent dynamic change, we chose three time points (14, 28 and 42 days of age) for examining histopathological injury, the alterations of apoptosis, mitochondrial membrane potential (MMP), apoptotic protein expression and mRNA expression levels.
In this study, a corn-soybean basal diet formulated by the National Research Council [57] was the control diet. NiCl2 (NiCl2·6H2O, ChengDu Kelong Chemical Co., Ltd., Chengdu, China) was mixed into the corn-soybean basal diet to produce the experimental diets containing 300, 600 and 900 mg/kg NiCl2, respectively.
The basis of doses (300, 600 and 900 mg/kg NiCl2) selection: Ling and Leach reported that dietary NiCl2 concentrations of 300 mg/kg and over resulted in significant reduction in growth rate. Mortality and anemia were observed in chicks receiving 1100 mg/kg nickel [58]. Weber and Reid found a significant growth reduction at 700 mg/kg NiSO4 and nickel acetate and over [59]. Chicks fed more than 250-300 mg/kg Ni in the diet exhibited depressed growth and reduced feed intake [60]. Bersenyi et al. [61] reported that supplementation of 500 mg/kg NiCl2 reduced weight gain (by 10%), feed intake (by 4%) and worse feed conversion efficiency (FCE) (by 5%) in growing broiler cockerels. According to the above-mentioned research results and our preliminary experiment, we chose the doses of 300, 600 and 900 mg/kg NiCl2 in this study for observing the does-dependent changes.
Our experiments involving the use of broilers, and all experimental procedures were approved by Animal Care and Use Committee, Sichuan Agricultural University (Approval No: 2012-024).

Histopathological Examination of Kidney
Five chickens in each group were humanely killed at 14, 28 and 42 days of age. Kidneys were removed, fixed in 4% paraformaldehyde, dehydrated in ethanol and embedded in paraffin. Serial slices at 5 mm thickness were prepared and stained with haematoxylin and eosin (H&E), and examined by light microscopy.

Detection of Renal Apoptosis by TUNEL
Five broilers in each group were humanely sacrificed at 14, 28, and 42 days of age. Kidneys were removed, fixed in 4% paraformaldehyde, dehydrated in ethanol and embedded in paraffin.
TUNEL analysis was carried out according to the manual of In Situ Cell Death Detection Kit (Cat: 11684817980, Roche, Mannheim, Germany). Briefly, tissue sections (5 μm thick) were rehydrated in a series of xylene and ethanol solutions and then rinsed in ddH2O, digested with 50 μL proteinase K (diluted in Tris·HCl pH 7.8) for 15 min, and incubated with 3% H2O2 in methanol for 15 min at room temperature to inactivate endogenous peroxidase. The sections were transferred to a reaction mixture containing biotin-dUTP terminal deoxynucleotidyl and incubated in a humidified chamber for 1 h at 37 °C, followed by washing in phosphate buffer saline (PBS), pH 7.2-7.4. Sections were incubated in Converter-POD (HRP) for 30 min at 37 °C. Reaction product was visualized with DAB kit (AR1022, Boster, Wuhan, China). After final washing in ddH2O, slices were lightly counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene and mounted.
Cells were observed with light microscopy (Olympus, Shimadzu, Japan). The nuclei of apoptotic cells containing DNA strand breaks were stained brown. The TUNEL positive cells (apoptotic cells) were counted by use of a computer-supported imaging system connected to a light microscope with an objective magnification of 1000×. Apoptotic cells were quantified by use of Image-Pro Plus 5.1 (Madia Cybernetics, Bethesda, MD, USA) image analysis software. Five sections in each chicken and five fields in each section were measured and averaged.

Detection of Mitochondrial Membrane Potential (ΔѰm) in the Kidney by Flow Cytometry
Mitochondrial membrane potential was measured with a mitochondrial membrane potential detection kit (Cat: 551302, Lot: 3242965, BD, Franklin lakes, NJ, USA).
At 14, 28 and 42 days of age, five broilers in each group were humanely killed, and the kidneys were immediately removed and ground to form a cell suspension, which was filtered through a 300-mesh nylon screen. The cells were washed twice with ice-cold PBS (pH 7.2-7.4) and suspended in PBS at a concentration of 1 × 10 6 cells/mL. One milliliter of the cell suspension was transferred to a 5-mL culture tube. The collected cells were incubated with JC-1 (5,5ʹ,6,6ʹ-tetra-chloro-1,1ʹ,3,3ʹ-tetraethylbenzimidalyl-carbocyanineiodide) working solution for 15 min in a 37 °C, 5% CO2 incubator. The staining solution was removed and the cells were washed twice with JC-1 staining buffer. The cell-associated fluorescence was measured with flow cytometry (BD, Franklin Lakes, NJ, USA). Normal ΔΨm produces red fluorescence for JC-1 aggregates, but loss of the ΔΨm results in the disaggregation of JC-1 and produces green fluorescence.

Detection of Protein Expression in the Kidney by Immunohistochemistry
Five chickens in each group were humanely sacrificed for gross examination at 14, 28 and 42 days of age. Kidneys were collected and fixed in 10% neutral buffered formalin, and then processed, trimmed, and embedded in paraffin wax.
The protein expression levels were measured using a computer-supported imaging system connected to a light microscope (OlympusAX70) with an objective magnification of 400×. The intensity of staining for each protein was quantified using Image-pro Plus 5.1 (Madia Cybernetics, Bethesda, MD, USA). Each group was measured in five sections and each section was measured using five views, and averaged.

Detection of mRNA Expression in the Kidney by qRT-PCR
The kidneys from five chickens in each group were taken at 14, 28, and 42 days of age and stored in liquid nitrogen. They kidneys were homogenized in liquid nitrogen with a mortar and pestle.
As described [21], total RNA was extracted from the frozen kidney powders with RNAiso Plus (9108/9109, Takara, Dalian, China) according to the manufacturer's protocol. Next, cDNA was synthesized with a Prim-Script™ RT reagent Kit (RR047A, Takara, Japan) according to the manufacturer's protocol. The cDNA product was used as a template for qRT-PCR analysis. Sequences for target genes were obtained from the NCBI database. Oligonucleotide primers were designed by use of Primer 5 software and synthesized at Takara (Dalian, China), as shown in Table 1. All qRT-PCR were performed by use of the SYBR ® Premix Ex Taq™ II system (DRR820A, Takara, Japan) with on a Model C1000 Thermal Cycler (Bio Rad, Hercules, CA, USA).
Chicken β-actin expression was used as an internal reference housekeeping gene. Gene expression values from control group subsamples at 14, 28, and 42 days of age were used to calibrate gene expression in subsamples from corresponding experimental subsamples. All data output from the qRT-PCR experiments were analyzed by use of the 2 −ΔΔCt method [62].

Statistical Analysis
The significance of difference among the four groups of broiler chicks was assessed with variance analysis, and results were presented as mean ± standard deviation (M ± SD). The variation was measured by use of one-way analysis of variance (ANOVA) test of SPSS 16.0 for windows. p < 0.05 was considered statistical significance.

Conclusions
In chicks, dietary NiCl2 in excess of 300 mg/kg was found to cause mitochondrial-mediated apoptosis by disruption of MMP and increased expression of AIF and EndoG mRNA and protein.
These results indicate that the underlying mechanism of MMP disruption involves Bcl-2 family protein modulation and PI3K/Akt pathway inhibition.


Biosensors: Recent Advancements in Tissue Engineering and Cancer Diagnosis

Biosensors are the devices, which integrate the biological recognition element and a small transduction unit for the purpose of detecting various analytes. Biosensors are being developed for various applications such as quality control of food, agriculture, medical and pharmaceutical field, clinical assay and disease diagnostics. Recently, field of tissue engineering and cancer diagnostic provide the new scope in biosensor development. Specially, Cancer diagnostics can provide the broad market for new biosensors. This review focuses on the general description of biosensors and their applications in the field of tissue engineering and cancer diagnostics. Biosensors: Recent Advancements in Tissue Engineering and Cancer Diagnosis

Introduction
1962, Assembly of world's first biosensor by Clark and Lyons begins a new era in the field of detection of various compounds with more specificity and sensitivity [1]. Biosensor developed by Clark and Lyon was actually a glucose sensor which measures the glucose level via utilizing glucose oxidase enzyme as recognition element and detect the oxygen reduction levels which further correlated with the glucose level in the body. The International Union of Pure and Applied Chemistry (IUPAC) defines a biosensor as "a device that uses biochemical reactions mediated by isolated enzymes, organelles or whole cells to detect the effects of chemical compounds by electrical, thermal or optical signals" [2]. Biosensors consist of three basic elements, biological recognition element, signal transduction unit and signal processing unit ( Figure  1). Antibodies, receptor proteins, nucleic acids, enzymes, cells and organelles can act as biological recognition element of the biosensor whose function is to detect the signal from environment through some biochemical reactions. Transducer converts the particular biochemical reaction into electric signal which further processed by the signal processing unit. Traditional analysis methods often include pH and conductivity analysis but their applicability is limited because of low specificity and sensitivity. Accurate analysis in these methods is expensive which usually necessitate the use of sophisticated instrumentation [3]. Biosensors have the ability to overcome such limitations because of their simple design, sensitivity and ability to provide the data in real time. Biosensors are being developed for various applications such as quality control of food, environmental and bioprocess control, agriculture, medical and pharmaceutical field, clinical assay and disease diagnostics [4]. The areas where biosensors have shown a particular importance are cancer diagnosis and tissue engineering. The objective of this article is to review the potential applications of biosensors in the field of oncology and tissue engineering.

Biosensors in Tissue Engineering
Tissue engineering involves the study of growth and development of cells, organs and engineered tissues for restoring the damaged or diseased tissues and organs. Biosensors have shown the tremendous potential in the field of tissue engineering. Concentrations of biomolecules such as glucose, adenosines, and hydrogen peroxide levels play major roles in the maintenance and development of three dimensional cell cultures and organs. Here, biosensors can apply their potential to detect such biological analytes in real time which further provide the insight into cellular activities [5].
Adenosine diphosphate (ADP) and adenosine triphosphate (ATP) plays important role in cellular metabolism and also has a role in the extracellular signalling. It is an important factor required for the tissue engineering experiments. Traditionally, luciferase based methods have applied for the adenosine detection but its use is restricted by the low sensitivity and resolution. Biosensors can be used for the specific and sensitive detection of the adenosines. Schneider et al. fix up a scanning tip, with the ATPase-containing S1 myosin fragment, to detect the sources of ATP release and its concentration as well [6]. Kirana Aran et al (2014) fabricates the biosensor system which poised of interdigitated microelectrodes (IDME) coated with a thin film of a stimuli responsive polymer which is sensitive to reactive oxygen species (ROS). The morphology of polymer changes as a result of ROS generated from the decomposition of hydrogen peroxides and it further generates a measurable electric signal which reveals the hydrogen peroxide concentration. This sensor can be further used to detect oxidative stress in cells [7]. Microfabrication technology leads to the development of new types of miniaturized electrochemical biosensors. Micro electrode arrays based biosensors can help to learnt the physiology of cardiomyocytes such as propagation and conduction Figure 1: A) Basic model of a biosensor represents three basic elements. B) Schematic of biosensor having VEGF specific aptamer as recognition element, aptamer is conjugated with conducting polymer nanotubes. Binding of VEGF to aptamer leads to the increase in conductance as further presented to the signal processing unit.
properties [8]. Natarajan et al. developed a modified Micro Electrode array to study the cardiac physiology formed from self-assembled monolayers (SAMs) with a photolithography method [9].

Role of biosensors in cancer diagnosis
Cancer is the major cause of deaths in the both developing and developed countries. Early diagnosis is crucial for the further successful treatment of the cancer and there is a better chance of cure. Tumor growth is accompanied by gene and protein changes mostly results from the mutations and these changes can act as the specific biomarkers for the cancer diagnosis (Table 1). Cancer biomarkers are potentially one of the most valuable tools for early cancer detection [10]. Traditionally, cancer is diagnosed by the techniques like solution hybridization, ELISA and PCR which are usually time consuming and sophisticated [11]. Biosensors have been developed in order to improve the diagnosis and treatment of various cancers. Aptamers, ssDNA, dsDNA, antibodies and specific antigens (p53 antigen) can be used as the biological recognition element in the biosensors. Medley et al. developed aptamer based biosensors conjugated to gold nanoparticles (Aptamer Conjugated Gold Nanoparticles) which uses the optical and resonance properties of gold nanoparticles which measure the variation in absorption spectra and scattering profiles when nanoparticles are near to each other [12]. Nanoparticles are come together on the cancer cell when specific aptamer is bounds to its target cell; it leads to the colour and absorption change of the sample. This technique can be used for the diagnosis of non-small cell lung cancer (NSCLC). In another study, Kwon et al combined the aptamers with conducting polymer tubes in a biosensor for the detection of an agent responsible for angiogenesis during cancer named as Vascular Epidermal Growth Factor (VEGF) and also revealed that tubes with smaller size shows the high degree of sensitivity during detection [13].
Leukaemia is one of the several cancers whose diagnosis is really difficult. Mostly, a more sophisticated real time PCR is used for the diagnosis purposes. Gorodkiewicz et al. developed a method of determining 20S proteasome levels using a surface plasmon resonance imaging (SPRI) biosensor which uses the highly selective interaction between the proteasome's catalytic subunit and immobilised inhibitors which acts as recognition element [14]. Here, gold chips have been utilized for the recognition of element. It can further be used for the quantitative data determination and helps to improve the early diagnosis of leukaemia.
Nanofibrous material based biosensors provide application for the detection of important cancer biomarker telomerase whose detection with conventional techniques is difficult due to its minute quantity present in the cell nuclei. Nanoscale biosensors with antitelomerase antibodies can detect the intracellular amount of telomerase [15].
In contrast to conventional biomolecular based nanosensors, Molecular imprinting based polymer scaffolds provide a new way for the development of nanosensors [16,17]. Dong Cai et al. developed the arrays of carbon nanotube tips imprinted with a non-conducting polyphenol polymer coating, which can recognize proteins i.e. human papilloma virus derived E7 protein with subpicogram per litre sensitivity using electrochemical impedance spectroscopy [18].

Conclusion
The use of biosensors in aid to cancer diagnosis and tissue engineering experiments would help clinical to make early therapeutic decisions and helps in real time monitoring of bioanalytes. Early and steak detection of molecules and portable use of biosensors makes an important step towards the personalized medicine and "in field detection of diseases".   [19][20][21].


s2fieldsofstudy: Biology Medicine

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Relation between ADIPOQ Gene Polymorphisms and Type 2 Diabetes

Objective: The manuscript investigates the relation between adiponectin gene (ADIPOQ) polymorphisms and type 2 diabetes mellitus (T2DM) in a Chinese population. Methods: We designed a case-control study involving 340 normal glucose tolerant (NGT) subjects and 340 type 2 diabetes patients. Three SNPs (rs182052, rs1501299, and rs7627128) were genotyped by TaqMan methods. Results: We found that rs7627128, rs1501299 and rs182052 were significantly associated with T2DM. Haplotypes analysis indicated that the frequency of the haplotypes A-A-T was frequent in T2DM patients (OR = 2.10; 95%CI: 1.44–2.90; p < 0.001), but G-A-T was more frequent in the control group than in the T2DM group (OR = 0.66; 95%CI: 0.54–0.81; p < 0.001). Conclusion: The ADIPOQ genetic polymorphisms were associated with type 2 diabetes in a Chinese population.

Introduction
The pathogenesis of type 2 diabetes mellitus (T2DM) remains unclear. However, previous studies suggested that T2DM is a complex disease resulting from the interaction between genetic polymorphisms and environmental factors [1]. Several genes have been identified as susceptible genes for T2DM, including the C5L2 gene [2], CYP2J2 gene [3] and CCR5 gene [4,5]. However, these susceptible genes can only explain a small fraction of the susceptibility to T2DM.
Recently, adipose tissue was considered to play an important role in the pathogenesis of diabetes, as well as obesity by secreting a variety of secretory proteins [6]. Adiponectin is one of the major adipocyte secretory proteins most abundantly found in human plasma, with potent roles in insulin sensitivity in muscle and liver, regulating energy homeostasis and glucose tolerance [7,8]. Adiponectin is a product of the ADIPOQ gene, which is located on human chromosome 3q27, where a region composed of three exons that span 17 Kb, identified as a susceptibility locus for metabolic syndrome and T2DM, has been reported [9,10]. T2DM is a complex heterogeneous group of metabolic disorders, including hyperglycemia and impaired insulin action and/or insulin secretion, and a detailed etiology underlying T2DM is still unclear [11,12]. Therefore, it is necessary to identify the pathogenesis of T2DM. Recently, the ADIPOQ gene polymorphisms have been suggested to be implicated in the risk for type 2 diabetes; however, association studies have reported conflicting results [13,14]. Therefore, we designed a case-control study to derive an association between the ADIPOQ gene polymorphisms and T2DM risk in a Chinese population.

Ethnics
The present study has been performed with the approval of the ethics committee of West China Hospital, Sichuan University, and is in compliance with the Helsinki Declaration. Informed consent for the study was collected from all of the candidate subjects.

Subjects
A total of 680 participants, including 340 T2DM patients and 340 healthy control subjects (normal glucose tolerant (NGT)), were selected for the present study from January 2010 to June 2014. Diabetes was confirmed according to the World Health Organization (WHO) consulting group criteria, for which an oral glucose tolerance test has a 2-h plasma glucose value ≥11.1 mmol/L (200 mg/dL); and the subjects with 2-h plasma glucose value <7.8 mmol/L (140 mg/dL) were labeled as NGT [15]. The characteristics of the case and control subjects are shown in Table 1.

Phenotype Measurements
We collected clinical data, such as weight, height, waist circumference and other data. The BMI was calculated as weight (in kg) divided by the square of height (in m). Fasting plasma glucose, serum cholesterol, serum triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were measured as described in a previous protocol [16]. Glycated hemoglobin (HbA1c) was estimated by high performance liquid chromatography using a Variant™ machine (Bio-Rad, Hercules, CA, USA). Serum insulin concentration was estimated using an enzyme-linked immunosorbent assay (Dako, Glostrup, Denmark). Total serum adiponectin was measured by radioimmunoassay.

Genetic Analysis
Although there are 683 SNPs for the human ADIPOQ gene listed in the National Center for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/SNP), we only selected three SNPs (rs182052, rs1501299, and rs7627128) in the present study according to the methods described previously [17]. These three SNPs are all tagSNPs of the ADIPOQ gene, which can represent the genetic information of the other SNPs in the ADIPOQ gene. Genomic DNA was extracted from the whole blood by the phenol-chloroform method of DNA extraction. Genotyping was confirmed by the TaqMan method as described previously [18].

Statistical Analysis
We used SPSS 17.0 for Windows (SPSS, Chicago, IL, USA) to perform the statistical analysis. Hardy-Weinberg equilibrium (HWE) was tested using a χ 2 test. Comparison of the means between the two groups was analyzed by Student's t test. The χ 2 test was used to compare the proportions of genotypes or alleles. We used the SHEsis software (http://analysis2.bio-x.cn/myAnalysis.php) [19,20] to perform the linkage disequilibrium (LD) analysis and haplotype construction. In the haplotype-based case-control analysis, haplotypes with a frequency of <0.03 were excluded. Statistical significance was established at p < 0.05.

Results
The genotype distribution of each SNP did not show a significant difference from the Hardy-Weinberg equilibrium values. As shown in Table 2 for total participants, the genotype and the allele frequency of rs182052, rs1501299 and rs7627128 were significantly different between the T2DM patients and the control subjects. According to the |D'| and r 2 values, we considered that these three SNPs (rs1501299, rs182052 and rs7627128) are located in one haplotype block. In the haplotype-based case-control analysis, haplotypes were established through the use of all three SNPs. As shown in Table 3, the haplotypes A-A-T was frequent in T2DM patients (OR = 2.10; 95%CI: 1.44-2.90; p < 0.001), but G-A-T was lower in the T2DM patient group than in the control group (OR = 0.66; 95%CI: 0.54-0.81; p < 0.001).

Discussion
In the present study, we found that the ADIPOQ gene rs1501299, rs182052 and rs7627128 polymorphisms were significantly associated with T2DM in a Chinese population.
T2DM is a complex disorder that may result in the interaction between genetics and environmental factors. There were many genes, such as Calpain 10, eNOS, the CRP gene, etc. [21][22][23][24][25], that have been found to be associated with T2DM. Previously, Ramya et al. found that the adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in a south Indian population [26]. Chung et al., reported that ADIPOQ genetic polymorphisms rs2241766 (+45T > G), rs1063537, rs2241767 and rs2082940 were correlated with the progression of diabetic nephropathy (DN) in Taiwanese male patients with T2D [27]. Du et al. observed that ADIPOQ gene polymorphisms (rs266729, rs1063539, rs16861205 and rs7649121) were associated with increased risk for the T2DM in a Chinese population [28]. Our study also indicated that ADIPOQ gene polymorphisms were associated with T2DM in a Chinese population.
In addition, we carried out a haplotype-based case-control study to investigate the association of ADIPOQ polymorphisms with T2DM, and we found that rs182052, rs1501299 and rs7627128 polymorphisms were significantly associated with T2DM. Haplotype analysis suggested that the haplotype A-A-T and G-A-T was associated the increased risk or decreased risk for T2DM, respectively.

Limitations
Several limitations in the present study should be mentioned. Firstly, we did not compare the difference in serum adiponectin levels among different genotypes. Secondly, we did not detect the HOMA-IR [HOMA-IR is an index used to evaluate an individual's level of insulin resistance. Calculated as follows: Fasting plasma glucose levels (FPG, mmol/L) × Fasting insulin (FINS, mIU/L)/22.5] in our study. Finally, the relatively small sample size may overestimate the OR value during the analysis of the risk for T2DM.

Conclusions
In conclusion, the present results indicate that T2DM is associated with the ADIPOQ gene polymorphisms. The A-A-T haplotype appear to be a useful genetic marker, and the G-A-T haplotype might be a protective factor from T2DM in Chinese people.


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Adaptive Immune Responses in a Multiple Sclerosis Patient with Acute Varicella-Zoster Virus Reactivation during Treatment with Fingolimod

Fingolimod, an oral sphingosine 1-phosphate (S1P) receptor modulator, is approved for the treatment of relapsing forms of multiple sclerosis (MS). The interference with S1P signaling leads to retention particularly of chemokine receptor-7 (CCR7) expressing T cells in lymph nodes. The immunological basis of varicella zoster virus (VZV) infections during fingolimod treatment is unclear. Here, we studied the dynamics of systemic and intrathecal immune responses associated with symptomatic VZV reactivation including cessation of fingolimod and initiation of antiviral therapy. Key features in peripheral blood were an about two-fold increase of VZV-specific IgG at diagnosis of VZV reactivation as compared to the previous months, a relative enrichment of effector CD4+ T cells (36% versus mean 12% in controls), and an accelerated reconstitution of absolute lymphocytes counts including a normalized CD4+/CD8+ ratio and reappearance of CCR7+ T cells. In cerebrospinal fluid (CSF) the lymphocytic pleocytosis and CD4+/CD8+ ratios at diagnosis of reactivation and after nine days of fingolimod discontinuation remained unchanged. During this time CCR7+ T cells were not observed in CSF. Further research into fingolimod-associated VZV reactivation and immune reconstitution is mandatory to prevent morbidity and mortality associated with this potentially life-threatening condition.

Introduction
Varicella-zoster virus (VZV) is a neurotropic α-herpes virus which can cause clinically distinct presentations. Primary infection with VZV occurs naturally during childhood and is characterized by a self-limiting generalized vesicular rash. Reactivation of latent VZV infection in cells of the cranial nerve, dorsal root and autonomic ganglia comprises the second manifestation [1]. The corresponding mechanism of action is the assembly of new viral particles and transport in anterograde direction. VZV reactivation most commonly manifests as localized, unilateral, and painful vesicular skin infection. This condition can be complicated by post-herpetic neuralgia, meningoencephalitis, and vasculopathy [2]. VZV-specific T-cell responses, in particular CD8+ effector memory T cells are considered the major host defense against symptomatic VZV reactivation from latency [3]. This antigen-specific cellular immune response, however, declines with age or immunosuppression, and exposes this group of individuals to an increased risk of VZV reactivation.
Fingolimod (FTY720, Gilenya) is a sphingosine-1-phosphate (S1P) receptor modulator, which is approved as oral therapy for relapsing forms of multiple sclerosis (MS). The drug inhibits the egress of C-C chemokine receptor 7 (CCR7)-positive lymphocytes from secondary lymphatic organs including lymph nodes [4]. As a consequence, naïve and central memory T (TCM) cells are reduced in the circulation, leading to a relative increase of effector and effector memory T (TEM) cell frequencies [5]. Despite the marked reduction in the peripheral lymphocyte count the incidence of most infections is comparable to patients receiving placebo or interferon-β-1a [6,7]. Treatment with fingolimod, however, is associated with increased rates of both primary and secondary VZV infections. Two fatal cases and severe VZV infections have been reported in the pivotal trials and post-marketing setting [6,[8][9][10]. The comprehensive analysis of 11915 patient years of treatment with fingolimod disclosed a nearly twice as high incidence rate of VZV infections compared to the placebo group. Post-marketing data showed a 2-3 times higher risk of VZV infection compared to other disease-modifying therapies [11]. It is, therefore, important to understand the immunological basis of why fingolimod selectively increases the risk of VZV reactivation. It may be assumed that fingolimod interferes with the function of lymphocyte subpopulations relevant for immune surveillance and memory immune responses specific for VZV in a subgroup of patients. Notably, cases of severe herpes virus infections have also been reported with other disease-modifying treatments including alemtuzumab and natalizumab.
Here, we report the time course of antibody responses against VZV and immune cell phenotypes in a patient with MS who developed herpes zoster reactivation during treatment with fingolimod. This report provides insights into the dynamics of systemic immune responses during VZV reactivation and immune reconstitution after treatment cessation and antiviral treatment.

Clinical Case Report
We report a 30-year-old man who presented with vesicular rash, erythema, pain and allodynia in the dermatome supplied by the first trigeminal nerve in May 2014. He had been suffering from severe holocranial headache for about one week and was taking oral valacyclovir (3.000 mg per day) and cefuroxime (1000 mg per day) since the previous day. Meningeal signs and fever were absent. The diagnosis of relapsing-remitting MS was made in April 2011 according to McDonald criteria after two relapses with dissemination in space [12]. Natalizumab treatment was initiated in September 2012 because of ongoing clinical and radiological disease activity on interferon-β-1b 8 Mio I.U. (International Units), s.c. every other day. The EDSS was 2.5 at this time. His JCV serology was negative on repeated testing (May 2012 and November 2013). Side effects in terms of fatigue and tiredness led to discontinuation of natalizumab after 16 monthly cycles (i.v., 300 mg). Fingolimod was started in March 2014 after a natalizumab wash-out period of 8 weeks (EDSS 2.5). At that time, he tested positive for serum VZV-IgG antibodies (1.700 mIU/mL). He did not report any history of recurrent VZV disease.
After two months of fingolimod treatment he developed symptoms of herpes zoster reactivation. Probable VZV meningitis was diagnosed on the basis of the clinical syndrome in combination with CSF findings. CSF analysis showed an increased albumin quotient (QAlb; 8.6, age-dependent normal value <6.0) and a lymphomononuclear pleocytosis of 7 cells/µL. Red blood cells (RBCs) were absent. PCR for the detection of herpes simplex virus (HSV) and VZV in the CSF was negative. Anti-VZV IgG was detected in the serum (3.000 mIU/mL) and CSF (290 mIU/mL), whereas the VZV antibody index (0.7) was negative. He tested negative for antibodies against HSV and Cytomegalovirus (CMV) and positive for anti-Epstein-Barr-Virus IgG in serum and CSF. Fingolimod was discontinued and antiviral treatment was changed to intravenous acyclovir (10 mg/kg body weight tid). Headache, pain and allodynia responded to a combination of acetaminophen and mefenamic acid. CSF re-tap 9 days later revealed persistent pleocytosis (11 cells/µL) and QAlb increase (7.7). Testing for oligoclonal bands (OCBs) was not performed. He was discharged with oral valacyclovir (3000 mg per day) for another two weeks and analgesics. At follow-up in 9/2014 he had remained clinically stable (EDSS 2.0), whereas MRI disclosed several new lesions when compared to April 2014. Treatment with dimethyl-fumarate was initiated in January 2015. An overview of the clinical course, sequential treatments, and sample collection time points is shown in Figure 1(Ae) Figure 1. Disease course, varicella zoster virus (VZV)-specific antibody response, and lymphocyte reconstitution after fingolimod discontinuation. (A) Schematic overview of the clinical course, sequential treatments, sample collection time points, and sample specimen; (B) VZV-specific serum IgG measured over a period of two years at defined time points including start and end of natalizumab treatment, start (T0) and four (T1) and eight (T2) weeks of fingolimod treatment, diagnosis of VZV reactivation (DxVZV), and two follow-up visits nine days (DxVZV/9d) and four months (DxVZV/4m) post VZV diagnosis. The dashed line represents mean and standard deviation of measurements from before DxVZV; (C) Changes of absolute lymphocyte counts (*10 3 /µL) and CD4+/CD8+ T cell ratios in response to fingolimod treatment after four weeks (T1), eight (T2) weeks, and at diagnosis of VZV reactivation, and after nine days of fingolimod discontinuation and antiviral therapy (DxVZV/9d) and four weeks (DxVZV/4w) and four months (DxVZV/4m) later. Abbreviations: CSF, cerebrospinal fluid; Dx, diagnosis; EDSS, expanded disability status score; IFN, interferon; PBMC, peripheral blood mononuclear cells; S, serum; VZV, varicella zoster virus. Big asterisk: time point of diagnosis of VZV reactivation; small asterisks: time points of sample collections.

Serial Analysis of Humoral and Cellular Immune Parameters
In our patient, changes in the humoral response to VZV reactivation were investigated from frozen serum samples collected on eight occasions. Anti-VZV serum IgG remained stable (mean 1.480 mIU/mL, standard deviation (SD) ± 259) over a period of almost two years until he developed zoster reactivation ( Figure 1B). At this time point, the titer had increased 2.3-fold within one week (T2: 1.300 mIU/mL versus DxVZV: 3.000 mIU/mL), and remained increased four months later (2.800 mIU/mL). Serum IgG, serum albumin, and total serum protein assessed from the same samples were unchanged (data not shown).
We observed a similar phenotype of CSF lymphocytes at diagnosis of VZV reactivation and after nine days of fingolimod discontinuation. The vast majority were T cells (91% versus 93%) with a CD4+/CD8+ T cell ratio of 2.4 and 2.7, respectively. B cells were below 3% and NK/NKT cells below 7% at both examinations. Subtyping according to the expression of CCR7 and CD45R0 was only performed at the control tap. CSF T cells consisted exclusively of TEM and effector cells. Nine days after discontinuation of fingolimod CCR7+ T cells were not detected in the CSF which was in contrast to the findings in peripheral blood (Figure 3).

Discussion
Fingolimod has been shown to attenuate the antiviral immune response in MS patients as evidenced by reduced numbers of IFN-γ-producing T cells and decreased VZV-specific proliferation of CD4+ and CD8+ T cells [13]. In addition, frequencies of VZV-specific memory T cells are reportedly low and amount to only half of HSV-and to less than a tenth of CMV-specific T cell frequencies in immune adults [14,15]. Together, this may add to a disrupted defense against VZV reactivation from latency. Indeed, subclinical reactivation as determined by detection of VZV in saliva is a common consequence of fingolimod treatment [13]. Particularly the elderly and immunocompromised individuals, including patients previously treated with immunosuppressive drugs, are at risk of developing clinical complications caused by VZV reactivation. This case illustrates adaptive immune responses associated with clinically evident VZV reactivation in a fingolimod-treated MS patient. These include relative enrichment of an effector CD4+ T cell subset, an only modest systemic VZV-specific IgG response, and a strikingly fast reconstitution of absolute lymphocyte numbers and CCR7 expressing lymphocytes after discontinuation of fingolimod.
Several studies indicate that T cell immunity, in particular circulating T cells are most critical for maintaining control of latent VZV [3,16]. CD8+ effector and effector memory T cells are considered key players in the control of virus infections [17,18] and fingolimod was shown to impair CD8 T cell effector functions [19]. We did not perform activation studies to investigate VZV-specific T cell responses, and did not encounter changes or differences in the frequencies of CD8+ T cells. We found, however, an increased percentage of presumably highly-differentiated effector CD4+ T cells in our patient compared to fingolimod-treated patients without evidence of VZV reactivation. Schub et al. reported that VZV-specific CD4+ T cells in peripheral blood of non-fingolimod treated patients with zoster infection showed a shift in cytokine expression toward interferon γ positivity, an increase in CTLA-4 and PD-1, and a decrease in CD127 expression [20]. This is in line with other studies which disclosed a role for CD4+ T cells in the defense of herpes virus infections by executing immune responses to viral glycoproteins, such as the VZV glycoprotein E [21]. These cells apparently are abundant at low levels but may expand in case of infection or profound alterations of the immune cell composition.
A mild to moderate inhibitory effect of fingolimod on T cell-dependent and -independent antibody responses was first demonstrated in healthy volunteers [22]. A recent randomized, placebo-controlled trial evaluated immune responses against novel and recall antigens in fingolimod-treated MS patients [23]. The study confirmed that these patients in principle can mount an immune response but to a lower extent than patients receiving placebo. The threshold defining a sufficient seroprotection was set at an increase in antibody titers above 4-fold at 3 and 6 weeks. Our patient developed an about 2-fold VZV-specific IgG response and did not meet this criterion for sufficient seroprotection. Other studies, however, used less stringent criteria (>2-fold increase of antibody titers) for the definition of a protective immune response.
Another key finding of this study was that after nine days on intravenous acyclovir and withdrawal of fingolimod lymphocyte counts had normalized to almost pretreatment levels and CCR7+ T cells had reappeared in the peripheral blood. This was faster than expected according to fingolimod's pharmacokinetic half-life data of about 7-9 days and studies on lymphocyte counts [22,24]. One possible explanation may be an accelerated fingolimod metabolism. We considered this unlikely as genetic polymorphisms associated with an altered drug metabolism of the cytochrome P450 (CYP450) enzyme 4F, by which fingolimod primarily is cleared, have not been described [25,26]. An accelerated fingolimod clearance caused by drug interactions related to acyclovir, acetaminophen, or mefenamic acid was unlikely as well as none of those compounds is listed as CYP450 enzyme inducer [27]. An alternative explanation for the fast peripheral immune reconstitution was an infection-related immunological trigger that overruled the declining impact of fingolimod on lymphocyte trapping. In fact, interferon-γ secreted from activated CD4+ T cells was shown to reduce the expression of CCR7 ligand CCL21 in lymph nodes of mice eight days post infection with viral or bacterial pathogens [28]. In the settings of such a Th1-mediated antiviral immune response declining fingolimod serum concentrations might be overruled and the balance between retention and exit signals allowing recirculation of CCR7+ lymphocytes disrupted. If so, this might have relevance concerning treatment decisions about continuing or pausing fingolimod in case of VZV infection. A rapid lymphocyte reconstitution of two to three weeks was reported by Gross and coworkers in a patient with cranial VZV polyneuritis during fingolimod treatment [8]. Follow-up MRI of their patient revealed several new lesions and a relapse after three and five months respectively after discontinuation of fingolimod, which they attributed to an immune reconstitution inflammatory syndrome (IRIS) due to the rapid restoration of immune surveillance. Similarly, follow-up MRI of our patient revealed several new brain lesions four months after cessation of fingolimod giving rise to speculations about MS-related disease versus IRIS. Another question is as to which extent peripheral immune reconstitution was important for containment of VZV reactivation. In contrast to the peripheral blood CCR7+ T cells were lacking in the CSF. Again, we do not know if any sustained other effect of fingolimod than S1P receptor modulation had interfered with reappearance of CCR7+ T cells in the CSF compartment, or if effector and memory effector T cells were the sole executers of the anti-VZV defense. Either way, control over the viral infection was established as evidenced by the clinical improvement.
VZV-induced disease is found most frequently within six months of initiation of fingolimod and does not increase as a function of time [11]. VZV reactivation in our case occurred eight weeks after initiation of fingolimod treatment. In this context, it needs to be clarified whether previous treatment with natalizumab might have played a role. We consider this unlikely, as the CD4+/CD8+ ratio in the CSF was almost 2.5 under fingolimod and thus even higher than the mean rates reported in a previous study [29]. Alternatively, latency of a highly neuroinvasive VZV strain could have been the cause of reactivation following fingolimod treatment. Indeed, recent studies implicate differences in viral neurotropism and polyclonal immune responses against VZV related to different antigens, as well as genetic susceptibility for VZV reactivation [30][31][32]. Treatment-associated increases of T regulatory cell (Treg) numbers [33,34], enhanced functional activity of Tregs [35], and reduced numbers particularly of VZV-specific T cells in combination of steroid treatment [36] are further factors potentially implicated in facilitating VZV reactivation in fingolimod-treated patients.
Reasons why we were not able to detect VZV DNA in the CSF of our patient are likely related to the short duration of clinical symptoms, the initiation of high-dose valacyclovir one day prior to the spinal tap, and a sensitivity of VZV PCR of only 80% in immunosuppressed individuals [37]. In regard to the minor extent of CSF pleocytosis, it needs to be taken into account that fingolimod efficiently sequesters immune cells from the CNS, and normalizes cell counts in CSF [29]. Fingolimod has limited impact on intrathecal IgG synthesis or antigen-specific adaptive immune responses [22,29,34]. Lack of intrathecal but rise of peripheral VZV-specific IgG at diagnosis thus may reflect an early stage of infection.
Results of our patient were compared at T0, T1, and T2 with those of four to five other fingolimod-treated MS patients (mean age 41 years; range 33-59) who had discontinued natalizumab treatment after mean 51 months (range 29-71) because of a positive JC virus (JCV) serostatus (controls). All patients participated in a local scientific study for monitoring immunological changes in MS (ethics committee of Salzburg 415-E/1612/2-2013) and gave written informed consent for publication of results.

Conclusions
Our findings show that VZV reactivation in fingolimod-treated patients is associated with a low level systemic VZV-specific antibody response and increased number of effector CD4+ T cells. The immune reconstitution upon fingolimod discontinuation was unexpectedly fast and may have been driven by infection-related triggers counteracting lymph node retention signals. If so, this is of relevance concerning treatment decisions for cessation of fingolimod in case of VZV infection. However, whether these findings from a single case can be generalized remains unclear. Further research into fingolimod-associated VZV reactivation and immune reconstitution dynamics are mandatory to prevent morbidity and mortality associated with this potentially life-threatening condition.