s2fieldsofstudy: Biology Medicine

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Potential Antitumor Effect of Harmine in the Treatment of Thyroid Cancer

Thyroid cancer is one of the most common types of cancer in endocrine system. In latest studies, harmine has been proved to inhibit the growth of several cancers in vitro and in vivo. In the current study, we evaluated the in vitro and in vivo anticancer efficiency of harmine against thyroid cancer cell line TPC-1. The in vitro cytotoxicity of harmine evaluated by XTT assay indicated that harmine suppressed the proliferation of TPC-1 cells in a dose- and time-dependent manner. Moreover, harmine dose-dependently induced apoptosis of TPC-1 cells through regulating the ratio of Bcl-2/Bax. The colony forming ability of TPC-1 cells was also time-dependently inhibited by harmine. The inhibitory effects of harmine on migration and invasion of TPC-1 cells were studied by wound scratching and Transwell assays. In vivo evaluation showed that harmine effectively inhibited the growth of thyroid cancer in a dose-dependent manner in nude mice. Therefore, harmine might be a promising herbal medicine in the therapy of thyroid cancer and further efforts are needed to explore this therapeutic strategy.

Introduction
Thyroid cancer is one of the most common kinds of cancer in endocrine system which includes four major types with papillary thyroid cancer (PTC) being the most common one [1]. Though most of PTCs respond well to surgery and chemoradiotherapy, part of them quickly develop resistance to chemotherapy, which generate an urgent demand of novel strategy to improve the efficacy of chemotherapeutics [2].
Recently a growing body of evidences demonstrated the promising antitumor efficiency of herbal medicine [3][4][5]. Several herbal medicines, such as harmine, resveratrol, and berbamine, have shown substantial tumor inhibitory effect against a series of cancers [6][7][8]. Among all these herbal medicines, harmine, originally isolated from the seeds of Peganum harmala, is a tricyclic compound belonging to the -carboline alkaloids [9]. It is reported that harmine possesses various pharmacological activities [10]. In latest studies, harmine has been proved to inhibit the growth of several cancers in vitro and in vivo [11,12]. For example, harmine induces the apoptosis of cancer cells by leading to DNA break as demonstrated in DNA fragmentation and tunnel study in melanoma cells [13]. Possible mechanisms include the block of homologous recombination, one of the major Double Strand Break (DSB) repair pathways and the inhibition of telomerase activity [9,12]. However, no related reports were found on the antitumor effect of harmine in thyroid cancer.
In the current study, we evaluated the in vitro and in vivo anticancer efficiency of harmine against thyroid cancer. The cell inhibition effect of harmine was measured by XTT assay. Apoptosis of thyroid cancer cell line TPC-1 was measured by DAPI staining. Related apoptotic protein expression was evaluated by western blotting. Influences of harmine on the migration and invasion of TPC-1 cells were also examined by wound scratching and Transwell assays. The in vivo antitumor effect was examined in a xenograft model of thyroid cancer.

Materials.
Harmine was purchased from Sigma Co., Ltd. (St. Louis, USA). Harmine was dissolved in DMSO to make a stock solution at a concentration of 10 mg/mL. Working solution was prepared by diluting the stock solution into medium with the final concentration of DMSO below 0.1%. Human papillary thyroid cancer cell line TPC-1 was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). TPC-1 cells were cultured in RPMI 1640 (w L-glutamate) completed by fetal bovine serum (10%) and penicillin/streptomycin (1%) at 37 ∘ C in a humidified atmosphere containing 5% CO 2 .
Male and female nude mice (nu/nu; 6-8 weeks old and weighing 18-22 g) were purchased from the Animal Center of Nanjing Medical University (Nanjing, China). The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

In Vitro
Cytotoxicity. TPC-1 cells were seeded in 96-well plates with a density of 10000 cells per well and allowed to grow 24 h before treatment. Cells were then incubated with escalated doses of harmine (2,4,8,16,32, and 64 g/mL) for 24, 36, and 48 h. The medium was replaced with 50 L XTT working solution and incubated for another 18 h. The optical density of each well was measured on a microplate reader at 560 nm (Bio-Rad, Hercules, USA).

DAPI Staining.
Cells were seeded on cover slips in a 24-well plate and allowed to grow 24 h. Cells were then exposed to three kinds of concentration of harmine (4, 8, and 16 g/mL) for 36 h. After being washed by PBS, cells were fixed by a mixture of cold methanol and acetone (1 : 1) for 5 min. 4 g/mL 4 ,6-diamidine-2 -phenylindole dihydrochloride (DAPI) was added to the well and allowed to incubate for 10 min at room temperature. Cells were imaged under a fluorescent microscope.

Western
Blot. TPC-1 cells were incubated with harmine (4, 8, and 16 g/mL) for 6 hrs. Cell total proteins were extracted and quantified according to the instruction of the kit. 50 g of proteins was loaded into each well during the electrophoresis. Protein expression was measured by western blot as reported previously. Primary antibodies (anti-Bax, anti-Bcl-2, and anti--actin) were purchased from Sigma Co., Ltd. (St. Louis, MO, USA).

Clonogenic
Assay. The first clonogenic assay was performed to study the inhibition of harmine on the colony forming ability of TPC-1 cells. Cells were treated with harmine (4, 8, and 16 g/mL) for 24, 36, and 48 h. Another clonogenic assay was performed to evaluate the radiosensitizing effect of harmine. TPC-1 cells were treated with 2 g/mL harmine and escalated doses of radiation singly or in combination. After each treatment, cells were trypsinized, collected, and set to the calculated concentration. After 10-14 days of incubation in 6-well plates, the 6-well plates were dyed by 0.5% crystal violet and counted as reported previously [14]. 2.6. DNA Fragmentation Assay. TPC-1 cells were seeded in a 24-well plate and allowed to grow for 24 h. Cells were then exposed to three kinds of concentration of harmine (4, 8, and 16 g/mL) for 36 h. Total DNA was extracted and run by gel electrophoresis as previously reported [15].

Wound Scratching Assay and Transwell
Assay. Cells were seeded in 6-well plate and allowed to grow for 24 h to form a monolayer. Then a tip was utilized to scratch in the middle to generate a clear space (wound). Immediately it is captured by the camera and was designated as 0 h. Then the medium was replaced with serum-free medium containing different concentrations of harmine (4, 8, and 16 g/mL). After 36 h, another image was captured to measure the width of the wound. The wound healing rate was calculated by the following formula: the average width of wound at 0 h − the average width of wound at 36 h/the average width of wound at 0 h.
Transwell assays were performed in 24-well Transwell chambers (Corning, Acton, MA, USA) as reported previously. Briefly, TPC-1 cells were seeded in the upper chamber in serum-free medium, while the bottom chamber was filled with complete medium as an attractant. 36 h later, the successfully invaded cells on the bottom chamber were stained by crystal violet, counted, and plotted.

In Vivo Antitumor
Efficacy. Briefly, 1.5 million TPC-1 cells were injected subcutaneously in the left axillary space of the mice. 10 days later the mice with the tumor nodule reaching a volume of 70-80 mm 3 were randomly divided into four groups with 6 mice per group. Escalated doses of harmine were administered by tail vein injection.
The mice were then treated with escalated doses of harmine with saline as the control. following formula: TV = ( 2 * )/2, where is the width of tumor nodule and is the length of tumor nodule.

Statistical Analysis.
Results were presented as mean ± SD. Statistical analysis was made by Student's -test or ANOVA. The value < 0.05 was considered as significant. Figure 1, escalated doses of harmine effectively led to the decrease of cell viability at different incubation times (Figure 1). The IC50 values of harmine against TPC-1 cells at 24, 36, and 48 h were 16.57 ± 1.4, 9.48 ± 1.1, and 5.51 ± 0.7 g/mL, respectively. It is noted that the IC50 values significantly lowered as the incubation time extended. Therefore, harmine inhibited the proliferation of TPC-1 cells in a time-and dose-dependent manner. As shown in Figure 2, it is obvious to locate the characterized morphology of apoptotic cells in harmine treated group with brighter DAPI staining with condensed chromatin forming crescent-shaped profiles around the periphery of the nucleus or separate globular structures (apoptotic bodies). Quantitative analysis indicated the significant differences among the three groups treated with elevated doses of harmine, which was in accordance with the results in cytotoxicity tests (Figure 3). Figure 4 indicated that harmine was effective to induce the fragmentation of DNA of TPC-1 cells in a dose-dependent manner, demonstrating the apoptosis inducing effect. to a significant decrease in the ratio of Bcl-2/Bax, which demonstrated the progression of apoptosis.

Effect of Harmine on Apoptotic Proteins and the Activity of
The activity of Caspase-3 in TPC-1 cells treated with a series of doses of harmine was evaluated by the caspase colorimetric protease assay kit. As one of the crucial mediators of apoptosis, Caspase-3 is considered as an apoptotic marker due to its significant role in catalyzing the specific cleavage of key proteins and generation of apoptotic bodies. Figure 6 showed that harmine was effective in elevating the activity of Caspase-3, which was in accordance with the results of apoptosis detection from Figures 2 and 3.   the antiproliferative effect of harmine was consistent with the results from cytotoxicity test. Figure 7(b) indicated that 2 g/mL harmine effectively enhanced the inhibitory effect of radiation on the colony forming ability of TPC-1 cells. There was a significant difference between the percent survival of radiation alone and radiation plus harmine, which demonstrated the potential radiosensitizing effect of harmine. Figure 8 indicated the effect of harmine on the wound healing rate of TPC-1 cells. Harmine dosedependently decreased the wound healing rate of TPC-1 cells, which demonstrated an inhibitory effect of harmine on the migration of thyroid cancer cells. Similarly, harmine inhibited the invasion of TPC-1 cells in a dose-dependent manner as shown in Figure 9. Statistical analysis indicated significant differences among the groups treated with different doses of harmine. Similarly, the number of invading cells decreased significantly as the concentration of harmine increased. Results from wound scratch and Transwell assays demonstrate that harmine is effective in restraining the mobility of TPC-1 cells in a dose-dependent manner, which makes it a potential drug for the reverse of tumor metastasis.

In Vivo Antitumor Evaluation of Harmine against TPC-1 Xenograft.
In vivo antitumor effects of harmine were examined in a xenograft model of TPC-1 cells in nude mice ( Figure 10). It is obvious that harmine delayed the growth of thyroid cancer in a dose-dependent manner. However, though the tumor of the mice receiving the lowest dose of harmine grew more slowly than that of the mice in the control group, there was no significant difference between the two groups. On the contrary, the other two relatively high doses of harmine significantly inhibit the growth of tumor when compared with the control group. Among all the three groups, the highest dose of harmine generated the greatest antitumor effect. Moreover, 40 mg/kg harmine restrained the tumor growth more significantly than 20 mg/kg harmine. In Drug concentration ( g/mL) Figure 8: Wound healing ability of TPC-1 cells exposed to a series of doses of harmine. Quantification of cell migration using the monolayer wound scratching assay. * represents < 0.05. * * represents < 0.01. addition, there was no obvious toxicity (such as weight loss or behavior) in all the mice during the whole experiment.

Discussion
Here we show the cytotoxic and antiproliferative effect of harmine on the in vitro and in vivo model of thyroid cancer. As reported in previous studies, harmine showed cytotoxic and antiproliferative effect against several kinds of cancers [11,16]. Several intracellular targets have been identified such as cyclin proteins and interference with DNA [17]. However, no report was about the antitumor effect of harmine in thyroid cancer.
In the current study, we demonstrated a time-and dosedependent cytotoxicity of harmine against thyroid cancer cell * * * * * * Drug concentration ( g/mL) Figure 9: Cell invasive ability of TPC-1 cells exposed to harmine. Quantification of the numbers of invaded cells exposed to a series of doses of harmine. Each data point represents the mean ± SD from three independent experiments. * * represents < 0.01.
line TPC-1. Both XTT and clonogenic assay showed that the antiproliferative effect of harmine was mainly dependent on the concentration and incubation time of harmine. Several studies have reported the dose-dependent cytotoxicity of harmine against other kinds of cancers. For example, Zhang et al. reported the time-and dose-dependent antiproliferative effect of harmine against two kinds of gastric cancer cells [11]. A recent study from South China also demonstrated the inhibitory effect of harmine derivative on the proliferation of gastric cancer cells [7].
Though most of the studies focused on the antiproliferative effect of harmine, few researchers evaluated the influence of harmine on the colony forming ability of cancer cells as well as the radiosensitizing effect. As introduced previously, loss of reproductive integrity and the inability to proliferate indefinitely is a key feature to cell death [14,18]. Cells with integrity but unable to divide and produce a large number of colony are considered dead [19]. Therefore, cells with both the integrity and the ability to proliferate are referred to as "clonogenic," which is pivotal to the proliferation of cancer cells and regarded as a marker of cancer malignancy [20]. Here we showed that harmine exerted a time-and dosedependent inhibition on the clonogenic ability of TPC-1 cells, which means that harmine could effectively disrupt the reproductive integrity and lead to the inability of proliferation of thyroid cancer cells. Moreover, the current study reported for the first time that harmine showed the potential to be a promising radiosensitizer.
The following DAPI staining of cancer cells indicated that harmine was efficient to induce apoptosis of TPC-1 cells. It is known that apoptosis, also called programmed cell death, is characterized by the specific morphology, such as chromatin condensation, membrane blebbing, internucleosome degradation of DNA, and apoptotic body formation [21]. It is obvious that cells treated with harmine underwent some characteristic phenomenon of apoptosis. It is reported in earlier studies that the Bcl-2 family, including proapoptotic proteins and antiapoptotic proteins, plays an important role in apoptosis [22,23]. Among all these proteins, the ratio of Bcl-2 and Bax is a key indicator of apoptosis [24]. Figure 4 showed that the treatment by harmine dose-dependently decreased the expression of Bcl-2 while simultaneously increased the expression of Bax, which led to an obvious decline in the ratio of Bcl-2 and Bax. Though the effect of apoptotic induction by harmine was also reported by other groups, different mechanisms underlying the antitumor effect of harmine have been identified such as cell cycle arrest and telomerase activity inhibition [9]. The anticancer effect of harmine was confirmed in TPC-1 xenograft model, which, together with other studies focusing on harmine, proved the efficacy of harmine in the treatment of cancer.
The ability of migration and invasion is the characteristic of malignancy, which often leads to the failure of cancer therapy [25]. The efficient inhibition of tumor migration and invasion is an effective way to control the metastasis of tumor. Since few studies examined the antimetastatic effect of harmine, we evaluate the ability of harmine on cell migration and invasion through wound scratching and Transwell assays. Results from the two experiments demonstrated the dosedependent inhibitory effect of harmine in vitro. Moreover, animal experiment on metastatic xenograft models in the author's lab is ongoing to further evaluate the in vivo antimetastasis efficacy of harmine.
As mentioned above, evidences from our report not only demonstrate the potential antitumor effect of harmine, but also provide a feasible way to counteract the metastasis of thyroid cancer. Moreover, further studies focusing on the chemosensitization effect of harmine are ongoing in the author's lab to explore more potential application of such alkaloids. It is undoubtedly, however, that the development of herbal medicine, especially alkaloids, warrants more intensive research in order to evaluate the feasibility and advantages of clinical applications.
In summary, the current study demonstrates the antitumor effect of harmine in the treatment of thyroid cancer. In vitro cytotoxicity tests indicate that harmine possesses a doseand time-dependent cell inhibitory and apoptosis inducing effect on TPC-1 cells. In addition, harmine effectively inhibits the migration and invasion of TPC-1 cells in a dosedependent manner. Moreover, in vivo evaluation shows that harmine significantly delays the growth of thyroid cancer in a dose-dependent manner in nude mice. Therefore, harmine might be a promising herbal medicine in tumor therapy and further efforts are needed to explore this therapeutic strategy.


FGF2 Attenuates Neural Cell Death via Suppressing Autophagy after Rat Mild Traumatic Brain Injury

Traumatic brain injury (TBI) can lead to physical and cognitive deficits, which are caused by the secondary injury process. Effective pharmacotherapies for TBI patients are still lacking. Fibroblast growth factor-2 (FGF2) is an important neurotrophic factor that can stimulate neurogenesis and angiogenesis and has been shown to have neuroprotective effects after brain insults. Previous studies indicated that FGF2's neuroprotective effects might be related to its function of regulating autophagy. The present study investigated FGF2's beneficial effects in the early stage of rat mild TBI and the underlying mechanisms. One hundred and forty-four rats were used for creating controlled cortical impact (CCI) models to simulate the pathological damage after TBI. Our results indicated that pretreatment of FGF2 played a neuroprotective role in the early stage of rat mild TBI through alleviating brain edema, reducing neurological deficits, preventing tissue loss, and increasing the number of surviving neurons in injured cortex and the ipsilateral hippocampus. FGF2 could also protect cells from various forms of death such as apoptosis or necrosis through inhibition of autophagy. Finally, autophagy activator rapamycin could abolish the protective effects of FGF2. This study extended our understanding of FGF2's neuroprotective effects and shed lights on the pharmacological therapy after TBI.

Introduction
Traumatic brain injury (TBI), the leading cause of death and disability nowadays, is a major health problem all over the world [1,2]. Even mild TBI can cause delayed physical and cognitive deficits [3]. Although lots of randomized controlled trials (RCTs) were done in recent years, no intervention had shown to be beneficial [4][5][6]. Thus, it is imperative to further elucidate the complicating pathophysiological mechanisms of TBI and develop effective pharmacological intervention targets. It is generally acknowledged that TBI has two injury phases-primary injury and secondary injury. The primary injury is directly caused by trauma itself, while the secondary injury is more complex with a series of pathological responses, including blood-brain barrier disruption, oxidative stress, neuroinflammation, autophagy, apoptosis, and necrotic cell death. These processes in the secondary injury are directly related with long-term neurological deficits and also provide us multiple therapeutic targets in TBI management.
Autophagy is a lysosomal degradation pathway that protects organisms against diverse pathologies [7]. In most circumstances, autophagy could promote cell survival by maintaining cellular homeostasis, but there are a number of studies that demonstrated that autophagy could also trigger cell death in certain pathological situations [8][9][10]. Researchers had already observed autophagy's existence in TBI model several years ago, and modulation of this process could result in neurological improvements [11][12][13]. However, up until recent years, whether inducing or inhibiting autophagy can result in neuroprotection remains controversial [14][15][16]. The relationship between autophagy and other forms of cell death such as apoptosis after TBI is worth studying.
Fibroblastic growth factors (FGFs) are small polypeptide growth factors which play a pivotal role in morphogenesis [17]. Fibroblast growth factor-2 (FGF2), also known as basic fibroblast growth factor (bFGF), is an important member of this family. FGF2 is highly expressed in the central nervous system and exhibits early decline during the course of aging [18]. Previous studies showed that it had a variety of neuroprotective effects, including supporting neural stem and progenitor cell proliferation in vitro and in vivo [19,20], maintaining vascular integrity and angiogenesis [21,22], and helping cognitive recovery [20]. Studies have demonstrated the neuroprotective roles of FGF2 in various pathological conditions in the central nervous system, such as TBI, spinal cord injury (SCI), ischemic brain injury, subarachnoid hemorrhage (SAH), and neurodegenerative diseases [21,[23][24][25][26]. Most of the previous studies focused on neurogenesis effects of FGF2, but several recent studies have discovered FGF2's novel effect as an autophagy inhibitor, in which the activation of PI3K/Akt/mTOR signaling pathway may take an important part [27,28]. As the molecular mechanisms of FGF2 in treatment of TBI has not been fully understood, we hypothesized that FGF2 could also inhibit autophagy and attenuate cell death in TBI treatment.
In the present study, we found that FGF2 could act as a neuroprotective agent after rat mild TBI, alleviating brain edema, reducing cerebral lesion volume, and promoting functional recovery. Meanwhile, FGF2 also inhibited autophagy and decreased neural apoptosis and necrotic cell death. Furthermore, autophagy activator rapamycin could abolish the protective effects of FGF2. These results provide us a new perspective about FGF2's neuroprotective role after TBI.

Animals and Study
Design. Adult male Sprague-Dawley rats (250-300 g) obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China) were used in this study. The animals were maintained under controlled temperature and humidity conditions at a 12 hr light/dark cycle place. All procedures involving animals were strictly conformed to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.

CCI Model.
The procedure details used for creating CCI model were described previously [29]. Briefly, after the rats were anesthetized with pentobarbital (40 mg/kg) through intraperitoneal injection, the head of them was fixed on a stereotaxic frame. A 5 × 5 mm craniotomy was performed approximately midway between the bregma and lambda on the right frontoparietal cortex after a midline incision. The bone flap was then removed carefully without disturbing the underlying dura. Then the CCI was performed using a PinPoint™ Precision Cortical Impactor (Cary, NC, USA) perpendicular to the brain surface (12°from the vertical). The impact tip was 4 mm in diameter, with an impact velocity of 3 m/s, a duration time of 120 ms, and a deformation depth of 2 mm below the dura, to mimic a mild focal TBI in rats. The bone flap was put back and sealed immediately, and the wound was sutured. Rats in the sham group received the same surgical procedure only without CCI. During the surgery, body temperature was monitored and maintained at 37.0 ± 0.5°C with a rectal probe coupled to a heating pad. For arterial pH, pO 2 , pCO 2 , and blood glucose level monitoring catheterize the right femoral artery before the procedure. The rats were returned to their home cages after completely recovered from anesthesia in a heated chamber.

Experimental Design
To explore the effects of FGF2 in rat TBI models, ninety rats were randomly assigned into three groups: the sham group (n = 30), TBI + vehicle group (n = 30), and TBI + FGF2 group (n = 30). A dose of 250 μg/kg recombinant human FGF2 (PeproTech Inc., Rocky Hill, NJ, USA) was administrated intranasally 1 h before TBI induction in the TBI + FGF2 group. Intranasal administration could deliver FGF2 directly to the brain through nasal epithelium [30,31]. The dosage and time point of FGF2 pretreatment were based on a previous study [22]. The sham group and TBI + vehicle group received the same volume of sterile saline intranasally at the same time point before TBI induction. The endpoints of the experiments were 6 h (PI staining) and 48 h (other experiments) after TBI induction, based on a previous study [32]. Six rats in each group were used for brain water content assessment, lesion volume assessment, Western blot, immunofluorescence staining, and PI staining.

Experiment 2.
To explore whether autophagy mediates FGF2 effects, fifty-four rats were randomly assigned into three groups: the TBI + vehicle group (n = 18), TBI + FGF2 group (n = 18), and TBI + FGF2 + Rap group (n = 18). The dosage and time point of FGF2 pretreatment were the same as in experiment 1. A dosage of 2 mg/kg rapamycin (Selleck Chemicals, Houston, TX, dissolved in 2% DMSO) was administrated intraperitoneally 30 min after TBI induction in the TBI + FGF2 + Rap group based on a previous study [33]. The TBI + vehicle group received same volume of sterile saline intranasally 1 h before TBI induction. The TBI + vehicle group and TBI + FGF2 group received the same volume of 2% DMSO intraperitoneally 30 min after TBI induction. Six rats in each group were used for Western blot, immunofluorescence staining, and PI staining. The endpoints of the experiments were the same as in experiment 1.

Evaluation of Neurological Deficits.
To examine the effects of FGF2 on the neurological deficits of animals after TBI, the modified neurological severity scores (mNSS) were used. The neurological scores of the animals in each group were evaluated by an independent observer at 48 h after TBI induction using motor, sensory, reflex, and balance tests. A total score ranging from 0 to 18 was calculated by adding the scores together, and a higher score indicated a worse neurological function [34]. Most mNSS of the rats were lower than 6, which indicated a mild injury after TBI. Foot fault test was examined in experiment 2 to evaluate the cognitive functional deficits by an independent observer at 48 h after TBI induction. The rats were put on an elevated grid and allowed to move freely for 5 minutes or until 50 steps were taken with the left forelimb. The total number of steps and fell time of the left forelimb were counted, and the percentage of left forelimb foot faults was calculated.
2.5. Brain Water Content Measurement. Rats were sacrificed under deep anesthesia after 48 h of TBI. The brains were removed, and the right hemispheres were collected immediately. After weighing for the wet weight, the samples were then dried at 105°C for 24 h to obtain the dry weight. The brain water content was calculated using the following formula: wet weight − dry weight /wet weight × 100% 2.6. Hematoxylin and Eosin (HE) Staining and Lesion Volume Assessment. At 48 h after TBI, the rats were sacrificed and perfused intracardially with 0.1 mmol PBS (pH, 7.4) and 4% paraformaldehyde (pH, 7.4). The brains were then removed and immersed in the same perfusate at 4°C for 72 h. Coronal sections started from one millimeter which was anterior to the lesion margin, to the posterior lesion margin, were obtained and then paraffin embedded. The lesion volumes were calculated by integrating 10 sections (3 μm thick) with 300 μm intervals. After HE-staining with standard methods (0.5% hematoxylin, 25°C, 5 min and 0.5% eosin, 25°C, 1 min), the brain sections were imaged using a stereomicroscope (Olympus BX51, Olympus, Tokyo, Japan) in a brightfield illumination and measured using Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). Lesion volume was calculated as follows: Σ A n + A n+1 × d/2, where A is the ventricular area and d is the distance between sections, according to a previously published work [35].

Western Blot.
Western blotting was performed as previously described [36]. Briefly, the cortical regions of the brains were collected and homogenized. The samples were centrifuged at 1000g for 10 min at 4°C. The supernatant was further centrifuged, and the protein content was measured using a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA). Equal quantities of protein (40 μg) from each samples were resuspended in loading buffer, denatured at 95°C for 5 min, and loaded into the walls of the sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred onto polyvinylidene fluoride membranes. The membranes were blocked with nonfat dry milk buffer for 2 h and subsequently incubated overnight at 4°C with the following primary antibodies: LC3 (1 : 1000, Cell Signaling Technology CST#4108), Beclin-1 (1 : 1000, Abcam ab62557), p62 (1 : 1000, Abcam ab56416), and β-actin (1 : 2000, Santa Cruz SC-47778). The membranes were processed using the appropriate secondary antibodies (1 : 5000) for 1 h at room temperature. The protein band densities were detected using X-ray film, and the densitometric signals were quantified using ImageJ software (NIH, Bethesda, MD, USA).

TUNEL and Immunofluorescence
Staining. The rats were sacrificed at 48 h after TBI induction in deep anesthesia and perfused intracardially with 0.1 mmol PBS (pH, 7.4) and 4% paraformaldehyde (pH, 7.4). The brains were then removed and immersed in the same perfusate at 4°C for 72 h and then dehydrated with 30% sucrose solution until they sank to the bottom (about 2 days). Coronal sections (7 μm) were collected from the right hemisphere. The brain sections were incubated with 10% normal goat serum and 0.3% Triton X-100 to prevent nonspecific binding for 1 h at room temperature. Then the sections were incubated at 4°C overnight with the primary antibodies: NeuN (1 : 250, Millipore MAB377) or LC3 (1 : 200, Cell Signaling Technology CST#4108). After washing with 0.01 mmol PBS several times, the sections were incubated with the secondary antibodies for 2 h at 4°C in the dark. A TUNEL staining kit (In Situ Cell Death Detection Kit, Fluorescein, Roche, Switzerland) was used to analyze apoptotic cell death. The brain sections were incubated with TUNEL reaction mixture (90 min) at 37°C. After washing with PBS again, the sections were mounted onto slides with Fluoroshield™ with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Immunostaining was observed using a fluorescent microscope (Olympus, Tokyo, Japan). The total number of NeuN-positive, LC3-positive, or TUNEL-positive cells were counted in three different slices per animal around the lesion area or in the CA1 and CA3 regions of the ipsilateral hippocampus by an independent observer.
2.9. Propidium Iodide (PI) Labeling. At 5 h after TBI induction, PI (10 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was diluted in normal saline and injected to rats intraperitoneally at a dose of 30 mg/kg. After 1 h, the rats were sacrificed and perfused intracardially with 0.1 mmol PBS (pH, 7.4) and 4% paraformaldehyde (pH, 7.4). Then the brains were removed and immersed in 4% paraformaldehyde for 72 h. After dehydrated with 30% sucrose solution, coronal sections (7 μm) were collected from the right hemisphere and PIpositive cells were quantitated around the lesion area or in the CA1 region from 200x cortical fields in three brain sections per rats. The counting task was also done by an independent observer.

Statistical Analysis.
To facilitate comparisons between three groups, the Western blotting results were expressed as relative density of the band as compared β-actin and then normalized to the mean value of the sham group. The data were expressed as mean ± SD. SPSS Statistics (version 22.0) was used for statistical analysis. All the data were performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Statistical significance was inferred at P < 0 05.

FGF2 Alleviates Neurological Deficits and Brain Edema.
The representative pictures of the brain samples in each group are presented in Figure 1(a). Significant neurological function deficits were observed 48 h after TBI induction (P < 0 05, Figure 1(b)). The mNSS were significantly lower in the TBI + FGF2 group than in the TBI + vehicle group (P < 0 05, Figure 1(b)). The brain water content of the injured hemisphere was also significantly increased at 48 h post-TBI, pretreatment with FGF2 could attenuate this increase (P < 0 05, Figure 1(c)).

FGF2 Prevents the Loss of the Brain Tissue and Promotes
Neuronal Survival after TBI. The cerebral lesion volume and the neuronal survival were assessed at 48 h post-TBI. The representative coronal sections in each group are shown in Figure 2(a). The lesion volumes of the TBI + FGF2 group were significantly smaller than those of the TBI + vehicle group at 48 h (P < 0 05, Figure 2(b)). The numbers of NeuN-positive cells in the ipsilateral cortex and the CA1 and CA3 regions of the ipsilateral hippocampus were significantly decreased 48 h after TBI, and this process could be significantly reversed when pretreated with FGF2 (P < 0 05, Figures 2(c) and 2(d)).

FGF2 Prevents the Increase of LC3-Positive Cell
Numbers after TBI. Autophagy takes an important part in brain injury after TBI, but the role of FGF2 in regulating autophagy after TBI has not been studied. As LC3 is an ideal biomarker for autophagy activation, we chose LC3 to mark the autophagic cells. The immunofluorescence staining showed that LC3-positive cells could be hardly observed in the cortex of the control group but significantly increased 48 h after TBI induction (P < 0 05, Figures 3(a) and 3(b)). In the TBI + FGF2 group, the number of LC3-positive cells was less than that in the TBI + vehicle group, which was statistically significance (P < 0 05, Figures 3(a) and 3(b)).

FGF2
Regulates the Autophagy-Related Protein Levels. To confirm the inhibition effects of FGF2 on autophagy post-TBI, we further measured the protein levels of Beclin-1 and p62 and the ratio of LC3II/I in each group. As shown in Figure 4, the protein levels of Beclin-1 was significantly upregulated in the injured cerebral cortex 48 h after TBI (P < 0 05, Figures 4(a) and 4(b)), and FGF2 pretreatmet significantly decreased the Beclin-1 level compared with the TBI + vehicle group (P < 0 05, Figures 4(a) and 4(b)). TBI significantly decreased the protein level of p62 at 48 h (P < 0 05, Figures 4(a) and 4(c)), while FGF2 could significantly prevent this process (P < 0 05, Figures 4(a) and 4(c)). FGF2 pretreatment also significantly upregulated the ratio of LC3II/I compared with the TBI + vehicle group (P < 0 05, Figures 4(a) and 4(d)). These results indicated that FGF2 pretreatment could inhibit the level of autophagy at 48 h after TBI.
3.6. FGF2 Prevents Cell Apoptosis. TUNEL staining was used to detect the apoptotic cell death in the injured cortex and CA1 region of the ipsilateral hippocampus. In the sham group, TUNEL-positive cells barely exist ( Figure 5(a)). TBI significantly increased the apoptotic index compared with the sham group, while pretreatment of FGF2 could significantly prevent the increase of the apoptotic index compared with the TBI + vehicle group (P < 0 05, Figures 5(a)  and 5(b)).

FGF2 Decreased the Number of Necrotic Cells after TBI.
To evaluate the effect of FGF2 in protecting cells from injury, PI was used to identify the plasma membrane disrupted cells. As plasma membrane disruption is one of the key hallmarks of necrotic cell death, PI-positive cells could represent necrotic cell death. The number of PIpositive cells was significantly increased in the injured cortex and CA1 region of the ipsilateral hippocampus in the TBI + vehicle group 6 h after TBI (P < 0 05, Figures 6(a) and 6(b)). Pretreatment with FGF2 could significantly prevent the increase of the percentage of PI-positive cells (P < 0 05, Figures 6(a) and 6(b)).

Rapamycin
Abolished the Neuroprotective Effects of FGF2. To explore whether autophagy mediates FGF2 effects, rapamycin was used to counteract the autophagy inhibition caused by FGF2. The mNSS and foot faults were significantly lower in the TBI + FGF2 group than those in the TBI + vehicle group (P < 0 05, Figures 7(a) and 7(b)). The increase of the neurological function with FGF2 management was significantly suppressed by rapamycin (P < 0 05, Figures 7(a)

Discussion
In the present study, we explored the neuroprotective role of FGF2 in rat mild TBI model and studied the potential mechanisms. We found that pretreatment with FGF2 had neuroprotective effect in rat mild TBI by alleviating neurological deficits, decreasing brain water content and reducing lesion volume. FGF2 also had the ability to increase the number of surviving neurons at the site of injury. Furthermore, we  The quantification of brain water content of the right hemisphere. The bars represent the mean ± SD. n = 6. * P < 0 05 versus sham, # P < 0 05 versus TBI + vehicle.   As previously mentioned, the long-term consequence of TBI was dominated by the secondary injury, so the key to successful treatment had to focus on how to alleviate secondary damage after TBI [37]. Brain edema and the increased intracranial pressure in the early stage of TBI are the main causes of mortality, even a minor increase of brain water content can lead to a significant increase of intracranial pressure and the poor outcome [38]. Thus, alleviate brain edema is a promising pharmacological therapeutic direction in TBI management.
The concentration of FGF2 significantly increases in the interstitial space in the first week after TBI [39]. FGF2 binds to the fibroblast growth factor receptor 1 (FGFR1) which is located on the cell membrane of neurons [40] and stimulates the neural stem/progenitor cell proliferation and differentiation [20]. However, the fact is that for TBI patients, spontaneous improvement of neurologic function is limited and almost all of them will keep a stable condition after 12 months. Exogenous FGF2 had been used as a pharmacological intervention method in experimental TBI since many years ago [41]. However, most of the previous studies focused on its delayed effect of stimulating neurogenesis [20,21], and to our knowledge, there is only one research about FGF2's effects in the early stage of TBI that is focused on its effect of alleviating blood-brain barrier disruption through activation of PI3K/Akt/Rac1 pathway [22]. Although disruption of blood-brain barrier permeability is the main cause of vasogenic edema [37], no direct evidence about FGF2's effect in reducing brain water content after TBI could be found in the literature. So, in this study, we tested FGF2's neuroprotective effects in the first 48 h after TBI and found that it could significantly alleviate brain water content of the injured hemisphere and promote mNSS of the injured rats. These results might be due to the alleviation of blood brain barrier disruption and were consistent with previous studies about FGF2's neuroprotective effects after intracerebral hemorrhage and ischemic brain injury [24,42]. We also found that FGF2 could prevent tissue loss and increase the number of surviving neurons in injured cortex, CA1, and CA3 regions of the ipsilateral hippocampus in the early stage of TBI. These data indicated that FGF2 could assist in preventing neuronal cell death after TBI. Several years ago, researchers had discovered that FGFs could regulate autophagy in cardiac stem cell differentiation and mouse embryonic fibroblasts [43,44]. Recently, Wang et al. discovered that FGF2 could regulate autophagy and ubiquitinated protein accumulation in myocardial ischemia mice and spinal cord injury rats [27,28]. In this study, we further explored the relationship between autophagy and the neuroprotective function of FGF2 in the early stage of TBI. The immunofluorescence study showed that the immunoreactivity of LC3 was downregulated by FGF2, suggesting that FGF2 pretreatment could suppress autophagic cell death. LC3 I and LC3 II are the two forms of LC3, and the ratio of LC3 II to LC3 I is generally used as a biomarker of autophagy [45]. Western blot showed that FGF2 pretreatment can prevent this ratio from increasing. Beclin-1, which played an important role in autophagic cell death, was also downregulated by FGF2. p62 links ubiquitinated protein bodies to LC3 and mediates the autophagic protein degradation, and its accumulation inversely related with autophagic activity [46]. In this study, p62 level was found to be significantly decreased after TBI, while FGF2 could reverse this change. These results suggested that FGF2 could act as an autophagy inhibitor in TBI management.
Autophagy takes an important part in many neurological conditions, including the secondary damage after TBI. Appropriate autophagy can eliminate the aberrant cell components and protein aggregates, so as to maintain cellular homeostasis [14]. In some situations such as subarachnoid hemorrhage, autophagy can play a neuroprotective role and activation of autophagy may protect cortical cells against apoptosis [47]. In TBI, however, whether inducing or inhibiting autophagy can result in neuroprotection is still controversial. On the one hand, the autophagy activator rapamycin or melatonin can protect the brain from TBIinduced damage [13,48], but on the other hand, the autophagy inhibitor 3-MA or resatorvid could also have similar neuroprotective effects [15,16]. In the present study, FGF2 could inhibit autophagy and at the same time promoting neurological function, alleviating brain edema, reducing lesion volume, and promoting neuronal survival. This result tallied with previous studies about the autophagy inhibitor 3-MA's neuroprotective effects after TBI [15]. Although the autophagy activator rapamycin may exert a neuroprotective effect after TBI, the results in our experiment showed that coadministration of FGF2 and rapamycin cannot exert a synergistic effect. On the contrary, rapamycin could reverse the benefit effects of FGF2. This result may be related with the dose of rapamycin, and worth further study. Apoptosis and necrosis are the two major ways of cell death after TBI, and both of them have been linked to autophagy, playing either a prosurvival or prodeath role [49]. Autophagy and apoptosis are the two main types of programmed cell death, and there are closed relations between autophagy and apoptosis. Beclin-1, the essential mediator of autophagy tested in this study, could also be inhibited by antiapoptotic proteins Bcl-2 or Bcl-xl [50,51]. In addition, autophagy-related gene 5 (Atg5) could increase the susceptibility to apoptotic stimuli [52], and caspase-mediated cleavage of Atg5 and Beclin-1 could switch autophagy into apoptosis [49]. In our study, pretreatment with FGF2 could decrease the number of apoptotic cells and plasma membrane-disrupted cells in injured cortex. These results The quantification of the PI-positive cells as the necrosis index as a percent of the total DAPI+ cells. The bars represent the mean ± SD. n = 6. * P < 0 05 versus sham and # P < 0 05 versus TBI + vehicle.
indicate that FGF2 could alleviate autophagy, apoptosis, and necrosis thus protect cells from various forms of death, at least in rat mild TBI models. Further studies are needed to explore the crosstalk among autophagy, apoptosis, and necrosis after TBI.
In this study, we explored the neuroprotective effects of FGF2 in the early stage of rat mild TBI and discussed the potential mechanisms. Nevertheless, there are several limitations. First, FGF2 is not a specific inhibitor of autophagy, actually it might play neuroprotective roles through various ways such as alleviating blood-brain barrier disruption, as mentioned previously. However, at least, the inhibition of autophagy might take part in FGF2's antiapoptotic and antinecrotic effects. Second, FGF2 was administrated 1 h before TBI, which limited the translational relevance. Whether FGF2 could exert the same effects when delivered after TBI needs further study. Finally, we only investigated the role of FGF2 on autophagy in the first 48 h after TBI, whether FGF2's effect of inhibiting autophagy is associated with its main function of stimulating neural stem/ progenitor cell proliferation and differentiation still requires further research.
In summary, the results of this study indicated that FGF2 plays a neuroprotective role in the first 48 h of rat mild TBI The relative band densities of Beclin-1 and p62. The densities of the protein bands were analyzed and normalized to β-actin. The data are expressed as a percentage of the sham group. The bars represent the mean ± SD. n = 6. * P < 0 05 versus TBI + vehicle, # P < 0 05 versus TBI + FGF2. (f) The band density ratio of LC3 II to LC3 I. The bars represent the mean ± SD. n = 6. * P < 0 05 versus TBI + vehicle and # P < 0 05 versus TBI + FGF2. through alleviating brain edema and neurological deficits. Pretreatment with FGF2 could also protect cells from various forms of death such as apoptosis or necrosis through inhibition of autophagy. This study extended our understanding of FGF2's neuroprotective effects after TBI. As FGF2 can both attenuate cell death in the early stage after TBI and stimulate neural regeneration and functional recovery later, it may be a promising pharmacological intervention in TBI management.

Conflicts of Interest
The authors declare no conflict of interest.

Acknowledgments
This study was supported by the Natural Science Foundation of Ningbo City (2015A610192) and Science and Technology Plan Projects of Cixi City (CN2015022).  The bars represent the mean ± SD. n = 6. * P < 0 05 versus TBI + vehicle and # P < 0 05 versus TBI + FGF2.


extfieldsofstudy: Biology Medicine

text:
The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery

Legionella pneumophila infects human alveolar macrophages and is responsible for Legionnaire’s disease, a severe form of pneumonia. L. pneumophila encodes more than 300 putative effectors, which are translocated into the host cell via the Dot/Icm type IV secretion system. These effectors highjack the host’s cellular processes to allow bacterial intracellular growth and replication. Here we adopted a multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown function. The crystal structure of the N-terminal domain at 1.7 Å resolution comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The C-terminal domain (residues 365–524) is sufficient to pilot the effector to acidified LAMP1-positive lysosomal compartments, where WipB interacts with the v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing and the control of energy metabolism during Legionella infection.

Supplementary Figure 1.
Structure-based sequence alignment of the WipB 25-344 with WipA (PDB entry code 5N6X), PP2B (PDB entry code 5C1V), PP2A (PDB entry code 2IAE) and PP1 (PDB entry code 5INB). Similar structures search was performed using the Dali server 1 , selected top-scores aligned with PDBeFold 2 and the resulting alignment was rendered with ESPript 3 . The WipB secondary structure elements are shown above the aligned sequences. Conserved phosphatase motifs are labeled DXH, GDxxDR, and NHE, and color-coded orange, green and blue, respectively.

P S H R L T A K E V F D N D G K P R V D I L K A H L M K E G R L E E S V A L R I I T E G A S I L R Q E K N L L D I D
. . . . . . . . . L N I D S I I Q R L L E V R G S K P G K N V Q L Q E N E I R G L C L K S R E I F L S Q P I L L E L E A 3 3 4 3 5 3 6 3 7 3       and Asp331 (396 in WipB) are involved in catalysis while Arg369 of WipA was hypothesized to be involved in pTyr recognition. In WipB, the equivalent residue of Arg369 is Arg310, which is conformationally restrained away from the active site and therefore cannot assume a role in substrate recognition. We hypothesize that this is the reason why WipB is inactive against pTyr-containing peptides (see main text).

Supplementary Figure 4: Localisation of GFP-WipB in cultured cells
HeLa cells expressing GFP-WipB (green) were fixed and stained with an anti-giantin or anti-calreticulin antibody (red), prior to fixation with Mitotracker (red), or loaded with transferrin (magenta). Scale bars, 10µm.

Figure 3d
The membrane was cut in 3 horizontal strips around the molecular weight of the protein of interest (v-ATPase A, 68kDa; v-ATPase B, 57kDa; LAMTOR1, 17kDa). Each strip was incubated with the corresponding primary antibody, then the secondary HRP-conjugated antibody, and finally the ECL reagents. The 3 strips were then re-assembled carefully before exposure.

Figure 3e
The membrane was cut in 5 horizontal strips around the molecular weight of the protein of interest (v-ATPase A, 68kDa; v-ATPase B, 57kDa; LAMTOR1, 17kDa; GFP 86kDa and 30kDa). Each strip was incubated with the corresponding primary antibody, then the secondary HRP-conjugated antibody, and finally the ECL reagents. The 5 strips were then re-assembled carefully before exposure.

Figure S3a
The membrane was cut in 2 horizontal strips around the molecular weight of the protein of interest (GFP, from 20kDa to top, Sec61 from 20kDa to bottom). Each strip was incubated with the corresponding primary antibody, then the secondary HRPconjugated antibody, and finally the ECL reagents. The 2 strips were then reassembled carefully before exposure.

Figure S5b
The membrane was cut in 2 horizontal strips around the molecular weight of the protein of interest (GFP, from 20kDa to top, Sec61 from 20kDa to bottom). Each strip was incubated with the corresponding primary antibody, then the secondary HRPconjugated antibody, and finally the ECL reagents. The 2 strips were then reassembled carefully before exposure.


text:
The therapeutic HIV Env C5/gp41 vaccine candidate Vacc-C5 induces specific T cell regulation in a phase I/II clinical study

Background Levels of non-neutralising antibodies (AB) to the C5 domain of HIV Env gp120 are inversely related to progression of HIV infection. In this phase I/II clinical study we investigated safety of Vacc-C5, a peptide-based therapeutic vaccine candidate corresponding to C5/gp41732–744 as well as the effects on pre-existing AB levels to C5/gp41732–744, immune activation and T cell responses including exploratory assessments of Vacc-C5-induced T cell regulation. Our hypothesis was that exposure of the C5 peptide motif may have detrimental effects due to several of its HLA-like features and that enhancement of non-neutralising anti-C5 AB by vaccination could reduce C5 exposure and thereby chronic immune activation. Methods Thirty-six HIV patients on effective antiretroviral therapy were randomised to one of three dose levels of Vacc-C5 administered intramuscularly with Alhydrogel or intradermally with GM-CSF as adjuvant through initial immunisation and two booster periods over 26 weeks. Vacc-C5-specific AB were measured by ELISA and T cell responses by both IFN-γ ELISPOT and proliferative assays analysed by flow cytometry. Immune regulation was assessed by functional blockade of the two inhibitory cytokines IL-10 and TGF-β in parallel cultures. Non-parametric statistical tests were applied. Results Vacc-C5 was found safe and well tolerated in all patients. Only marginal changes in humoral and cellular responses were induced, without any effect on immune activation. Overall, anti-Vacc-C5 AB levels seemed to decrease compared to pre-existing levels. Whereas Vacc-C5-specific CD8+ T cell proliferative responses increased after the first booster period (p = 0.020; CD4+, p = 0.057), they were reduced after the second. In contrast, Vacc-C5-induced T cell regulation increased after completed vaccination (p ≤ 0.027) and was lower at baseline in the few AB responders identified (p = 0.027). Conclusions The therapeutic HIV vaccine candidate Vacc-C5 safely induced only marginal immune responses, whereas Vacc-C5-induced T cell regulation markedly increased. Our data support further attention on immune regulation during therapeutic HIV vaccination studies. Trial registration NCT01627678. Electronic supplementary material The online version of this article (doi:10.1186/s12879-017-2316-x) contains supplementary material, which is available to authorized users.

Background
More than 30 years after its discovery, Human Immunodeficiency Virus type 1 (HIV) remains a global challenge with more than 36 million people infected worldwide [1]. Despite the achievements of effective antiretroviral therapy (ART), patients with access to ART control viremia but are not cured. Chronic immune activation is reduced, but persists [2] and is associated with non-AIDS morbidity and mortality [3,4], an increasing concern in the long term care of HIV patients. Search for alternative treatment strategies are therefore warranted.
Vacc-C5 is a peptide-based therapeutic vaccine candidate that aims to induce non-neutralising antibodies (AB) to the 5th constant domain (C5) of the HIV-1 envelope glycoprotein gp120 in conjunction with part of the transmembrane glycoprotein of gp41. The presence of AB towards the C5 region has previously been shown to correlate with a slower disease progression [5][6][7]. Analysis of serum from treatment naïve patients has demonstrated higher levels of non-neutralising anti-Vacc-C5 AB levels in HIV natural viral suppressors with moderate viral loads (<10,000 copies/ml) than in patients with higher viral loads (>10,000 copies/ml). Furthermore, longitudinal analysis of HIV patients has revealed an inverse correlation between anti-Vacc-C5 AB levels and disease progression [8]. The C5 domain has sequence similarity to peptide binding sites on HLA molecules [9] and can bind both defined class II and class I restricted peptides, bindings that are blocked with anti-C5 monoclonal AB [10]. It has therefore been proposed that exposed C5 could amplify chronic immune activation, either directly by crosssignalling via HLA-like motifs or by presentation of peptides that facilitate alloactivation-like responses [11]. Our hypothesis was that any detrimental effects of C5 explain the inverse relationship between anti-C5 AB levels and clinical progression, and that enhancement of non-neutralising anti-C5 AB by vaccination with Vacc-C5 could reduce exposure of C5.
Against this background, a phase I/II trial was conducted with the primary objective to evaluate safety of Vacc-C5 at three dose levels administered either intradermally or intramuscularly with two different adjuvants while on effective ART. Local low dose granulocytemacrophage colony-stimulating factor (GM-CSF) facilitates dermal dendritic cell maturation and migration to the lymph nodes for antigen presentation [12,13] and has been used in former studies with intradermal administration of the peptide-based HIV Gag p24 vaccine Vacc-4× [14,15] and with peptide-based cancer vaccines [16]. Alhydrogel, the only adjuvant approved and marketed for worldwide use (e.g. pneumococcal vaccine Prevnar and HPV vaccine Gardasil), was applied in the intramuscularly administered vaccine.
The secondary objectives were to explore humoral responses to Vacc-C5, HIV-associated immune activation and T cell immunogenicity. T cell responses to HIV antigens, such as C5, may be strongly regulated in chronic HIV infection [17][18][19], and immune regulatory mechanisms to HIV vaccine antigens may therefore be important to consider before and during therapeutic HIV vaccination [20,21]. We have previously assessed an exploratory parameter for vaccine antigen-induced T cell regulation, based on simultaneous blockade of the two potent, soluble cytokines downregulating HIV-specific T cell proliferation, a key feature of effector T cells [22,23]. Our approach does not intend to differentiate the mechanistic pathways of immune regulation, but rather measure the net impact on proliferative capacity of effector T cells to simplify implementation in a clinical trial setting.
This parameter of immune regulation was addressed also in the present study, and whereas only marginal cellular and humoral immune responses were induced, vaccine-specific regulation markedly increased after completed vaccination. These results may illustrate the complexity of therapeutic HIV vaccination in general and may also hopefully encourage further attention on assessments of immune regulation.

Study design and immunisation schedule
Thirty-six HIV-infected patients (male n = 35; female n = 1) from the Outpatient Clinic, Department of Infectious Diseases, Oslo University Hospital were enrolled in this open, dose escalating phase I/II trial (Fig. 1). Patients between 18 and 55 years of age, infected for at least one year, on effective ART for the last six months with viral load <50 copies/ml, CD4 + T cell count >400/μl and nadir CD4 + count >200/μl were included. Exclusion criteria were AIDS-defining illnesses, malignancies, chronic active infections, immunosuppressive therapy or unacceptable general biochemical and hematological parameters. Pregnant or breastfeeding women were excluded.
The participants were openly and sequentially randomised to three dose groups with 12 patients in each group, and included to the allocated dose within three months after screening.
Within each dose group, patients were randomised to either intradermal injections (n = 6) of Vacc-C5 given 10 min after administration of the local adjuvant GM-CSF, or intramuscular injections (n = 6) of Vacc-C5 with Alhydrogel as adjuvant.
During the study period of 26 weeks, the participants received a total of seven doses of Vacc-C5 at either low (LD, 100 μg), medium (MD, 300 μg) or high dose (HD, 900 μg) administered at week 1, 2, 4 (primary immunisation), 12, 13 (first boost), 21 and 22 (second boost). Dose Review Group Decision meetings were regularly held to evaluate safety before the higher dose level was administered.
The study was approved by the Norwegian Regional Ethics Committee South-East and the Norwegian Medicines Agency (EudraCT 2012-000710-11) and registered at www.clinicaltrials.gov (NCT01627678). Written informed consent was obtained from all participants. The trial was carried out in accordance with the International Conference on Harmonisation Guideline for Good Clinical Practice.

Peptide design and adjuvants
Vacc-C5 (Bionor Pharma, Oslo, Norway), a single heterodimeric peptide corresponding to the C5 domain on gp120 501-512 and the external domain of gp41 732-744 , was supplied as sterile vials of freeze-dried powder and administered at doses of 100 μg, 300 μg or 900 μg reconstituted in water.
In the intradermal group, 0.1 ml of GM-CSF (Leukine™) (Bayer HealthCare pharmaceuticals LLC, Seattle, WA) at 0.60 mg/ml was given intradermally superficial to the left deltoid muscle 10 min before 0.1 ml of Vacc-C5 at the same site. In the intramuscular group, Alhydrogel (Apotek production & Laboratorier AB, Umeå, Sweden) was added to Vacc-C5 at a ratio of 1:1.5 in a total volume of 0.25 ml and administered in the left triceps muscle.
The vaccine was prepared by the Oslo University Hospital pharmacy on the day of administration.

Monitoring and blood sampling
Adverse events were monitored, and clinical examination performed at each visit. Blood samples for safety (general biochemistry and hematology), CD4 + and CD8 + T cell counts and HIV RNA were analysed by approved methods. Peripheral blood mononuclear cells (PBMC) were obtained from CPT tubes (Becton Dickinson Biosciences, San Jose, CA) (BD), processed and stored at −150°C until further analysis. Serum was processed and stored at −70°C on site before being sent to Huntingdon Life Sciences (Huntingdon, Cambridgeshire, England) to assess anti-Vacc-C5 AB levels. Blood samples drawn at a day of vaccination were collected prior to immunisations.
Raw data were processed in Watson LIMS, using a 5PL (auto-estimate) algorithm with a weighting factor of 1/Y. Limit of detection was 390.7 ng/ml, and levels below were set to 390 ng/ml. A responder was defined as a patient with an increased AB level from baseline to end of study.

Vacc-C5-specific T cell responses by ELISPOT
The IFN-γ enzyme-linked immunosorbent spot (ELI-SPOT) assay was performed on PBMC, collected at week 1 (baseline), 6, 15 and 26 (end of study), as instructed by the manufacturer (Mabtech, Nacka Strand, Sweden). The plates were washed with sterile PBS, conditioned with albumin-containing PBS and incubated at room temperature for 30 min. Cryopreserved PBMC were thawed and rested overnight before stimulated in triplicate wells with pooled Vacc-C5 15-mer overlapping peptides offset by 1 amino acid at 2 μg/ml/peptide; 200,000 PBMC/well in 200 μl medium (Gibco AIM V, Invitrogen/Life technologies, San Diego, CA). Staphylococcal enterotoxin B (SEB)-stimulated (S-A) and unstimulated cells were positive and negative controls, respectively. The plates were incubated overnight at 37°C and 5% CO 2 before they were washed and 7-B6-1-ALP conjugate added. After 2 h of incubation at room temperature the plates were washed and the substrate solution added. Spots developed for 12 min before the plates were washed extensively in tap water to stop the process.
IFN-γ spot forming units (SFU) were counted by an AID ELISPOT reader with AID ELISPOT v5 Scanner software (AID GmbH, Strassberg, Germany). Net SFU were determined by subtracting corresponding negative controls for each sample. No cut-off for positive response was defined. Median values of triplicates were used for analysis, and data given as SFU per 10 6 input cells. All four time-points for a given patient were run on the same day to avoid inter-assay variation.

T cell proliferation and regulation
Fifteen patients were selected for T cell proliferation and regulation assays at week 1, 6, 15 and 26. These included the six patients with a specific T cell ELISPOT responder profile at some time-point, defined by at least twice the negative control, regardless of their AB responses. Additionally, nine patients with varying AB responses were selected. They had either an increase in Vacc-C5 AB from baseline to end of study (n = 4), an increase in AB at some time-point (n = 3) or no AB response at all (n = 2). Every dose group (n = 6: 3: 6, LD: MD: HD, respectively) and both adjuvants (GM-CSF n = 6; Alhydrogel n = 9) were represented.
Cryopreserved PBMC were thawed and rested overnight before they were labelled with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen Molecular Probes, Eugene, OR) at 2 μM for 5 min and subsequently stimulated with pooled overlapping Vacc-C5 15-mer peptide panels at 2 μg/ml/peptide. SEB (S-A) at 0.5 μg/ml was used as positive control. 250,000 PBMC were cultured in 200 μl serum-free culture medium (Gibco AIM V, Invitrogen/Life technologies, San Diego, CA) at 37°C and 5% CO 2 in 96-well tissue-culture plates (Nunc, Roskilde, Denmark). At day 6, cells were harvested and stained with CD3 V450, CD8 APC-H7 and to exclude nonviable cells, 7-aminoactinomycin (7AAD) (BD). Vacc-C5specific proliferative T cell responses were defined as percentages of live (7AAD − ) CFSE dim CD8 + or CD4 + (i.e. CD8 − ) CD3 + T cells. The cut-off for the proliferated CFSE dim T cell subset was set by median fluorescence intensities equal to or below the second proliferated generation. Net responses were found by subtracting background proliferation in the corresponding unstimulated control cultures. All four time-points for a given patient were run on the same day.
Inhibitory monoclonal antibodies (mAbs) to soluble interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were added to parallel antigen and control cultures at 10 μg/ml final concentration according to the manufacturer's instructions (anti-IL-10 clone 23,738, anti-TGF-β clone 1D11; R&D Systems Europe, Abingdon, UK). Antigen-induced cytokine-mediated net regulation was defined as the difference in proliferative responses between antigen cultures with and without mAbs to IL-10 and TGF-β. Fold increase was the ratio between Vacc-C5 proliferative responses with and without blocking mABs.
For both T cell proliferation and activation markers flow cytometry data were obtained with BD FACS Canto II™ with BD Diva software v6.1.

Analyses of soluble markers
The soluble pro-inflammatory chemokine interferongamma inducible protein 10 (IP-10) and cytokine IL-6 were analysed in snap frozen serum from all 36 patients at week 1, 6, 15 and 26 by Quantikine ELISA kits (R&D Systems, Abingdon, England) according to the instructions by the manufacturer. IP-10 samples were diluted 1:5. All samples for each individual were run in duplicates on the same plate.

Statistical methods
Statistical analyses were performed by Statistica v7 (Stat-Soft, Tulsa, OK) using non-parametrical statistics. Mann-Whitney U test or Kruskal-Wallis was applied to compare two or more groups. Wilcoxon matched pairs test was used for dependent variables, Spearman rank for correlations and Fisher exact test to test proportional differences. All data are presented as medians (interquartile ranges, IQR). p-values <0.05 were considered significant. Graphics were done by GraphPad Prism v6 (GraphPad Software, La Jolla, CA).

Safety and tolerability
All 36 patients completed the study. Their baseline characteristics are presented in Table 1. For more details, see Table 2. Vacc-C5 was safe and well tolerated, although many patients in the intradermal vaccination group experienced transient (seconds) pain/discomfort at the injection site during administration. No serious adverse event related to study drug was reported, and no significant changes in clinical status, vital signs, laboratory parameters, HIV RNA, CD4 + or CD8 + T cell counts were observed.

Anti-Vacc-C5 antibodies
Anti-Vacc-C5 AB were detected in 24 patients (67%) at baseline. AB levels were generally low (median 1433 ng/ ml), but comparable to previous assessments (with another ELISA assay) in 102 cross-sectional sampled HIV patients on ART (median 1160 ng/ml) where 80% of the sera carried <10,000 ng/ml (data not shown). Despite randomisation, AB levels were significantly higher in the HD group compared to the LD group at baseline (Fig. 2b), but vaccine induced changes were evaluated relative to the individual's own baseline AB level in the following.
From baseline to end of study, AB levels slightly decreased in the entire vaccinated group (p = 0.004) (Fig. 2a). When data for both adjuvants were combined in dose groups, AB levels were reduced in the MD (p = 0.025) and possibly the HD group (p = 0.059) whereas no changes were observed in the LD group (p = 0.327) (Fig. 2b).
Changes in the anti-Vacc-C5 AB levels over time did not differ between the intradermal and the intramuscular group (data not shown). However, when we divided the cohort further into groups based on both dose and administration, AB responses tended to decrease in the intradermal HD group (p = 0.068).
A baseline anti-Vacc-C5 AB limit of 2000 ng/ml was set according to subanalysis of a previous trial where patients with higher pre-existing levels (> 2000 ng/ml adjusted for assay differences) of anti-Vacc-C5 AB experienced improved viral outcome following Vacc-4× immunisation compared to placebo [24]. Patients with baseline anti-Vacc-C5 AB levels >2000 ng/ml (n = 15) showed a significant decrease in AB levels (p = 0.003), whereas no change was observed in patients with levels <2000 ng/ml (n = 13) (p = 0.859) (data not shown). Participants with undetectable AB levels throughout the study (n = 8) were excluded. Apart from the baseline AB levels, the two groups were comparable.
Even if the overall changes in anti-Vacc-C5 AB levels were small, individual differences were found. From baseline to end of study, increased AB responses were seen in six (17%) of the 36 vaccinated patients, a modest increase of 5-65% (median 26%) compared to their baseline levels. Four out of these six patients had detectable baseline levels. The six AB responders represented all three dose groups (n = 3: 1: 2, LD: MD: HD, respectively), and four had received intramuscular injections. Anti-Vacc-C5 AB responders (n = 6) and nonresponders (n = 30) were comparable in all baseline characteristics except for the CD4 + /CD8 + T cell ratio

IFN-γ ELISPOT responses to Vacc-C5
At baseline, no significant differences in Vacc-C5 IFN-γ responses were observed between any of the three dose groups (Fig. 3b) or between the two modes of administration (data not shown).
Only small changes in IFN-γ producing cells were seen throughout the study, with a trend towards an overall decrease in SFU (p = 0.090) (Fig. 3a). A significant HIV RNA (copies/ml) 0 b (0 -<20) 0 (0 -<20) 0 (0 -<20) 0 (0 -<20) No differences between the groups a Median (interquartile range) b 0 implies undetectable PCR product reduction was found in the HD group (p = 0.015) (Fig. 3b) and the HD Alhydrogel subgroup (p = 0.028), whereas we observed no changes within the LD or MD groups. Analysing all vaccinated, the Alhydrogel and GM-CSF groups were comparable (data not shown). Nevertheless, 13 (36%) of the study population showed an increase in IFN-γ producing cells in response to Vacc-C5 stimulation from baseline to end of study (n = 5: 6: 2, LD: MD: HD, respectively). These responders were characterised by a significantly lower CD8 + count at baseline as compared to the non-responders (p = 0.005) and a tendency of higher CD4 + /CD8 + T cell ratio (p = 0.055) (data not shown).

Vacc-C5-specific proliferative CD8 + and CD4 + T cell responses
From baseline to end of study, Vacc-C5-specific proliferative T cell responses tended to increase in the CD8 + T cell subset (p = 0.069). After the first booster period, CD8 + T cell responses increased significantly (p = 0.020), but were subsequently reduced after the second booster period (Fig. 4a, upper panel).
In general, small changes in proliferative T cell responses were seen, although some patients developed strong responses at different time points (Fig. 4b). At end of study, improved responses in both T cell subsets were found in nine patients (60%) including four (67%) HIV RNA (copies/ml) of the six with enhanced ELISPOT responses at some time point.
As a validation of the T cell assays, we noted that the overall changes in Vacc-C5 specific responses by ELISPOT were positively correlated with the overall changes in proliferative responses from baseline to end of study (CD8 + r = 0.67, p = 0.006; CD4 + r = 0.51, p = 0.050) (data not shown).

Vacc-C5-induced immune regulation
No Vacc-C5-induced cytokine-mediated T cell regulation was detected at baseline (Fig. 5, left panel). In contrast, there was a significant difference in Vacc-C5-specific proliferative responses between cell cultures with and without blockade of IL-10 and TGF-β after vaccination (p = 0.006, both T cell subsets) (Fig. 5, right panel). Vacc-C5-induced cytokine-mediated net regulation increased in both the CD8 + (p = 0.017) and CD4 + (p = 0.027) T cell subsets, mainly after the last booster period (Fig. 6a). Overall, there was a three-fold increase in regulation in the CD8 + T cell subset (p = 0.027) (Fig. 6b). Notably, no correlations between proliferation and regulation were seen at end of study.
Five out of the six who increased in anti-Vacc-C5 AB were among the 15 selected for T cell proliferation and regulation assays. Interestingly, these five AB responders had a significantly lower baseline regulation in the CD8 + subset as compared to the ten non-responders (p = 0.027) (data not shown).

T cell activation markers HLA-DR and CD38
Immune phenotyping was performed on unstimulated PBMC from all patients. Some differences between groups were found at baseline as the fraction of CD8 + T cells expressing HLA-DR/CD38 was higher in the Alhydrogel group (median 14.8% [IQR 11.8-18.3%]) than in the GM-CSF group (11.5% [9.4-14.2%]) (p = 0.037). However, from baseline to end of study, no changes in T cell activation markers expressed as HLA-DR/CD38 were observed.

Soluble markers IP-10 and IL-6
To further investigate the possible influence of Vacc-C5 on immune activation, analyses of serum IP-10 and IL-6 were carried out, representing soluble markers that have been associated with immune activation in HIV infection. In the total cohort, the levels of the proinflammatory chemokine IP-10 were strongly associated with the activation marker HLA-DR/CD38 on CD8 + T cells at all four time points (r = 0.37-0.52, p = ≤ 0.025). In contrast, no correlations were found for IL-6 (data not shown). No differences between the dose groups or modes of administration were seen for IP-10 and IL-6

Discussion
In this phase I/II trial the therapeutic vaccine candidate Vacc-C5 was administered over six months with initial immunisation and two booster periods in the presence of ART. The primary objective was to evaluate safety of Vacc-C5 at three dose levels with two different adjuvants, while the secondary objectives were to explore humoral responses to Vacc-C5, HIV-associated immune activation and T cell immunogenicity. Immune regulation of T cell proliferative responses was an optional exploratory objective. Vacc-C5 was well tolerated, and all 36 patients completed the study. Despite rather low preexisting anti-Vacc-C5 AB levels at baseline, only marginal changes in humoral responses to Vacc-C5 were found. Overall, both AB levels and T cell IFN-γ ELI-SPOT responses decreased throughout the study. The two modes of administration were comparable, with no significant differences in Vacc-C5-induced responses between them. The higher doses appeared disadvantageous as AB levels declined in the MD group, and a reduction in IFN-γ responses was seen in the HD group.
Although intra-and inter-assay variations might cause some of the minor changes in anti-Vacc-C5 AB levels, the main conclusion is that marginal changes were induced with the doses, schedule and adjuvants tested. It should be noted that Vacc-C5 has been shown to induce robust humoral responses in two classical animal models; in sheep using Alhydrogel and in rabbits using rh-GM-CSF as adjuvants (Bionor Pharma, data on file). Although not tested in HIV seronegative volunteers for regulatory and ethical reasons, Vacc-C5 was expected to induce humoral responses in humans as well. Moreover, if consistent enhancement of Vacc-C5 AB levels had been observed, additional characterisation of Vacc-C5specific Ig isotype and subclass responses could have been assessed, although no reference to such information is available from reports on anti-C5 AB levels and slow disease progression [5][6][7].
Despite the minimal changes in anti-Vacc-C5 AB levels, some interesting data regarding T cell responses were observed. It should be noted that the T cell responses to Vacc-C5 were generated by pooled peptides representing the Env vaccine heterodimer, and did not differentiate responses between the two short Env regions. Overall, Vacc-C5 induced only small changes in cellular responses from baseline to end of study. After the first booster period, however, CD8 + T cell responses significantly increased, but were subsequently reduced after the second booster period. Strikingly, Vacc-C5induced cytokine-mediated immune regulation was the only parameter that markedly increased during the trial. We assessed a parameter for regulation by blocking the two inhibitory cytokines IL-10 and TGF-β in Vacc-C5stimulated cell cultures. In previous studies, this parameter has been associated with rapid disease progression [25] and inconsistent boosting effects of the HIV p24based Vacc-4× [22]. In a recent study, where Vacc-4× was administered intranasally, immune regulation could explain most cases of T cell anergy towards HIV p24 as well as the variable final responses [23]. In the present study, increased immune regulation might partially explain the coinciding declining T cell responses after the second booster period (Figs. 4a and 6a) and possibly the lack of anti-Vacc-C5 AB responses as well. Our data find support in a recent primate prime and boost vaccination model which has shown that a SIV vaccine can induce suppressor cells and thereby dampen protective CD8 + T cell responses [26].
Immune regulation may be quantified in different ways. In previous papers [22,23,25], we have defined antigen-induced cytokine-mediated regulation as a parameter which also included control cultures with and without mAbs to IL-10 and TGF-β. Net regulation is a more simplified assessment, as reported by others [27] and illustrated in Fig. 5. Net regulation might be easier to comprehend, and is probably a more precise term to use in the discussions of this phenomenon. A variant of net regulation is fold increase, i.e. the ratio between proliferative responses in stimulated cultures with and without blockade. Here we explored immune regulation in either way with generally identical results.
Our approach is supported in part by Brockman et al. [27] who reported that blockade of the IL-10 pathway increased proliferative T cell responses to HIV antigens in viremic patients, whereas elite controllers and patients on effective ART failed to respond to the blockade. After immunisation with Vacc-C5, we found that blocking of both IL-10 and TGF-β significantly increased the proliferative capacity to Vacc-C5 in these non-viremic patients. We have previously shown that this parameter of T cell regulation is HIV antigenspecific and not an inhibitory response to T cell activation in general. In an HIV positive individual, regulation may be high to one specific HIV antigen but low or absent to other HIV antigens and non-HIV antigens. Moreover, regulation induced by different HIV antigens are not correlated in a particular sample [25,28]. Therefore, it is likely that the regulation induced by Vacc-C5 is vaccine-specific and not a global T cell regulation induced by the vaccine.
We chose to assess regulation by blocking two inhibitory cytokines and quantify the total inhibitory effect on Vacc-C5-specific T cell proliferation. Since proliferation is a fundamental feature of effector T cells, the impact of regulation on T cell proliferative capacity may provide additional information, relevant for choosing patients expected to benefit from therapeutic vaccination.
In the era of an HIV cure, there has been a renewed interest in immunotherapy [29,30]. Efficient HIV-specific CD8 + cells able to eliminate reactivated latently infected cells are essential to put HIV into remission [31], but these cells are hampered in chronic HIV infection [32,33]. Immunotherapy may therefore be required for an effective functional HIV cure, but a possible challenge may be induction of immune regulation.
Both proliferative responses and immune regulation differed considerably from one patient to another throughout the study. Consistent with previous results [22,23], our data highlight the importance of individualised immunotherapy. Moreover, they support the view that both proliferative responses and regulation should be monitored in therapeutic immunisation trials, because T cell proliferation and immune regulation induced by a defined HIV antigen are generally not correlated [25,28].
This dose escalation study, with safety as primary objective, has limitations, particularly small size of each adjuvant group. Regardless, it was possible to analyse secondary endpoints in subgroups, but with the risk of type II statistical errors. Moreover, the study was planned and approved with no unimmunised or adjuvant only control groups included. Another limitation may be the selection of patients for proliferative assays. Although proliferation and regulation were not tested in all participants, these patients were comparable to the rest of the cohort regarding clinical characteristics, dose and administration as well as AB levels and IFN-γ ELISPOT responses at baseline and end of study. The regulatory assays could have been expanded to explore the individual contribution by the two inhibitory cytokines and to include other well characterised regulatory pathways by blockade of T cell receptors like CTLA-4 and PD-1. Still, all the above mentioned regulatory pathways are recently addressed by our group, and the combined blockade of IL-10 and TGF-β was found to best identify net regulation induced by Env antigens [28]. Finally, more direct methods to assess regulation might have provided even more definitive data, e.g. separation of T regulatory cells by cell sorting and add-back experiments with the sorted cells. However, these in-depth analyses would require substantial PBMC and resources unrealistic to implement in a clinical trial setting.
The development of Vacc-C5 was based on the hypothesis that the C5 region is involved in chronic immune activation and that blocking this domain with non-neutralising AB may lead to reduced immune activation and disease progression [10,11]. However, in this study including patients on ART, increased levels of anti-Vacc-C5 AB were not induced, and the activation markers HLA-DR/CD38 and the two chosen pro-inflammatory mediators IP-10 and IL-6 remained unchanged. Since the C5 domain shows sequence similarity to HLA-DR, the HLA phenotype of study participants may potentially have influenced the results. Finally, it might be possible that Vacc-C5 administered with another adjuvant or with a carrier molecule could have generated AB with effects on HIV-associated immune activation.

Conclusions
In this study investigating safety and immunogenicity of the therapeutic vaccine candidate Vacc-C5, only marginal immune responses were safely induced, whereas vaccine-specific regulation clearly increased after completed vaccination. It is said that we learn more from failure than success (Samuel Smiles 1812-1904, Scottish author and reformer). Despite the absence of enhanced immune responses, this study might still provide insights to improve future therapeutic vaccine designs and immunotherapy and encourage further focus on immune regulation in therapeutic vaccination.


Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

Introduction
Bacteria are key microbes in marine microbiology that play fundamental roles in the ocean environment. Apart form being the base of the food chain, supplying more than half of the world's oxygen, controlling the role of marine nutrients, and being major processors of greenhouses gases, they are also rapidly responsive to changes in their ecosystem, making bacteria an ideal indictor of marine change [1] [2]. One such case is the microbialization of bacteria in contaminated waters over the past decade. The growing pathogenicity of marine microbes can cause disease in both marine and human life. Upon contact or consumption of specific bacteria, humans can experience cramps, nausea, diarrhea, tuberculosis, legionella pneumonia, typhoid, and cholera [3]. Bacterial diseases of fish include-but are not limited to: hemorrhagic septicemia, vibriosis, and columnaris [4]. The susceptibility of infection from pathogenic bacteria reinforces the importance of water purification before industrial, agricultural, or domestic applications. It is also ideal that water treatment processes use purification methods that limit leftover waste, as brine sediments often contain pathogens not killed off during treatment, and disposing them back into the ocean reintroduces the harmful bacteria into the aquatic environment in abundance.
There is growing interest in marine bacteria in the scientific world because pathogenic, or otherwise, their and identification serves many purposes. Firstly, extremophiles can be used as a measure of water pollution levels or aquatic condition because these species only grow in extreme environments. For example, halophilic bacteria only thrive in waters of high salt concentration, and are an indicator of overly high salinity. Next, being aware of pathogenic bacteria and locations were specific genera reside allows researchers, scientists and doctors to better prevent disease that could be contracted in the area-some of which can be very dangerous. Treatment plants can also better allocate purification methods for more effect water treatment with an understanding of the bacterial genera residing in specific marine regions. For example, bacteria killing methods of purification-such as electrochemical remediation, will be more efficient in treating water samples containing pathogens than other methods that do not eliminate bacteria. Employing target specific ways of water purification is more effective, efficient, and economical. Finally, bacterial identification is also important because many bacterial species such as: Marinobacter hydrocarbonoclasticus, Carboxydothermus hydrogenoformans, and Sphingomonas harbor properties that can be industrially useful in pharmaceutical, biotechnological, agricultural, chemical, and environmental fields. The purpose of this study is to investigate the diversity and properties of marine bacteria species from Salwa Beach (N1), North West (N2) and Sulaibikhat Beach (N3) by directly culturing seawater samples, PCR amplification of 16S rRNA gene, and DNA sequencing.

DNA Growth and Preparation
Bacteria from each beach was first grown by spreading 3μL of each sample onto separate marine agar plates, then incubated at 28°C in an bacteriological incubator for 24 hours. Grown bacteria cultures were then collected and mixed into cell pellets of 10μL of sterile distilled water. 48 samples of bacteria were collected from each beach.
Cultivation was also attempted on MacConkey, and nutritional agar plates. However, results indicated that bacteria could not be cultured on said plates, which made the identification of fecal coliforms based on culturing conditions and microscopical morphologies impossible.

PCR
Bacterial 16S rDNA was amplified from the extracted genomic DNA by using the following universal eubacterial 16S rRNA primers: forward primer 5' AGAGTTTGATCCTGGCTCAG 3' and reverse primer 5' ACGGCTACCT TGTTACGACTT 3'. An ABI PCR System 9700 was used to perform the PCR protocol in a volume of 24 μL of bacteria strain identification solution mix containing 10 x 2.5μL buffer, 1.25μL magnesium chloride, 1μL dNTP, 0.05μL primer 1, 0.05μL primer 2, 0.1μL enzyme taq, and 19.05μL of distilled water per 1μL of template bacteria. Thermal cycling consisted of a 10 minute initial denaturation at 95°C, 30 cycles of denaturation at 94°C for 30 seconds, 30 seconds of annealing at 60°C, and extension for 2 minutes at 72°C with a 10 minute final maintenance at 4°C [5].

Agarose Gel Electrophoresis
From each PCR product, an aliquot of 15μL was subject to 1.0% agarose gel electrophoresis containing 0.5 x TBE buffer (pH 8.0), and 5μL of ethidium bromide was used to stain the gel. Electrophoresis was conducted at 100 Volt, 400mA for 20-30 minutes with 0.8μL of 100bp DNA marker. After, the DNA bands were observed under ultraviolet light using gel documentation system [5].

Sequencing of 16S rRNA Genes
Sequencing reaction was carried out using ABI PRISM 310 Genetic Analyzer (PE applied Biosystems). For the sequencing reaction, Big Dye Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer) was employed.

Data Analysis
Sequence analysis was performed with sequences in the NCBI database using BLAST as well as with sequences available with Ribosomal Database Project (RDP)  infections [7]. N1-021 Spirometra erinaceieuropaei: is a tapeworm that causes a parasitic infection called sparganosis [8]. N1-023 Hydatigera taeniaeformis: a parasitic tapeworm with cats as their primary definitive hosts [9]. N1-025 Schistosoma rodhaini: commonly known as blood-flukes, they are parasitic flatworms that cause schistosomiasis in humans-an infection considered by the World Health Organization to be the second most socioeconomically devastating parasitic disease infecting hundreds of millions worldwide [10]. N1-030 Xanthomonas oryzae pv. Oryzae: causes bacterial blight, a serious disease for rice, grass, and sedges worldwide [11]. N1-038 Diphyllobothrium latum: is the largest human tapeworm and the principle species of Diphyllobothrium causing diphyllobothriosis It infects humans through the somsuptiono of raw or undercooked seafood, but can also affect fish and other mammals [12]. N1-041 Schistosoma rodhaini: refer to N1-025 N1-045 Spirometra erinaceieuropaei: is a tapeworm that infects humans and domestic animals. In humans, is causes sparganosis [8]. N1-047 Plasmodium falciparum: is a protozoan parasite that is the deadliest of five human malaria species, being responsible for more than 85 % of all malaria cases, and has the highest mortality rate amongst all malaria species [13]. It can be inferred that Salwa beach is home to an abundance of tapeworms, as 6 out of 8 pathogenic bacteria identified were of such. Tapeworms, which generally reside in areas of inadequate sanitation is an indication of the area's contamination levels. Salwa beach is also a harbor. Many tapeworms could dwell inside the intestines of fish, so extra persuasions should be taken when eating undercooked seafood to prevent infections. Table.3. Bacteria with industrial uses found in Salwa Beach isolates after data analysis.

Isolate
Description N1-020 Sphingomonas: typically found in environments polluted with toxic compounds, they have the ability to utilize the contaminants as nutrients. Their biodegradative and biosynthetic capabilities have been utilized for numerous biotechnological applications including bioremediation of environmental contaminants for the production of extracellular polymers such as sphingans [7]. It is also effective in degrading degrade alkylated polyaromatic hydrocarbon (2-methylphenanthrene), commonly known as polystyrene plastic [14]. N1-031 Pseudoalteromonas sp. F497: capable of synthesizing biologically active molecules that produce compounds beneficial to eukaryotic organisms [15]. N1-046 Tetrapisispora phaffii: killer yeast that secretes a glycoprotein known as Kpkt that has antimicrobial activity with spoilage yeasts under winemaking conditions. It can be used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and to develop novel antimycotics for the treatment of human and animal fungal infections in the medical field [16]. There is no pattern to the useful bacterial species identified in Salwa beach, but the particularly useful Sphingomonas was cultured and identified. Its unique ability to synthesize and degrade toxic compounds in water could be applied as an effective organism in water purification-a function that can be utilized in Kuwait's many water treatment plants.

North West Beach
Total bacteria counts: 112 should be taken into consideration, as it is the closest to a direct polluting source (industrial power plant) with notably less fish than in popular fishing regions such a Salwa Beach. This could possibly explain the sample's lack of pathogenic bacteria, as many species of pathogens reside in marine life. Table.6. Bacteria with industrial uses found in North West Beach isolates after data analysis.
Isolate Description N2-006 Marinobacter hydrocarbonoclasticus: are important to bioremediation because they can effectively degrade hydrocarbons-especially those found in alkanes and major components of oil as they are tolerant of high salinity and can grow aerobically and anaerobically [18]. The marinobacter hydrocarbonoclasticus' distinctive ability makes it ideal for treating recalcitrant pollutants [19]. N2-015 Bacillus pumilus: is used as an active ingredient in agricultural fungicides because its growth on plant roots prevents rhizoctonia and fusarium spores from germinating [20]. Bacillus pumilus proteases are also used in chemical, detergent, food, and leather industries as antimicrobials and antifungals. N2-019 Marinobacter hydrocarbonoclasticus: refer to N2-006 N2-029 Erythrobacter citreus: is a potential candidate for removing poisons because it can resist and reduce the toxic compound tellurite [21]. N2-030 Marinobacter hydrocarbonoclasticuss: refer to N2-006 3 isolates of Marinobacter hydrocarbonoclasticus was found out of the 48 bacteria samples taken, suggesting that the species is abundant in North West Beach waters. Its properties are very useful in purifying Kuwaiti water, as it is able to degrade hydrocarbons found in oil. Oil is an abundant resource in Kuwait that frequently pollutes the county's water sources, but is difficult to remove once contaminated. Therefore, the potential of using bacteria to purify this recalcitrant pollutant is exciting and could perhaps be more cost efficient than currently employed methods.

Isolate
Description N3-001 Carboxydothermus hydrogenoformans: is an extremely thermophilic bacteria that feeds almost entirely on toxic carbon monoxide and water, and produces hydrogen gas as part of its metabolism [27]. Its ability to rapidly and efficiently conduct this conversion makes it highly valuable for the future where hydrogen gas is being studied as potential biofuel [28]. N3-013 Exiguobacterium undae: According senior research engineer Wei-Min Wu in his report "Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms. 2. Role of Gut Microorganisms", injections of exiguobacterium undae into the gut of mealworms allows them to survive solely on a diet of polystyrene, and thus, can significantly help in the biodegradation and mineralization of plastic [29]. N3-014 Exiguobacterium undae: refer to N3-013 N3-015 Exiguobacterium undae: refer to N3-013 N3-034 Vibrio sp. B2-15-2 16S: is capable of producing biosurfactant that can be applied into environmental, technical, and potentially pharmaceutical industries [30].
A wide diversity of environmentally useful bacterial species was identified in Sulaibikhat Beach. The most abundant was Exiguobacterium undae. Its property of reverting mealworms to feed solely on polystyrene has enormous potential as an eco-friendly way of degrading plastic. Next, Carboxydothermus hydrogenoformans's diet of only toxic carbon monoxide and water, which later produces hydrogen gas, could provide an alterative for oil. Lastly, a useful species of Vibrio, sp. B2-15-2, was identified amongst its pathogenic counterparts and demonstrates the impressive biodiversity of marine bacteria and their respective properties.

Conclusion
The data analysis revealed that Kuwaiti water samples harbor extremely diverse bacteria belonging to over 53 bacterial genera. 24 genera were isolated in Salwa Beach, 17 in North West Beach, and 12 in Sulaibikhat Beach. In Salwa Beach samples, identified genera consisted primarily of Alteromonas (17.5% out of 40), North West Beach samples consisted of prominently Marinobacter (26.8% out of 41 sequences), and Sulaibikhat Beach samples consisted of mostly Vibrio (46.3% out of 41 sequences). Alteromonas and Marinobacter strains were found to be abundant and distributed in both Salwa and North West Beach, while none were cultured and identified in Sulaibikhat Beach.
Each sampled location exhibited specific patterns of cultivated pathogen bacterial species that reveals information regarding each beach's unique marine condition and life. In Salwa Beach, most pathogens were tapeworms, only Schistosoma mattheei were isolated from North West Beach, and 3 pathogenic species of Vibrio was found in Sulaibikhat Beach. Since tapeworms and Vibrio species are ubiquitous in aquatic environments copious in marine life, the intense mariculture in Salwa and Sulaibikhat Beach may be partially accountable for the high occurrence of these pathogens.
On the other hand, identified industrially useful bacteria demonstrated no pattern in accordance to sampled location, but similarities in valuable property. While a wide range of diverse, unrelated bacterial cultures were isolated in each beach, the majority of all had biodegradative and biosynthetic capabilities. For example, Sphingomonas found in Salwa Beach can utilize toxic compounds and contaminants as nutrients, Marinobacter hydrocarbonoclasticus found in North West Beach can effectively degrade hydrocarbons, and Carboxydothermus hydrogenoformans found in Sulaibikhat Beach can feed almost entirely on toxic carbon monoxide and water, while producing hydrogen gas. Upon further study, these species-along with the others identified, potentially offer incredible scientific breakthroughs in the usage of microorganisms in pharmaceutical, biotechnological, agricultural, chemical, and environmental fields.
In summery, the purpose of this study was to cultivate, isolate, identity, and determine properties of marine bacterial species in Kuwait from Salwa Beach (N1), North West (N2) and Sulaibikhat Beach (N3) through PCR amplification of 16S rRNA gene, and DNA sequencing of bacteria grown from collected water samples. The findings indicate towards a high level of macrobiotic diversity, and shed light on the ecological functions of both pathogenic and useful bacterial isolates-information that can be used towards precautionary causes or further inquiry and useful application.