Indigenous knowledge of zootherapeutic use among the Biate tribe of Dima Hasao District, Assam, Northeastern India

Background The present study addresses the use of zootherapy in the traditional healthcare system of the Biate tribe of Dima Hasao district, Assam, India. It sought to identify the different species used for zootherapeutic use with the detailed methods of usages to create awareness and contribute to the conservation and sustainable utilization of the resources. Method 15 Biate villages within the district of Dima Hasao were surveyed through semi-structured questionnaires and informal interviews. Detailed information on the uses of each animal was recorded. Species were identified using standard literature. Fidelity level (FL) was calculated to demonstrate the percentage of respondents claiming the use of a certain animal for the same major purposes. Result The study documents 34 species for the treatment of about 34 different ailments. The largest number of species reported was mammals with17 species. Maximum number of species has been reported for the treatment of diabetes and its high fidelity levels warrants in-depth studies to establish its pharmacological activity. The usages documented herein are unique to the Biate tribe. Very often, these animals are hunted and sold openly at the local markets in the lure of quick money. A 300 gm live Gekko gecko may fetch a sum of 2,50,000 Indian Rupees (INR), and smoked meat of Hoolock hoolock cost approximately 250–300 INR per kg. Animals are also hunted for its hide. The unrestricted hunting of species like Capricornis sumatraensis has almost wiped out the population within the district. Some species are also reared as pets while some are used for display as a sign of expertise in hunting. The present study has documented the usage of at least 15 animals listed in the IUCN Red List. Conclusion The study illustrates the in-depth knowledge of the Biate tribe on zootherapy. Systematic investigation to identify the active ingredient may lead to the development of new drugs, which would prompt protection of these valuable resources.

Background
Animal parts have been used as a source of medicine in the traditional healthcare system since ancient times, and have played a significant role in the healing practices. In the modern societies, zootherapy constitutes an important alternative among many other known therapies practiced worldwide [1]. The World Health Organization estimates that as many as six billon people rely primarily on medicines of animal and plant origin [2]. Although plant and plant derived materials are more commonly used in traditional medical systems than animal derived products, the latter also constitute an important element in most of the traditional healthcare systems of the world. As for example, Traditional Chinese Medicine (TCM) recorded more than 1,500 animal species to be of medicinal use [3]. A list of 60 different species of insects used to treat a wide range of disabilities and illnesses in Japan has been published [4] and 24 animal species were identified, whose byproducts were used therapeutically by the Tamang people of Nepal [5]. However, in spite of such worldwide prevalence, research on medicinal animals has often been neglected in comparison to medicinal plants [6]. Pieroni et al. [7], for example, has pointed out that studies on drugs of animal origin are still rare in the scientific literature.
India is known for its age-old heritage of traditional medicine. It is a goldmine of well-recorded and traditionally practiced knowledge of herbal medicine such as the Ayurveda, Siddha and Unani medical system. India officially recognizes over 3,000 plants for their medicinal value and estimated that over 6,000 plants are in use in traditional, folk and herbal medicine, representing about 75% of the medicinal needs of the third world countries [8]. Nonetheless, as far as zootherapeutic uses is concerned, very few researches has been done so far, especially in the northeastern states of India. Northeastern India harbors over 130 major tribal communities. Most tribal communities still largely depend on their traditional system of medicine. Because of their scattered and far-flung settlements, and problems arising due to transportation and communication, traditional medicine has remained as the most affordable and easily accessible source of treatment [9]. Although some of the traditional medicines have already been documented, tried, tested and even incorporated in the modern systems of medicine, much larger number of these folk medicines is still undocumented and remains endemic to certain pockets of the area. Each tribe have their own unique way of traditional healthcare system that has been handed down from generation to generation orally, but with the steady development of rural areas coupled with the lure for a more profitable job, the interest of the younger generations in this tradition is declining. The traditional ecological knowledge including the healing practice is gradually being corroded from their culture. Therefore, ethnobiologist in this region have a greater responsibility not only in inventorizing the traditionally used biological resources but also in conserving and revitalizing the traditional beliefs, so that these age-old cultures are not lost [10].
Because of their rich traditional knowledge, different tribal communities of northeastern India have been a subject of several intense and eye-opening ethnozoological studies. Solanki [14] while Kalita reported the usage of 4 species from Assam [15]. Kakati and Duolo reported 23 species from Nagaland [16] while Kakati reported 25 species used by the Ao Naga [17]. The district i.e. Dima Hasao of Assam, formerly North Cachar Hills district, has also been subjected to several ethnobotanical studies [18][19][20][21][22][23][24][25][26]. Nevertheless, till date, no comprehensive report on the ethnozoological wealth and indigenous knowledge of zootherapeutic use is available from the district. The present study area remains to be explored in this aspect. It is on this backdrop that the present research work has been taken-up for the first time to document the indigenous zootherapeutic knowledge of the Biate tribe of Dima Hasao district of Assam, northeastern India.
The study area Dima Hasao district, covering an area of 4890 km 2 (92°37 / E -93°17 / E longitude and 25°3 / N -25°27 / N latitudes) is a place of immense interest and potential for ethnobotanist, ethnozoologist as well as anthropologist ( Figure 1). More than 12 ethnic tribes namely Dimasa, Zeme, Biate, Jaintia, Hrangkhol, Hmar, Kuki, Vaiphei, Khelma, Rongmei, Lushai, Karbi etc., live harmoniously together within the district with several non tribal groups like the Assamese, Bengali, Nepali, etc. who are mostly government employees, traders, living in urban and semi-urban areas. It has a total population of 213,529 and the density of population is 44 individuals per square kilometers which is the lowest in the state of Assam according to the census of India, 2011 [27]. Most of the villages are situated far from modern conveniences and inaccessible by road or rail. Thus, the tribal villagers have deep faith in their traditional healthcare system and in most cases prefer them to the modern system of medicine.

The Biate tribe
The Biate tribe ( Figure 2) belonging to one of the branches of Mongoloid stock of race is among the prominent inhabitants of the district occupying the Kharthong constituency spreading into the other side of the Kopili River in the Saipung constituency of Jaintia Hills of Meghalaya [28]. Shifting cultivation is the traditional means of agriculture practiced by the villagers. Besides these, they raise livestock such as cows, goats, pigs, chicken and also grow a variety of both wild and cultivated plants in their residential compounds.
In olden days, the Biate people believe in naturalism, animism and animalism. Besides them, they believed that there is the high God called Chung Pathian which means Heavenly Father. When any disease or fatal epidemic comes through the village, the priest (Puithiam or Thiampu) performs the rituals calling upon the Chung Pathian to protect the village from the epidemic and the entire village is out of bounds to outsiders for five days or more [29]. However, today, with the advent of Christianity and majority of them being Christians, most of these rituals have been discarded and with it scores of traditional knowledge including ethnomedicine has been forgotten.

Methodology
The present study is based on a carefully planned, field survey on 15 Biate villages within the district of Dima Hasao. Surveys were carried out from November 2010 to March 2013 with a minimum of three visits to each village, to gather information on a maximum number of species. Surveys were conducted through semistructured questionnaires and in cases where the respondents were uncomfortable with the questionnaires, discussion and informal interviews were employed, and in the process information on different zootherapeutic uses were noted and documented [30,31]. A total of 300 individuals were interviewed. The age of the respondents ranges between 27 years to 78 years and the number of male respondents was higher (67%) as compared to the female respondents (33%). The respondents were selected randomly and Prior Informed Consent (PIC) was obtained for all interviews conducted. Information on the uses of each animal for disease treated, method of preparation, use and dosage, side effects, spiritual aspects linked to its use, parts used as medicine, local name, whether dried or fresh, whether rare or abundant, changes in abundance of the animal for the last 10 years, food value with market rate, efficacy of the remedies and how this knowledge was acquired by the respondents themselves were recorded. The scientific name and species of animals were identified by using relevant and standard literature.

Data analysis
For the data analysis, Fidelity Level (FL) calculated that demonstrates the percentage of respondents claiming the use of a certain animal for the same major purpose, was calculated for the most frequently reported diseases or ailments as, FL (%) = Np × 100/N. Where Np is the number of respondents that claim a use of a species to treat a particular disease, and N is the number of respondents that use the animals as a medicine to treat any given disease [31].
The range of fidelity level (FL) is from 1% to 100%. high use value (close to 100%) show that this particular animal species are used by large number of people while a low value show that the respondents disagree on that species to be used in the treatment of ailments.

Results and discussion
The present study documents 34 species used for the treatment of different ailments. Table 1 summarizes the scientific names of the medicinally used species, their vernacular names, the part(s) of the species used, the diseases or ailments the animal derived medicines thought to be effective for, and the ways the treatments are carried out. Table 2 summarizes the present conservation status of the species mentioned in Table 1 as zootherapeutically important. These species are distributed among at least 31 zoological families. The taxonomic Class with the largest number of species were Mammalia (17 species), followed by Aves (7 species) and Reptilia with five species (Figure 3). In similar studies carried out around the world, mammals and birds also recorded the highest use as part of local folk medicines [32][33][34][35]. Surveys other than our own research from northeastern India indicates the same [14,16,17]. Other Class mentioned by the interviewees belonged to Gastropoda (2 species), Insecta (2 species) and Malacostraca (1 species). Different researchers from India have already contributed to the knowledge of 351 ethno medicinal uses of animal and its parts (Table 3) [ [11][12][13][14][15][16][17][36][37][38][39][40][41][42][43][44]. The present study, with the usage of 34 species recorded for the first time from the district is an addition to the above figure.
Fidelity Level (FL) demonstrates the percentage of respondents claiming the use of a certain animal for the same major purpose. The uses of animals that are commonly known by the respondents have higher fidelity level than less common known. The bone of Hoolock hoolock used to relieve rheumatism has the highest FL (97%) followed by Paratelphusa sp for the treatment of jaundice (96%) and the gall bladder of Melursus ursinus used for easy delivery of a child (93%). The use of the fur of Lutrogale perspicillata to treat burns and Periplaneta americana for tuberculosis has the lowest FL of 2% each ( Table 1). The FL reveals that the treatment for frequently reported ailments has the highest FL value and those with low number of reports have low FL value. About 34 different ailments have been reported to be treated in the present study ( Figure 4). While some of      the ailments reported here are common ones such as burns, worms, hair care etc., but most of them are serious ailments such as jaundice, diabetes, malaria, impotency, hernia, epilepsy etc. Maximum number of species has been reported for the treatment of diabetes ( Figure 4). The high fidelity levels for the treatment of diabetes suggest high frequency of report and thereby warrant in-depth studies to establish its pharmacological activity.
In several cases, medicine is prepared singly or in combination with herbs and very often cooked with vegetables to enhance the taste. While some are prepared fresh, some are prepared after it is sun dried or smoked dried. Fifty three percent of the animal parts are used after drying either after sun dried or smoked dried ( Figure 5). Different parts of the animals are utilized for different ailments. In some cases, the sought after body parts does not always come from the same species. For example, the gall bladder from six different species has been reported as treatment for diabetes (Table 1). Maximum number of animal parts used for medicine comes from the flesh, followed by the gall bladder and fat ( Figure 6). Very few side effects have been reported except for a few species.
The study has revealed that the Biate tribe possesses vast knowledge on the traditional usage of animal parts for medicine. This traditional knowledge is a guarded secret passed on from generation to generation through oral tradition. The reluctance of the traditional healers to reveal their secrets is due to the belief that revealing the properties and secrets renders the medicine ineffective. Although some of the species has already been documented elsewhere in India, the usages documented herein are unique to the Biate tribe. As for example, fats of Buceros bicornis is used by the Nyishi and Galo tribe of Arunachal Pradesh to treat body pain [13], while in the present study area, the fats of the same species is used in the treatment of arthritis. Flesh and fats of Hystrix sp are used in the treatment of body ache, rheumatic pain etc. [14] however the flesh of the same species is used to facilitate easy delivery of a child amongst the Biate tribe. Tooth of Elephas maximus is used for conjunctivitis and pimples [38], whereas it is used for toothache by the Biate tribe. Similarly, the use of Python molurus flesh for seizure, Cimex lectularius Table 3 Ethnomedicinal uses of animals reported from different parts of India  Figure 3 Proportional representation of total number of species and the percentage of contribution amongst each Class.
for malaria, flesh of Upupa epops for gall bladder stone etc. are all unique to the Biate tribe. It has been observed that most of the animal parts are usually procured manually which involves hunting of these animals and birds. Very often, animal-derived medicines and preserved animal body parts are sold openly at the local markets. Commercialization of animals for medicinal purposes is a widespread phenomenon; with significant implications for their conservation and sustainable use [45]. Local villagers very often hunt these animals in the lure of quick money. For example, a 300 gm live Gekko gecko may fetch a sum of 2,50,000 INR and scales of Manis crassicaudata cost approximately 10,000 INR (Table 2). Thus, the unchecked and unmonitored sale of wild animal products in the local markets has a significant impact on the population of the wild fauna.
The present study has documented the usage of at least 15 animals listed under Endangered, Vulnerable and Near Threatened category of the IUCN Red List ( Table 2). While several are listed under Least Concern in the IUCN Red List, the present study observes that there has been a drastic change in the abundance of the species even within the last ten years ( Table 2). Species like Gekko gecko, Gallus gallus, Canis aureus, Macaca assamensis, Nycticebus coucang are less abundant now as compared to the last ten years (Figure 7).
The present study has revealed that out of the 34 species documented herein, at least 24 are also hunted for its meat as much as it is hunted for its medicinal value.
Smoked meat of Sus scrofa, Trachypithecus pileatus, Hoolock hoolock etc. are in high demand and cost approximately 250-300 INR per kg (Table 2). Wild edible plants and animals form a part of the basic source of food amongst the tribal community in the study area. Traditionally, most of the Biate villages have an age old system of sustainable utilization which involves protecting or closing a section of the village forest area for several years and then open it for several years while another section is protected. The village chief is the supreme head and owner of the entire land and it is he who ensures the protection of an area along with his council. However, presently, with the lands coming under the Autonomous District Council, the village chiefs are often appointed by the district council and doesn't follow the clan system as earlier and although the village chief is still the head, his powers are limited and the sense of ownership is gradually declining leading to unimpeded exploitation of the forest resources by the villagers and also by outsiders. Elephas maximus, Capricornis sumatraensis and Hoolock hoolock has almost been obliterated in the study area due to its excessive hunting. This is a fact of great concern because those species directly involved in traditional medicines and food should be amongst those of the highest priority for conservation [46].
Besides its medicinal and food value, these animals are also hunted for other uses. In a Biate community, as in  Arthritis Burns  most of the tribes of the Chin-Kuki-Mizo clan of the district, the traditional drum known as 'Khuang' plays an important role while performing rites and rituals in the early days. Even in the present day, after the advent of Christianity, the 'Khuang' is still an integral part of the church music. The hide of Capricornis sumatraensis is considered one of the best raw materials for making the 'Khuang', and almost every household has one. The unrestricted hunting of this particular species for its meat and hide has almost wiped out the population within the district. Species like, Melanochelys trijuga, Macaca assamensis, Nycticebus coucang, Hoolock hoolock etc. are also reared as pets while skull and feather of Buceros bicornis, Upupa epops etc. are used as decoration and as a sign of expertise in hunting. The spine of Hystrix indica is used in weaving (Table 2). Thus, it is important to point out that although medicinal use of animals is one of the main cause of threats to wild populations, it cannot be considered the only threat to the conservation of the species [47]. Medicinal use of animals must be considered together with other anthropogenic pressures, such as habitat loss, hunting for food etc. In most cases, the medicinal products of animals are by products from animals hunted for other purposes; thus, these multiple uses (including medicinal) of fauna and their impact on animal populations must be properly assessed and taken into consideration when implementing recovery plans for these species, especially those that are highly exploited [48].
It was observed that most of the people prefer this traditional cure to the modern pharmaceuticals, as it is less expensive and claimed to be more effective. Thus, zootherapy is an important and integral part of the traditional healthcare system of a tribal community, however overexploitation and lack of regulation and monitoring to safeguard for sustainable utilization is a point to consider seriously from the conservation point of view. Active participation by stakeholders at various levels in any management strategies, especially youths and the younger generation could help guarantee the sustainable use of these zootherapeutic resources. Spreading awareness coupled with a holistic approach to in situ and ex situ conservation, clinical trials and multidisciplinary studies to establish the efficacy of the products could come a long way in safeguarding the traditional knowledge and the resources as well.

Conclusion
The study provides a comprehensive account of the vast wealth of traditional knowledge and healthcare system of Biate tribe inhabiting the Dima Hasao district of Assam. Careful scientific scrutiny and screening of this traditional knowledge could lead to the development of newer and safer drugs as well as spur conservation and sustainable utilization of such a unique habitat and resources. The study area warrants further studies to document the traditional knowledge of the rest of the tribes inhabiting the area, which is the most important aspect in determining practical approaches to the management and sustainable use of the local flora and fauna.


extfieldsofstudy: Biology Medicine

text:
Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing

Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.

* to whom correspondence should be addressed.

Chimeras
We detected unexpectedly high levels of chimeras when we examined the TP-tag junctions in the raw data. Our first hypothesis was that they were caused by intermolecular ligation during circularization. We lowered amounts and compared to blunt end ligation and cre recombinase based systems using data from another study (Akan P., et al, manuscript submitted). In this study blunt-end ligation circularization had a chimera frequency of around 2% and cre recombinase < 1%. Further the fragment concentration during circularization did not affect the chimeric frequency found in this protocol. We therefore concluded that the circularization was not the major contributor of this phenomenon. We suspect that the library PCR causes the elevated levels of chimeras, with the hypothesis being that the secondary structure of the circularization hairpin obscures the activity of the polymerase, causing it to stall or dislodge.

Bias of the transposase
Some interesting aspects of the transposase based library preparation (Nextera, Epicentre now Illumina) was discovered in this study. The transposase in the kit is a variant of the Tn5 transposase 1 and has been characterized for high throughput sequencing applications 2 . We noticed a clear preference in binding to the circularization adapter. This is highly undesirable as those fragments will not produce the information incorporated in the library. Roughly, the average length of the dsDNA circles to be prepared for sequencing is 1500 bp for targets of 3000 bp, assuming a uniform distribution. The adapter is 46 bp, and digestion inside the adapter should by chance happen in approximately 2-4% of the sequences. Instead 12% of the sequences begin with the circularization adapter, and the pattern of digestion is clearly non-stochastic ( Figure S4). The reported recognition sequence for Tn5 is A-GNTYWRANC-T 1 and if we align this motif to our adapter, the best match (82%) is indeed the highest noted insertion site (position 22, Figure S4). This is contrasted to a previously noted low bias of transposition. When we compare to a human shotgun sample generally a low bias is seen, as is the case for all the raw data in this study ( Figure S5). Although a general low bias is seen per position on a global scale, particular sequences can be quite strongly biased. Table S1. Tile-seq throughput.

Supplementary tables
The theoretical output is based on a typical amount of reads obtained from one lane of Illumina HiSeq2000 sequencing, one of the 2x100 bp reads is used for classifying the construct (reading indices). The current throughput is approximated based on the mapped non-duplicated reads of the data, average read length and average amount of bases that could be generated in a typical run.  Table S2.

Comparison of Tile-seq and Sanger sequencing
Cost comparison between Tile-seq and Sanger sequencing. This is a simplified example of a sequencing project containing 96, 3kb amplicons, either sequenced by Tile-seq as described in this report, or by 600 bp Sanger reads in both directions. This example is included to provide an overview of the protocol as compared to the established protocol of Sanger sequencing. Polymer consumption of Sanger sequencing is not considered in this example. If the same amplicon is prepared, 107 oligos (96 forward, 1 reverse, 2 universal and 8 adapters) is needed for Tile-seq and 8 for Sanger (each used as sequence primers). This example assumes low complexity samples (e.g. two alleles), known reference sequence within the amplicon, and unproblematic sequence (e.g. no long homopolymer stretches). If these criteria would not be fulfilled, cloning or sequence primer iterations will be required for Sanger but would not impact the Tile-seq protocol. Tile-seq would produce approximately 40x average coverage, and the Sanger protocol is used to read five 600 bp regions in both directions (2x). The manually intensive steps of Tile-seq is the first PCRs, and for Sanger sequencing setting upp the Big Dye reactions and the subsequent ethanol preparations (960 in total). The purifications of Tile-seq, in total 244, are automated and cost approximately $0.1 per reaction.         Figure S1. Unidirectional exonuclease III degradation. 1% agarose gel analysis of exonuclease III degradation of differently prepared constructs. 1. Unidirectional degradation expected by uracil incorporation and subsequent USER treatment. 2. Same construct as 1 but no USER treatment (degradation in both ends expected). 3. Uracil incorporated in both ends and USER treatment (no degradation expected). 4. SacI restriction site incorporated in 1 end and SacI treatment (unidirectional degradation expected). Figure S2. Time-point tag occurrence after mapping to the lambda amplicon references. The mapped position of each respective TP-tag detected of each amplicon is plotted using a Gaussian kernal density approximation (R). Plots indicate amplicon dependent variation but generally a distinguished order. TP8 omitted due to generally low detection levels. Figure S3. Frequency (y-axis) of reads beginning with the adapter in the raw data, counted per starting position (x-axis) in the adapter. The plot shows a transposase induced bias in adapter fragmentation. Dots indicate variable positions due to the TPtag, and the last 11 bp were not included in the query due to the rise of unspecific hits. Figure S4. Weblogo plots on the base distribution of the 12 first bases of raw data from Nextera prepared libraries. Left and middle shows a typical human shotgun sample with expected random distribution, right shows Tile-seq data. Left plot shows probability over base position, and middle and left shows bits (0.0-0.3) over base position. All plots indicate slight bias supporting the recognition sequence of Tn5.


Implementation of Good Clinical Laboratory Practice (GCLP) guidelines within the External Quality Assurance Program Oversight Laboratory (EQAPOL)

The EQAPOL contract was awarded to Duke University to develop and manage global proficiency testing programs for flow cytometry-, ELISpot-, and Luminex bead-based assays (cytokine analytes), as well as create a genetically diverse panel of HIV-1 viral cultures to be made available to National Institutes of Health (NIH) researchers. As a part of this contract, EQAPOL was required to operate under Good Clinical Laboratory Practices (GCLP) that are traditionally used for laboratories conducting endpoint assays for human clinical trials. EQAPOL adapted these guidelines to the management of proficiency testing programs while simultaneously incorporating aspects of ISO/IEC 17043 which are specifically designed for external proficiency management. Over the first two years of the contract, the EQAPOL Oversight Laboratories received training, developed standard operating procedures and quality management practices, implemented strict quality control procedures for equipment, reagents, and documentation, and received audits from the EQAPOL Central Quality Assurance Unit. GCLP programs, such as EQAPOL, strengthen a laboratory's ability to perform critical assays and provide quality assessments of future potential vaccines.

Introduction
The External Quality Assurance Program Oversight Laboratory (EQAPOL) is a contract awarded by the National Institutes of Health/National Institute of Allergy and Infectious Diseases/Division of AIDS (NIH/NIAID/DAIDS) to support the development of external proficiency testing programs for flow cytometry-, ELISpot-, and Luminex bead-based assays (cytokine analytes). The EQAPOL Program is comprised of a Central Management Team, Central Quality Assurance Unit (CQAU), Statistical Group, Data Management Group, Biorepository, Central Laboratory, A3R5 Neutralizing Antibody Assay Validation Program, and three EQAPOL Oversight Laboratories (EOLs) described in detail in this issue of Journal of Immunological Methods (see Ferrari et al. for ELISpot; Staats et al. for ICS by Flow Cytometry; Sempowski et al. for cytokine-based Luminex). In addition to proficiency testing, EQAPOL is also tasked with creating a diverse panel of high-titer (approximately 10 9 copies/mL), HIV-1 viral culture supernatants grown in PBMC from seed stocks (i.e., from plasma samples and other source material) using a Viral Diversity Core (see Sanchez et al. in this issue) and in validating immunogenicity assays (see Sarzotti-Kelsoe et al. in this issue).
The EQAPOL Laboratory Teams (EQAPOL Viral Diversity Core, Biorepository, Central Laboratory, A3R5 Neutralizing Antibody Assay Validation Program, and each of the EOLs) are required to operate under Good Clinical Laboratory Practices (GCLP), since this is a set of standards designed to facilitate uniform and consistent data generation and reporting. GCLP encompasses both quality assurance (QA) and quality control (QC) principles into its standards. QA proactively and periodically reviews the various components of the research process to assess adherence to standard operating procedures (SOPs) and policies and to determine the accuracy of research records. QC measures are continuous and carried out on all records (QC logs, worksheets, etc.) by the Laboratory Teams. While external laboratories, participating in the EQAPOL proficiency testing programs, are not required to operate under GCLP, many of these laboratories are already GCLP-compliant and perform clinical trial related work. It is for this reason that the program operates in GCLP compliance as it ensures the quality, integrity, and validity of the test data. GCLP was initially designed by the British Association of Research Quality Assurance (BARQA) in 2003 and later expanded upon by the NIH/NIAID/DAIDS in 2008 to provide a regulatory framework to laboratories performing endpoint assays for HIV-1 human clinical trials (Stiles et al., 2003;Ezzelle et al., 2008). The two sets of GCLP guidelines were harmonized in 2009 in order to provide a single set of recommendations for laboratories to utilize (Sarzotti-Kelsoe et al., 2009). The process of converting laboratories into GCLPcompliant entities includes initial laboratory assessments and GCLP training; establishment of SOPs, Quality Management Systems and Study Plans; quality controlled equipment and reagents; optimization and validation of applicable assays; and laboratory audits and corrective action programs. The EQAPOL CQAU, which has over 10 years of experience in performing audits, document control and study monitoring in GCLP compliance, was tasked with implementing these standards for EQAPOL (Sarzotti-Kelsoe et al., 2009;Ozaki et al., 2012;Todd et al., 2012).
External Quality Assurance (EQA) Programs serve three purposes according to GCLP guidance: 1) provide a way for laboratories to ensure that data generated are timely, accurate, and clinically appropriate; 2) provide sponsors with assurance that data generated are of the highest quality; and 3) ensure that human specimens from clinical trials will be tested accurately and reliably (Ezzelle et al., 2008). Although GCLP is a robust set of guidelines governing the conduct of endpoint assays for clinical trials, there are no specific statements regarding the management of external proficiency testing programs. The International Organization for Standardization (ISO) and International Electrotechnical Commission (IEC) have created a set of guidelines/requirements (ISO/IEC 17043) for external proficiency testing programs to follow, so as to provide extra assurance to participants that the program is operated competently (ISO/IEC, 2010). ISO/IEC 17043 requirements primarily apply to management, planning and design, personnel, quality assurance, and confidentiality (ISO/IEC, 2010). The EQAPOL CQAU implemented GCLP, along with many aspects of ISO/IEC 17043, in an effort to make the program compliant to the most appropriate quality standards.
In addition to the proficiency testing programs, EQAPOL was also charged with establishing and characterizing clade-specific HIV-1 viral culture panels representing world-wide genetic diversity. These HIV-1 viral diversity panels are created from HIV-1 positive plasma specimens received from collaborators or from currently existing viral culture supernatants.
Finally, as an option exercised by the NIAID contract, EQAPOL was also charged with performing formal validation of specific immunogenicity assays to be employed as endpoint assays for HIV vaccine clinical trials. The Neutralizing Antibody Assay for HIV-1 in A3R5 cells was optimized and formally validated (Sarzotti-Kelsoe, et al. in this issue) under the oversight of the EQAPOL CQAU.
This report describes the process by which GCLP compliance was established for the entire EQAPOL Program.

Laboratory assessments
Prior to implementing GCLP throughout the EQAPOL Program, the CQAU performed an overall assessment of each EQAPOL Laboratory Team. Fig. 1 illustrates the approach taken by the EQAPOL CQAU to bring each EQAPOL Laboratory Team into GCLP compliance. The laboratory assessment process did not apply to the A3R5 Validation Program as it was already operating under GCLP-compliance. The CQAU began the assessment process with an analysis of the organizational structure of the laboratory, provided documentation of critical areas that needed to be addressed in order to achieve GCLP compliance, created a general plan of action, and conducted GCLP training for all staff members of the laboratory. This introductory training to the GCLP guidelines discussed the requirements for organization and personnel training, equipment maintenance and calibration, reagent and specimen maintenance, document control, assay optimization and validation, and corrective action plans. Each EQAPOL Laboratory Team started the program with different levels of adherence to the principles of GCLP, thus laboratory-specific strategies were developed by the CQAU to address and close gaps in compliance based on previous experience from implementing GCLP in domestic and international laboratories Gilmour et al., 2007). Successful establishment of GCLP compliance for the Laboratory Teams ranged from a minimum of three months for those with more quality experience to approximately two years.

Quality Management Systems
In order to effectively implement document and version control of SOPs, and manage training, equipment and calibration/maintenance records, the CQAU adopted a Quality Management System (QMS) (Q-Pulse, Gael Limited, Scotland) that allowed all of the aforementioned information to be captured and maintained electronically. The software package allows laboratory personnel to access SOPs on any computer with internal server access, which eliminates the need to have paper copies of SOPs present in the laboratoriesfurther mitigating the risk of expired/uncontrolled procedures being used in the laboratory. The use of electronic copies also allows for better version control as paper copies may be altered while in use in the laboratory. In addition, the QMS allows the CQAU to electronically distribute SOPs to laboratory staff, maintain version control of documents, and manage SOP revision and training records.
The QMS has also been instrumental in streamlining the audit process by allowing auditors to complete a report and present findings electronically to each EQAPOL Program Team. The use of tablet computers running QMS-specific applications allows audits to be conducted in a more efficient manner in BSL-3 containment environments where removal of paper is restricted. Laboratory managers are able to view all audit findings and address them via corrective action entries. In order to ensure 21 CFR, Part 11 compliance (http:// www.access.gpo.gov/nara/cfr/waisidx_05/21cfr11_05.html.), the QMS software utilizes an electronic signature package allowing users to enter their username and password to sign-off on training records, audit findings, and equipment maintenance records, which provides an audit trail.

Personnel organization and training
Formal reporting structures must be in place, in compliance with GCLP, to describe the relationships between the EQAPOL Management and the EQAPOL Laboratory Teams. Sollecito and Johnson describe communication as "necessary not only within the team but also between the team and the larger external environment, including other teams (Sollecito and Johnson, 2012)." In addition, errors or lack of communication and coordination have been identified as the key factors that lead to poor performance and detractors from quality (Sollecito and Johnson, 2012). EQAPOL Management created an organizational chart illustrating that relationship and each EQAPOL Laboratory Team also developed a chart describing the intra-laboratory management structure. Fig. 2 provides an example of an organizational chart that clearly delineates the reporting structure within EQAPOL.
According to GCLP, personnel must successfully complete required institutional, general safety and pathogen-specific training before performing laboratory assays. EQAPOL utilized the Duke University Occupational and Environmental Safety Office's on-line training program for laboratory, biological shipping, and other critical training (Chosewood et al., 2009;OSHA, 1992). Additionally, any laboratory personnel performing GCLPcompliant work must be able to document successful completion of assay training and provide evidence that he/she is competent by meeting pre-defined acceptance criteria. The EQAPOL CQAU helped laboratories design and implement SOPs for personnel training to assess initial and ongoing competency of the operators. Competency of operators must be assessed on a routine basis through annual intra-laboratory testing.

Equipment maintenance and calibration
Equipment must be verified that it is "fit for purpose" (Stiles et al., 2003) and must be maintained at high standards for use in GCLP-related activities. Documentation must be present to show that the equipment is properly installed, operated, inspected, cleaned, maintained, tested, and calibrated to ensure that all results are of optimal quality (Ezzelle et al., 2008;ISO/IEC, 2010). An essential component of the initial laboratory assessments was to identify the pieces of equipment that needed to meet these requirements based on risk assessment. For those items that needed to be formally validated, the CQAU assisted laboratory management in developing validation plans according to ICH guidelines (Guideline, 2010) and approving the validation before execution and upon completion. Validation of equipment helps assure that the item behaves accurately and consistently over time. Each piece of equipment used in EQAPOL receives a unique inventory number for quick reference in the equipment database. All EQAPOL Laboratory Teams manage their equipment in the QMS which captures the unique inventory number, model number, serial number, manufacturer, owner, equipment type, and location of the equipment. For the seven EQAPOL Laboratory Teams, approximately 250 pieces of equipment are currently catalogued and maintained under GCLP conditions. Additionally, equipment maintenance and calibration schedules (dependent upon the manufacturer's recommendations) can be entered for each equipment item. The QMS sends reminders to designated personnel for upcoming and/or overdue service for equipment. Data loggers are used by the EQAPOL Central Laboratory to monitor the environment of all temperature-sensitive shipments and provide assurance of the kit's quality to participating sites.

Standard operating procedure development
An elaborate SOP structure was developed for all of the EQAPOL Programs to help ensure that all procedures were conducted in an identical manner to guarantee the quality and integrity of generated data. GCLP guidelines require that SOPs be written in a standard format similar to the one recommended by the Clinical and Laboratory Standards Institute (CLSI) (Ezzelle et al., 2008). An SOP framework was already present in several EQAPOL Laboratory Teams, facilitating the process of implementation. SOPs are reviewed on a biannual basis with the EQAPOL CQAU managing version control. With an SOP system in place, the majority of SOPs that needed to be written were program-specific. For example, each program within EQAPOL developed SOPs on: • how to conduct the assay, • training for the assay, • proper instrument use and maintenance, • reagent bridging, and • data analysis.
In addition to program-specific SOPs, EQAPOL Management developed SOPs that detailed how external proficiency (EP) kits are assembled and ordered using the EQAPOL web portal. While facilitating the control of SOPs, the QMS notified users of the effective dates for new SOPs and sent reminders when documents were due for review.
When conducting assays, EQAPOL laboratory operators are required to complete assayspecific checklists to document who performed each step. Assay checklists are associated to version controlled SOPs and accessible through the QMS. Checklists have been used in many industries as a way to improve quality and safety (Sollecito and Johnson, 2012).
Recently, healthcare has also implemented checklists throughout many of its practices and an increasing amount of evidence has begun to show their importance (Sollecito and Johnson, 2012).

Study Plans
In order to conduct a GCLP compliant study and proficiency testing program, the Study Director needs to write a complete Study Plan outlining the scope and conduct of the study. Although EQAPOL is not fully adherent to ISO standards, the Study Plan incorporates many of ISO's concepts. EQAPOL Study Directors, Leadership, and the Statistical Group prepare program-specific Study Plans for each round of testing under the CQAU oversight. The EQAPOL Study Plans for proficiency testing program include the following: • a detailed list of the program's organizational and management structure, • introduction and background of the program, • requirements for participation, • number and type of program participants, • proficiency panel contents, • assay specifics, • timeline for completion and upload of data, • methods for data reporting, • confidentiality, and • methods for statistical analysis and grading.
EQAPOL Study Plans are approved and finalized by the EQAPOL Study Directors, Leadership, the CQAU Director, and the Statistician prior to the implementation of a particular EP send-out. Study Plans provide evidence that the EQAPOL EP was planned thoroughly and received Central Leadership approval prior to the conduct of the study. The Study Plans are not shared with the laboratories participating in the proficiency testing programs, as they contain information that could unblind their testing.

Audits
The EQAPOL CQAU performs internal and in-process audits of each EQAPOL Laboratory Team (at least annually) as required by GCLP guidelines to identify any potential gaps in compliance and/or deviations from established protocols/SOPs/Study Plans. Audits are performed by the EQAPOL CQAU with the assistance of other QA personnel not involved in the EQAPOL contract, to eliminate potential bias. Checklists are used throughout all audits and reports are prepared with any findings that need to be addressed. Laboratory personnel then respond to the audit findings with appropriate corrective actions to address the issues. The CQAU oversees the proficiency test kit assembly, reagent preparation, and shipment by the EQAPOL Central Laboratory, for at least one send-out per year for each program. As these are often the only proficiency testing materials that clinical trial immunogenicity endpoint assay laboratories receive, it is crucial that all aspects of the sendout are subject to CQAU oversight. At the conclusion of the audits, the CQAU prepares a report summarizing the kit preparation, send-out process and congruence with what is described in the Study Plan. Throughout the first series of audits, common findings existed for many of the EQAPOL Laboratory Teams. These non-conformances typically included a lack of a reagent bridging SOP, assay-specific training SOP, internal competency testing program, and SOPs for all pieces of equipment used during the conduct of the assay (Table  1).

Assay optimization, qualification, and validation
In previously reported studies, performed by the CQAU, it was demonstrated that it is easier to transfer an assay from one laboratory to another when the assay had been optimized and validated prior to the transfer . This process led to high reproducibility of results when inter-laboratory comparison was performed using a proficiency testing program . Validating an assay consists of analyzing the assay parameters recommended by the ICH-Q2 (R1) guidelines (Guideline, 2010): specificity, accuracy, precision, detection and quantitation limits, linearity, range, and robustness. As an option exercised by the NIAID contract, EQAPOL was charged with performing a formal validation of the Neutralizing Antibody Assay for HIV-1 in A3R5 cells. The process began with numerous optimization experiments to help establish pre-set acceptance criteria for the formal validation experiments (Fig. 3) (Sarzotti-Kelsoe et al., 2009). Applicable ICH parameters of validation were selected and a validation plan, inclusive of a statistical analysis plan, was written and authorized by the CQAU. The assay validation was performed, data statistically analyzed and a validation report was written and approved by EQAPOL Leadership, Study Director, Statistician and CQAU, and is reported in this issue (see Sarzotti-Kelsoe et al. in this issue). This validated assay is being transferred to and implemented by multiple laboratories and inter-laboratory reproducibility will be determined by developing a proficiency testing program for the A3R5 assay as a part of EQAPOL.

Reagent Biorepository Management
EQAPOL developed a Biorepository to manage all of the reagents and samples used in the External Quality Assurance (EQA) Programs, virus panel development, assay development, and validation. All materials are recorded electronically in the EQAPOL Application webportal, which captures the reagent name, lot number, expiration date, and storage location.
The EQAPOL Biorepository uses an electronic, wireless temperature surveillance system for constant monitoring of all freezers, refrigerators, and incubators. Repository staff members receive alerts upon deviations from established temperature ranges for all monitored equipment. With this temperature monitoring software, the Biorepository manager and CQAU auditors are able to view and determine when out-of-range temperature excursions occurred, the operator who handled the excursion, and the corrective action taken to address the deviation. This information is stored permanently on the EQAPOL server which is backed up nightly and the back-ups are stored off-site. A Laboratory Data Management System (LDMS) is utilized for the inventory of peripheral blood mononuclear cells (PBMCs) that were processed from fresh leukapheresis blood units and all source material and viruses received/cultured as part of the EQAPOL Viral Diversity Program.
In accordance with GCLP guidelines, all new lots or batches of reagents and samples are parallel tested with previous lots to document their compatibility in the assay prior to being placed into use. Each EOL defined in SOPs the critical reagents required to be parallel tested as well as pass and fail criteria for their eligibility to be placed in proficiency testing kits. Examples of such reagents include PBMCs, Fetal Bovine Serum (FBS), and Streptavidin. Reagents that did not pass parallel testing were not permitted for use in the EQA proficiency test kits.

EQAPOL ordering, shipping and data reporting: web-based portal
As another aspect of the EQAPOL system, a user-friendly and comprehensive web-based portal was developed to secure all of the electronic data generated from the program. The web-based portal was developed specifically for the needs of EQAPOL by SciMed Solutions and operates in compliance with the Federal Information Security Management Act of 2002 (FISMA, 2006). Within the system, laboratories and individual users are given roles that are used to define which aspects of the system they can view. This is of particular importance for the EQA programs where sites should only see their own data.
The EQAPOL web portal is used to document all aspects of each EQA program from sendout of material information to data upload to EQA report retrieval. It maintains reagent and specimen inventory and location, as well as certificates of analysis for reagents. For each EQA send-out, the system records all samples/reagents sent to each site, shipment manifests, tracking information and shipment sent/receipt dates. For each EQA sendout, sites can download assay protocols and data reporting templates on the web-based system. Once a site completes an assay, the data reporting template is completed and returned using the webbased system. Sites also complete a post-assay questionnaire using the portal. As data are received from each site, the templates undergo data verification to ensure that the data are in the proper format prior to importing them into the EQAPOL database. Specific data verification instructions have been developed for each EQA program. The EQAPOL statisticians access all EQA data directly from this database ensuring data integrity throughout the system.
Once the statisticians complete all data analysis, site-specific reports are generated and uploaded to the web-based system with each site having access to their site report. Sites receive both a score out of 100 and a performance rating for each EP, with points awarded for different performance criteria (i.e., precision, accuracy, protocol adherence). These scores and performance ratings are entered into the web-based system to allow performance tracking over time. Finally, the web-based system can capture comments and files used as part of the remediation process. This comprehensive system allows for complete traceability of the EQA process.
The web portal also allows investigators to place orders for viral samples that are prepared by the EQAPOL Viral Diversity Core. Additionally, the portal captures virus culture and characterization data, and provides a means for approved laboratories to view virus inventory and order products. The system maintains an electronic inventory of all specimens and reagents for EQAPOL, and all applicable compliance information (Institutional Review Board, Material Transfer Agreement and Safety Compliance Forms) to ensure that sites only receive material which they are authorized to receive. These orders are then processed by EQAPOL Program Management and sent through an approval process.

Reporting to EQAPOL Leadership
A key aspect to maintaining GCLP-compliance within the EQAPOL Program is to keep Leadership abreast of key quality issues on a regular basis. The EQAPOL CQAU meets with the EQAPOL Steering Committee on a bi-weekly basis with updates on audits, SOP development, proficiency testing kit preparation and shipment status, and other key quality matters. The Steering Committee is comprised of the Program Director and Co-Director, Study Directors, IT Personnel, Finance, Regulatory Affairs Compliance, Statisticians, CQAU and other critical program managers. In addition, the CQAU presents annual progress updates to the EQAPOL External Scientific Advisory Board and receives feedback from the Board.

Archives
The EQAPOL CQAU provides archival functions for all study and proficiency testing materials. The materials are required to be kept in case a study needs to be investigated. Obsolete SOPs, study plans, raw data, and quality control documents are given to the EQAPOL CQAU and are stored in a space that has restricted access and is separated from the laboratory environment. Archived material is maintained indefinitely or until the Sponsor has requested destruction of the documents. In addition to on-site archiving, the CQAU has contracted with a third party sub-contractor to store documents off-site for extended periods of time.

Discussion
Implementation of GCLP into the EQAPOL Laboratory Teams began with an initial laboratory assessment, followed by GCLP training of laboratory staff, implementation of a QMS, equipment qualification, SOP document control, and audits by the CQAU. Converting EQAPOL into a GCLP-compliant operation has a number of added benefits that include increased credibility for the program and improved accuracy, integrity, and traceability of generated data. Having a CQAU in charge of monitoring the EQAPOL project has allowed for more stringent quality control measures of all processes within the program.
Implementing GCLP guidelines designed for clinical trial testing into a setting for proficiency testing was ultimately a challenge for the EQAPOL CQAU. Converting all of the EQAPOL Laboratory Teams into GCLP-compliant laboratories required approximately two years and multiple hurdles were encountered along the way. One of the primary challenges for the EQAPOL Program was unifying all of the programs under one system of SOPs and document control. Since EOLs were members of different departments within Duke, not all of them followed the same system of SOPs. To address this dilemma, the QAU utilized an electronic QMS which ultimately incorporated multiple sets of pre-existing SOPs. Additionally, the CQAU did eliminate SOP redundancies by creating an overarching system of SOPs that were to govern common processes across all programs.
SOP development for new GCLP assays was also a considerable hurdle because some of the laboratories lacked experience in the creation and usage of SOPs. Commitment from laboratories is typically weak at the beginning of GCLP conversion and leads to many delays in SOP finalization. The quicker a laboratory "bought-in" to the concept and importance of GCLP, the smoother the overall transition went. Throughout this two-year process, it became evident that those laboratories that had already implemented some of the key GCLP components into their operations were better prepared to become GCLPcomplaint.
The up-front cost of operating a laboratory under GCLP is often seen as a hurdle, yet mistakes are often more costly then preventative measures (Crosby, 1979). Utilizing the EQAPOL contract, it has been possible to estimate that full implementation of GCLP into a laboratory increases operating costs by close to 20% (G. Sempowski, personal communication). This figure is based on financial estimates from laboratories with preexisting quality control measures such as service contracts for equipment maintenance and calibration, standardized protocols, and reagent inventory systems. Pending funding approval, EQAPOL is considering ISO/IEC 17043 accreditation for the proficiency testing program. Although ISO/IEC accreditation is not a contractual requirement, it would add consistency and quality to all aspects of the program. Accreditation is a mechanism that would reassure external participants that quality and safety standards are demonstrated throughout the program (Sollecito and Johnson, 2012).
GCLP implementation and compliance in the EQAPOL laboratories have provided assurance that all processes are planned, performed, monitored, recorded, and reported in a reliable and consistent manner. Program participants can be assured that the kits are assembled with the utmost quality and detail and that all handling of data and statistical analysis had been thoroughly planned and executed. Ultimately, GCLP programs, such as EQAPOL, strengthen laboratory's ability to perform critical assays and provide quality assessments of potential vaccines in the future. Quality improvement within EQAPOL Laboratory Teams is an ongoing process and changes will continue to be made with future funding so that all aspects of the program can operate at the highest level of compliance possible. EQAPOL QAU approach to GCLP-compliance for EQAPOL Laboratory Teams. This schematic diagram illustrates the plan taken by the EQAPOL CQAU to assess the EQAPOL Laboratory Teams and bring them to GCLP-compliance. Example of organizational chart. This is an example of a GCLP-compliant organizational chart that represents the communication and reporting structure within the EQAPOL Program. Assay development to validation. This schematic diagram illustrates the steps necessary to validate an endpoint immunogenicity assay such as the A3R5 Neutralizing Antibody Assay.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
Fructus Gardenia Extract Ameliorates Oxonate-Induced Hyperuricemia with Renal Dysfunction in Mice by Regulating Organic Ion Transporters and mOIT3

The potent anti-hyperuricemia activities of Fructus Gardenia Extract (FGE) have been well reported. The aim of this study was to evaluate the uricosuric and nephro-protective effects of FGE and explore its possible mechanisms of action in oxonate-induced hyperuricemic mice. FGE was orally administered to hyperuricemic and normal mice for 1 week. Serum and urinary levels of uric acid, creatinine and blood urea nitrogen (BUN), and fractional excretion of uric acid (FEUA) were measured. The mRNA and protein levels of mouse urate transporter 1 (mURAT1), glucose transporter 9 (mGLUT9), ATP-binding cassette, subfamily G, 2 (mABCG2), organic anion transporter 1 (mOAT1), mOAT3, oncoprotein induced transcript 3 (mOIT3), organic cation/carnitine transporters in the kidney were analyzed. Simultaneously, Tamm-Horsfall glycoprotein (THP) levels in urine and kidney were detected. FGE significantly reduced serum urate levels and increased urinary urate levels and FEUA in hyperuricemic mice. It could also effectively reverse oxonate-induced alterations in renal mURAT1, mGLUT9, mOAT1 and mOIT3 expressions, as well as THP levels, resulting in the enhancement of renal uric acid excretion. Moreover, FGE decreased serum creatinine and BUN levels, and up-regulated expression of organic cation/carnitine transporters, improving renal dysfunction in this model. Furthermore, FGE decreased renal mABCG2 expressions in hyperuricemic mice, contributing to its beneficial actions. However, further investigation is needed in clinical trials of FGE and its bioactive components.

Introduction
Hyperuricemia, a key risk factor for the development of gout, has been involved in many diseases such as renal dysfunction, cardiovascular diseases, hypertension, hyperlipidemia, diabetes and metabolic syndrome [1,2]. Impaired renal excretion of uric acid rather than uric acid overproduction, however, was considered to be the major cause of hyperuricemia [3]. Urate transport-related proteins, such as organic anion transporters involved in renal urate secretion and re-absorption, play a pivotal role in maintaining urate homeostasis in vivo [4,5].
Renal tubular handling of uric acid is dependent on tubular transporters [6]. Some organic anion transpoters, such as urate transporter 1 (URAT1, SLC22A12) located at the apical membranes, have been reported to be responsible for proximal tubule transport of uric acid, accounting for urate re-absorption [7]. Glucose transporter 9 (GLUT9, SLC2A9) has two splice variants, both of which have been proposed to mediate urate re-absorption [8]. The two splice variants differ only in the length of their amino-terminal cytoplasmic domains, locating at the basolateral (hGLUT9) and apical sides (hGLUT9 N) △ of proximal convoluted tubules, respectively [9,10]. Two splice variants of mouse GLUT9 (mGLUT9a and mGLUT9b) share the same transport characteristics of hGLUT9, but mainly distributed in distal connecting tubules [11]. ATP-binding cassette, subfamily G, 2 (ABCG2) present at the apical membrane, has been proved to be involved in renal secretory transport of uric acid [12]. Moreover, organic anion transporter 1 (OAT1, SLC22A6) and OAT3 (SLC22A8) distributed at the basolateral membrane contribute to renal urate secretion by mediating urate transport across cell membranes in kidney proximal tubules [13]. Given altered expressions of these organic anion transporters observed in hyperuricemic rodents, the recovery of urate transpoter expressions may exert positive effects on hyperuricemia improvement. Recently, the oncoprotein induced transcript 3 (OIT3), also named liver-specific zona pellucida domain-containing protein (LZP), has been reported to maintain urate homeostasis by regulating the excretion and re-absorption of uric acid in renal tubule via cooperating with Tamm-Horsfall glycoprotein (THP) [14]. Nevertheless, there were few studies focusing on the role of OIT3 in renal urate excretion in hyperuricemic individuals. On the other hand, hyperuricemia is known to cause renal dysfunction, which can be indicated by renal tubular transport of organic cations mediated by renal organic cation and carnitine transporters (OCTs and OCTNs) [15,16]. The reduced expression of renal OCTs and OCTNs is observed in rats or mice with various renal injuries [17][18][19], therefore indicating that regulation of renal OCTs and OCTNs expressions might exert nephroprotective effects on hyperuricemia with kidney dysfunction.
Clinically, hypouricemic agents including uricosuric agents and xanthine oxidase inhibitors are available. Unfortunately, both of them could produce unacceptable adverse effects, such as allpurinol-induced hypersensitivity syndrome [20] and benzbromarone-induced hepatotoxicity [21]. Thus, it is necessary to explore safer and more effective hypouricemic agents. Fructus Gardenia, the dried ripe fruit of Gardenia jasminoides (Ellis.), is used clinically against inflammation, viral encephalitis, hepatitis, tonsillitis, tracheitis and high fever, as recorded in the State Pharmacopoeia of People's Republic of China (2010 edition). Our previous study suggested that FGE could attenuat oxonate-induced hyperuricemia in mice through regulating hepatic xanthine oxidase, nevertheless, the potential of FGE in down-regulating xanthine oxidase activities were not parallel with that in lowering serum uric acid levels [22]. Thus, further investigation is needed in clarifying the exact mechanisms of FGE in improving hyperuricemia. Here, we assessed the hypouricemic effects of FGE with respect to its possible mechanisms by determining the expression levels of renal urate transport-related genes, such as mURAT1, mGLUT9, mABCG2, mOAT1, mOAT3 and mOIT3. In order to evaluate the effects of FGE on renal function in the setting of hyperuricemia, we determined serum creatinine and blood urea nitrogen (BUN) levels, and creatinine clearance (Ccr). Simultaneously, the expression levels of renal organic cation and carnitine transporters, as well as THP levels were examined in FGE-treated hyperuricemic and normal mice.

Effects of FGE on Serum Uric Acid Levels, Renal Excretion of Uric Acid and Renal Function
As shown in Table 1, after seven consecutive days of 250 mg/kg/day oxnate administration, serum uric acid levels of mice were obviously elevated compared with the normal control group. Serum uric acid levels in hyperuricemic animals were significantly lowered by the treatment of FGE at 139, 278 and 556 mg/kg in a dose-dependent manner. In addition, positive control group allopurinol significantly decreased the levels in this model, even lower than that of normal group. On the other hand, potassium oxonate treatment remarkably reduced 24 h-urinary uric acid and creatinine excretion, and increased serum creatinine levels in mice. Notably, treatment of 278 and 556 mg/kg FGE and 5 mg/kg allopurinol recovered uric acid and creatinine excretion in 24 h, as well as serum creatinine levels in hyperuricemic mice. Moreover, FGE at 139 mg/kg also increased the decreased 24 h-urinary uric acid excretion in potassium oxonate-treated mice significantly. Fractional excretion of uric acid (FEUA), as a renal uric acid handling parameter, was remarkably decreased in potassium oxonate-induced hyperuricemic group in comparison with normal group, which could be restored by treatment of FGE and allopurinol at all doses. In addition, neither FGE nor allopurinol affected these biochemical parameters in normal mice, except that allopurinol at 5 mg/kg even significantly lowered uric acid levels in normal group, exhibiting potential side effects.

Effects of FGE on Renal mRNA and Protein Levels of Urate Transporters
The effects of FGE and allopurinol on mRNA and protein levels of renal mURAT1, mGLUT9, mABCG2 and mOAT1 in hyperuricemic mice were shown in Figure 1. Potassium oxonate significantly up-regulated expressions of mURAT1, mABCG2, mGLUT9 and down-regulated expression of mOAT1, but failed to alter expression of mOAT3 in mouse kidneys. In potassium oxonate-treated mice, FGE at the two higher doses significantly restored the mRNA and protein expression levels of these renal urate transporters, while it only succeeded in down-regulting renal mRNA levels of mURAT1 and mABCG2 at 139 mg/kg. Allopurinol at 5 mg/kg succeeded in recovering both mRNA and protein expressions of renal mURAT1, mABCG2 and mOAT1, but failed to down-regulate renal mGLUT9 expressions in hyperuricemic mice. In addition, neither FGE nor allopurinol affected mRNA and protein expressions of renal mURAT1, mABCG2, mGLUT9, mOAT1 and mOAT3 in normal mice.

Effects of FGE on Renal mRNA and Protein Levels of mOIT3 and THP Concentrations
As illustrated in Figure 2, potassium oxonate led to a remarkable up-regulation of renal mOIT3 expression compared to normal vehicle mice, which could be attenuated by FGE in a dose-dependent manner. Moreover, THP concentrations were significantly reduced in urine and increased in kidneys, while mRNA levels of renal THP were remarkably up-regulated in hyperuricemic mice. FGE at 139, 278 and 556 mg/kg effectively reversed oxonate-induced alternations of THP concentrations in mouse kidneys, while FGE at the two higher doses notably recovered renal THP mRNA levels and urinary THP concentrations in hyperuricemic mice. In addition, allopurinol at 5 mg/kg succeeded in down-regulating expressions of renal mOIT3 and modulating THP levels or expressions in kidney and urine of hyperuricemic mice. On the other hand, FGE and allopurinol had no significant effects on levels or expressions of mOIT3 and THP in normal animals.

Effects of FGE on Renal mRNA and Protein Levels of Organic Cation/Carnitine Transporters
As shown in Figure 3, the effects of FGE and allopurinol on mRNA and protein levels of renal mOCT1, mOCT2, mOCTN1 and mOCTN2 in hyperuricemic mice. The mRNA and protein levels of renal mOCT1, mOCT2, mOCTN1 and mOCTN2 were remarkably decreased in hyperuricemic mice compared with normal vehicle group. Treatment of FGE at the two higher doses significantly up-regulated both mRNA and protein expressions of renal organic cation/carnitine transporters, except that FGE at 278 mg/kg failed to reach significance despite increased renal protein levels of mOCTN1. In addition, FGE at 139 mg/kg could also significantly increase mRNA and protein levels of renal mOCT2 in hyperuricemic mice. Moreover, allpurinol at 5 mg/kg significantly up-regulated renal expressions of mOCT1, mOCT2, mOCTN1 and mOCTN2 in this model. On the other hand, FGE or allopurinol did not significantly alter the expression levels of renal mOCT1, mOCT2, mOCTN1 and mOCTN2 in normal mice.  Kidney cortex tissues were used for western blot analysis of mOCTN1, mOCTN2, mOCT1 and mOCT2 (E). The ratio of mOCT1 (F), mOCT2 (G), mOCTN1 (H) and mOCTN2 (I) density to α-tubulin density. Data were expressed as the mean ± S.E.M. for 4 mice. # p < 0.01, ## p < 0.01, ### p < 0.001, compared with normal vehicle animals. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with hyperuricemia vehicle animals.

Discussion
FGE, currently used as diuretic in traditional Chinese medicine, has not been used clinically in gout and hyperuricemia treatment. In the present study, we firstly observed that FGE exerted hypouricemic effects by cooperatively reducing urate reabsorption through down-regulating renal mURAT1, mGLUT9 and enchancing secretion through up-regulating mOAT1 in hyperuricemic mice. Of note, expressions of mOIT3 associated with alternations of THP concentrations were firstly explored in oxonate-induced hyperuricemic mouse kidneys, which could be restored by FGE treatment. On the other hand, FGE was found to restore the expressions of renal mABCG2, mOCT1, mOCT2, mOCTN1 and mOCTN2, improving oxonate-induced renal dysfunction in mice. These findings confirmed that FGE exhibited beneficial effects on oxonate-induced hyperuricemia with renal dysfuction.
In clinic, increased serum uric acid levels induced by renal urate underexcretion contribute to about 90% of hyperuricemia and gout [5]. The enhanced protein levels of URAT1 contributed to hyperuricemia in obesity/metabolic syndrome model mice [23]. Homozygous loss-of-function mutations of URAT1 or GLUT9 result in a total defect of uric acid absorption, leading to severe renal hypouricemia [24,25]. Genetic impairment of ABCG2 increased serum uric acid levels in humans [12]. The decreased expression level of renal rOAT1 was observed in hyperuricemic rats induced by combined administration of uricase inhibitor oxonic acid and uric acid [26]. Thus, renal handling of urate transport constitutes an important target for drugs to treat hyperuricemia and gout [27]. Consistent with our previous studies, administration of potassium oxonate for seven consecutive days induced dys-expresssion of renal uric acid transporters including mURAT1, mGLUT9 and mOAT1 in the present study, subsequently leading to hyperuricemia [27,28]. It was worth noting that up-regulation of renal mABCG2 was found in oxonate-induced hyperuricemic mice in the present study, which might be explained to be an adaptive response to accumulation of urate that have to be excreted into the urine through kidney [29]. Parallel with its enhancement of renal urate excretion and reduction of serum urate in hyperuricemic mice, FGE could recover expressions of these organic anion transporters, exhibiting beneficial effects on regulating uric acid excretion. The restoration of mABCG2 in hyperuricemic mice by FGE might be contributed to its uricosuric and nephro-protective actions. Importantly, we first observed the up-regulation of mOIT3 in this model. Consistently, it was reported that OIT3 deficiency impaired uric acid reabsorption in renal tubule, indicating that OIT3 over-expression might stimulate urate reabsorption and subsequently result in hyperuricemia. Moreover, THP co-localized with OIT3 in the renal thick ascending limb of Henle's loop was a useful marker of renal dysfunction in hyperuricemia [14], as demonstrated by some findings that mutations in THP gene lead to familial juvenile hyperuricemic nephropathy and develop kidney dysfunction in mice [30][31][32]. In the present study, obvious alternations of renal and urinary THP levels were observed in oxonate-induced hyperuricemic mice, which was in accord with dys-expression of mOIT3. Meanwhile, FGE treatment restored renal mOIT3 expressions as well as mTHP levels in kidney and urine, which might be partly due to that FGE disrupted the protein polymerization involving both mOIT3 and mTHP, promoting excretion of mTHP to the urine [14]. As those findings indicated, OIT3 provided us with some insights in exploring a new target for treating hyperuricemia. Furthermore, recent studies suggested that human OAT2 located at the basolateral membrane of proximal tubules played a role in renal uric acid uptake from blood as a first step of tubular secretion [33], while OAT4 and OAT10 in the apical membrane contributed to proximal tubular urate absorption in human kidneys [34,35]. Different from that in human, the roles of OAT2 and OAT10 in renal urate transport have not been identified while OAT4 even has no ortholog in mouse kidneys [36]. Therefore, further studies with human renal tubular epithelial cells are needed to confirm the exact mechanisms of FGE, involving OAT2, OAT4 and OAT10.
On the other hand, renal organic cation and carnitine transporters, being responsible for the uptake of endogenous substrates and detoxification of xenobiotics and chemotherapeutics, are considered to be related with renal dysfunction [37][38][39]. Many reports have touched upon the relationship between dys-expression of renal OCTs/OCTNs and renal dysfunction induced by cadmium, adenine or oxonate [17,[26][27][28]40]. In the present study, FGE restored renal mRNA and protein levels of mOCT1, mOCT2, mOCTN1 and mOCTN2 in hyperuricemic mice, restoring renal function characterized by decreased serum creatinine and BUN levels, as well as increased FEUA. Therefore, we concluded that the nephro-protective effects of FGE on oxonate-treated hyperuricemic mice might be partly attributed to the regulation of renal mOCT1, mOCT2, mOCTN1 and mOCTN2 expressions.
FGE has a variety of biological activities, but few studies have focused on its uricosuric and nephro-protective effects. Major effective constituents in FGE, such as flavonoids, geniposide, crocin and crocetin, have been demonstrated to exhibit hypouricemic effects or nephro-protective actions in animal models [27,28,41,42]. These observations suggested that, although the precise mechanisms of the uricosuric and nephro-protective effects of FGE remain incompletely understood, it might be attributed to the active principles by the regulation of renal organic ion transporters in hyperuricemia with kidney dysfunction. On the other hand, daily administration of 30g fructus gardenia resulted in hepatotoxicity, while oral administration of 1.62g/kg FGE in rats for three consecutive days led to hepatic function and morphology abnormalities [43]. In the present study, FGE treatment exhibited no side effects in normal mice at the effective doses which seemed to be much lower than the toxic dose, therefore, in consideration of the similarity of renal organic ion transporters between mice and human [11,44,45], FGE might have great potential in clinical application.

Reagents
Uric acid, allopurinol and potassium oxonate were purchased from Sigma (St. Louis, MO, USA). TRIzol reagent and reverse transcriptase moloney murine leukemia virus (M-MLV) used for cDNA synthesis were obtained from Invitrogen (Carlsbad, CA, USA). SYBR green qPCR mix was purchased from Bio-rad Laboratories (Hercules, CA, USA). Assay kits of BUN, creatinine and protein concentrations were obtained from Jiancheng Biotech (Nanjing, China), respectively. ELISA assay kit of THP was purchased from R&D (Minneapolis, MN, USA).

Plant Material
Fructus gardenia was purchased from HuangQingRenJian drugstore (No: 1010004), and was authenticated by Prof. X.W. Lai, Jiangxi University of Traditional Chinese Medicine, Nanchang, China.

Preparation of the Ethanol Extract
Fructus gardeniae, 100 g, after being chopped into small pieces and ground, was dipped in 60% ethanol (800 mL), and then boiled for 2 h. The extract was immediately filtered through a two-layer mesh. The residue was again boiled in 800 mL 60% ethanol for 2 h and left to stand for 1 h before filtration. The combined filtrate was then concentrated under reduced pressure and evaporated to dryness and the yield of the extract is 35.71%.

Animals
All animal studies were approved by the Animal Ethics Committee of China Pharmaceutical University. Male Kun-Ming strain of mice (20 ± 2 g) purchased from the Central Institute for Experimental Animals of Zhejiang were housed 5 per cage (320 × 180 × 160 mm). Mice were maintained in a room controlled at 22-24 °C with a relative humidity of 55 ± 5% and a 12:12-h light-dark cycle (6:00 a.m.-6:00 p.m.). They were given standard chow ad libitum for the duration of the study and allowed 1 wk to adapt to the laboratory environment before experiments.

Hyperuricemic Mice and Drug Administration
Experimental animal model of hyperuricemia induced by oral administration of potassium oxonate has been used in pharmacological studies [28,46]. Mice were divided into 10 groups: normal + vehicle, oxonate + vehicle, oxonate + 139 mg/kg FGE, oxonate + 278 mg/kg FGE, oxonate + 556 mg/kg FGE, oxonate + 5 mg/kg allopurinol (as a positive control drug), and normal + 139 mg/kg FGE, normal + 278 mg/kg FGE, normal + 556 mg/kg FGE, normal + 5 mg/kg allopurinol. Dosages of FGE and allopurinol were determined based on the adult dosages (Chinese Pharmacopoeia Committee, 2010) and our preliminary studies. According to State Pharmacopoeia of People's Republic of China (2010 edition), dosage of fructus gardenia for adults is 6 g (the total raw materials)/day, or 780 mg raw materials/kg/day calculated by the formula that converts dosage of human into that of mouse in terms of the respective body surface areas in accordance with the Chinese Medicine Pharmacology Research Technology (1994). With a yield of 35.71%, the dose of FGE was set at 278 mg/kg/d in the present study. Therefore, we used three doses of FGE at 139, 278, 556 mg/kg in this study.
For administration to the animals, oxonate, FGE and allopurinol were freshly dispersed in 0.9% CMC-Na solution, respectively. The solution concentrations were 16.67 mg/mL for oxonate, 9.27, 18.53, 37.07 mg/mL for FGE, and 0.33 mg/mL for allopurinol, respectively. The volume of drug administered was based on body weight measured immediately prior to each dose. Food, but not water, was withdrawn from the animals 1 h prior to the administration. Mice were administered in a volume of 15 mL/kg by gavage once daily with potassium oxonate (250 mg/kg) or 0.9% CMC-Na solution (vehicle) at 8:00 a.m. for seven consecutive days. FGE and allopurinol were orally initiated at 9: 00 a.m. on the day when the oxonate was given.

Blood, Urine and Tissue Sample Collection
From 24 h before final administration on the seventh day, 24 h urine sample for each mouse collected in a metabolic cage was centrifuged at 2,000 × g for 10 min to remove the particulate contaminants.
Whole blood samples were collected 1 h after final administration on the seventh day and centrifuged at 10,000 × g for 5 min to obtain serum. Serum and urine samples were stored at −20 °C until biochemical assays. Simultaneously, kidney cortex tissues were rapidly and carefully removed on ice-plate, and stored at −70 °C for assays.

Determination of Uric Acid and Creatinine Levels and Renal Urate Handling Parameters
Uric acid concentrations in serum (Sur) and urine (Uur) were determined by the phosphotungstic acid method [47]. Creatinine levels in serum (Scr) and urine (Ucr), as well as BUN levels were determined using trinitrophenol colorimetry or urease ultraviolet kits, respectively. FEUA was calculated using the formula: FEUA = (Uur × Scr)/(Sur × Ucr) × 100, expressed as a percentage [48].

Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted by the use of TRIzol reagent, and DNA was removed by the use of the DNA-free kit. Real-time PCR with SYBR Green involved the use of the SuperScript III Platinum Two-Step RT-PCR Kit on an ABI PRISM 7000 sequence detection PCR system (Applied Biosystems, Foster City, CA, USA). Sequences of primers were summarized in Table 2. Mouse kidney cortex for mURAT1, mABCG2, mGLUT9, mOAT1, mOAT3, mOIT3, mOCT1, mOCT2 and α-tubulin analysis was homogenized in 1 mL RIPA buffer using a Polytron set, and centrifuged at 3000 × g for 15 min (4 °C). The supernatant was centrifuged at 12,000 × g for 20 min, 4 °C to get tissue supernatant. Protein concentrations of kidney cortex supernatant were determined by the Bradford protein assay reagent (Jiancheng Biotech, Nanjing, China) with bovine serum albumin as standard. western Blot analyses for renal mURAT1, mABCG2, mGLUT9, mOIT3, mOAT1, mOAT3, mOCT1, mOCT2, mOCTN1, mOCTN2 and α-tubulin were described in our previous studies [27,42]. Primary antibodies were listed in Table 3.

Determination of THP Levels
Mouse kidney tissues were homogenized in 10 wt/vol of sodium chloride on ice and then centrifuged (10,000 × g) for 15 min at 4 °C. THP levels in serum, urine and kidney were analyzed by ELISA kits, respectively. Fifty µL standard, control or sample mixed with 50 µL assay diluent were added to each well and incubated at room temperature for 2 h. After removing unbound material by washing five times, 100 µL of conjugate was added to each well and incubated at room temperature for 2 h. After rinsing each well, 100 µL substrate solution was added and incubated at room temperature away from light. 30 min later, the reaction was stopped by adding 100 µL of stop solution to each well, and optical density was read at 450 nm within 30 min in enzyme-labeled instrument.

Statistical Analysis
All data were expressed as means ± SD. Statistical differences between means were determined using analysis of variance on ranks followed by a post hoc test (LSD procedures) as appropriate. A value of p < 0.05 was considered statistically significant. Figures were obtained by the Statistical Analysis System (GraphPad Prism 5, GraphPad Software, Inc., San Diego, CA, USA).

Conclusion
In conclusion, the present study has demonstrated for the first time that FGE exhibited potently uricosuric effects in hyperuricemic mice by down-regulating renal mURAT1, mGLUT9 expressions and up-regulating renal mOAT1 expression. Moreover, we firstly found that renal mOIT3 dys-expressions participated in the pathogenesis of oxonate-induced hyperuricemia, which could be reversed by FGE treatment. On the other hand, FGE improved renal dysfunction in hyperuricemia by recovering renal expression of mABCG2, mOCT1, mOCT2, mOCTN1 and mOCTN2 in mice. The molecular mechanisms by which FGE displays the uricosuric and nephro-protective actions in oxonate-treated mice provide some scientific support for the empirical use of Fructus Gardenia as a traditional Chinese medicine for hyperuricemia with renal dysfunction.


Dendritic cell-based immunotherapy in ovarian cancer

Worldwide, 80% of patients with ovarian cancer die of the disease. New treatments for this aggressive disease are therefore being intensively searched. Although dendritic cell-based vaccines against gynecological malignancies are in their infancy, this immunotherapeutic approach holds much promise. Here, we present our view on an optimal dendritic cell-based immunotherapeutic strategy against ovarian cancer.

Ovarian cancer is the second most common type of gynecologic tumor in women, with a wordwide incidence of 12.5/100,000 women. Ovarian cancer presents as a silent killer, usually metastasizing throughout the abdomen before causing symptoms. Consequently, in 63% of patients ovarian cancer is detected as FIGO stage III or IV disease. This is associated with a poor prognosis, median survival being 36-53 and 20 months for patients with FIGO stage III and IV disease, respectively. Surgery in combination with platin-based adjuvant chemotherapy remains the cornerstone therapeutic modality. If the tumor relapses within 6 months of the initial treatment with platin-based combination chemotherapy, the prognosis is very poor. 1 Eventually, 80% of patients that are diagnosed with ovarian cancer will die of the disease.
It is thus clear that new treatment modalities against ovarian cancer are urgently needed. Nowadays, the major focus of targeted anticancer therapies is on molecular changes and genetic alterations, that may offer patients a personalised treatment. However, the heterogeneity of neoplastic cells constituting primary neoplastic lesions and their metastases is an important issue in this respect. In an attempt to target rather universal mechanisms of tumorigenesis, for example vascular endothelial growth factor (VEGF)-dependent vascularization, passive forms of immunotherapy based on preformed antibodies have been devised and tested in several clinical Phase II trials, mainly resulting in a prolongation of progression-free survival, but associated with a non-negligible toxicity. 2 Active immunotherapy as a measure to treat gynecological malignancies has been neglected for a long time and still is in its infancy. This approach relies on anticancer vaccines that are able to elicit an immune response against tumor-associated antigens (TAAs) in the human body. Several technical aspects of this type of immunotherapy should be considered.
First, TAA-based immunotherapeutic strategies can be divided into 2 groups: one, those that rely on products derived from whole cancer cells, including whole cancercell lysates, dendritic cell (DC)/cancer cell fusions, total cancer cell RNA or mRNA; and two, those that rely on one or more specific TAAs. Both have specific advantages and disadvantages, though whole cancer-cell approaches are gaining momentum, mostly because they encompass a personalized and broad range of known and unknown tumor antigens (and hence may minimize the development of tumor escape variants) as well as many proteins shared with normal cells, including immunostimulatory cytokines. Moreover, this approach provides both MHC class I and II TAAderived epitopes. The same advantages do not all apply when specific TAAs are used. However, side effects should generally be milder with TAA-based vaccines than with whole cancer cell-based approaches. Over 60 ovarian cancer-relevant TAAs have been studied, including Wilms' tumor 1 (WT1), which is overexpressed by a relevant proportion of these tumors. 3 Second, TAAs can be administered as pure preparations, i.e., as naked peptides, proteins or DNA molecules, or via a carrier. DCs are one of the most common carriers used in this setting, offering a superior platform for stimulating TAAspecific immune responses in vivo. 4 One of the main obstacles against the use of synthetic peptides is that only patients bearing the HLA type for which the peptide is restricted can obtain benefits from this approach. One strategy that would circumvent this issue is the transfection of TAA-coding mRNA into DCs, resulting in transient TAA expression and subsequent presentation of antigenic TAA-derived epitopes on the DC surface. The work of several laboratories suggests that this approach is an effective, if not superior, method to generate immunostimulatory DCs. 5 However, in order to present TAA-derived epitopes in the context of both MHC class I and II molecules, TAA-coding mRNAs should be modified with a lysosome-associated membrane protein (LAMP) signal. 6 Table 1 gives an overview of all clinical studies testing DC-based immunotherapy in ovarian cancer patients till now. Although DC-based immunotherapeutic strategies hold great promise, significant clinical responses have yet to be achieved. Several studies are ongoing to optimize this approach. Our research group focuses on two types of tumor: high grade glioblastoma (HGG) 7 and pelvic gynecological malignancies. 8,9 In both these settings, DCs were chosen as carries of TAAs for vaccination. Since HGG has not yet been associated with a specific TAA, we chose to employ cancer cell lysates. Conversely, in uterine and ovarian cancer we chose to work with a specific TAA, namely, WT1. DCs were loaded with TAA-coding mRNA. Both the use of total tumor lysate and WT1-coding mRNA offered different insights in the tumor biology, behavior and immune monitoring. In contrast to the rather low clinical responses previously reported for DC-based immunotherapy in this setting, we achieved a significant improvement in the clinical outcome of HGG patients receiving DCs loaded with tumor lysate. Our studies with gynecological cancer patients receiving DCs loaded with WT1-coding mRNA generated profound insights into the immune responses elicited by the vaccine. Therefore, we believe that new studies testing DC-based immunotherapy in ovarian cancer patients should combine the whole tumor cell approach with the use 1 or 2 defined TAAs.
However, besides focusing on the development of immunotherapeutic strategies that elicit improved anticancer immune responses, it is crucial not to neglect the immunosuppressive mechanisms within the tumor microenvironment, which normally hamper antitumor immunity. Therefore, combinatorial approaches are likely to induce therapeutically relevant immune responses, as recently demonstrated by Kandalaft et al. (Table 1). Specific agents have been developed to limit immunosuppression by targeting for example regulatory T cells and myeloid-derived suppressor cells. In our opinion, it is more interesting to harness chemotherapeutic agents that are currently employed against metastatic ovarian cancer. Several of them have indeed been shown to exert immunomodulatory effects and to induce the immunogenic demise of cancer cells. 10 The doses and combination schedules of these immunochemotherapeutic regimens will however be crucial to achieve optimal anticancer immune responses.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.