extfieldsofstudy: Biology Medicine

text:
Green Tea Polyphenols Reduced Fat Deposits in High Fat-Fed Rats via erk1/2-PPARγ-Adiponectin Pathway

Objective Hypoadiponectinemia contributes to the development of obesity and related disorders such as diabetes, hyperlipidemia, and cardiovascular diseases. In this study we investigated the effects of green tea polyphenols (GTPs) on adiponectin levels and fat deposits in high fat (HF) fed rats, the mechanism of signaling pathway was explored as well. Methods and Results Male Wistar rats were fed with high-fat diet. GTPs (0.8, 1.6, 3.2 g/L) were administered via drinking water. Serum adiponectin and insulin were measured by ELISA, mRNA levels of adiponectin and PPARγ in visceral adipose tissue (VAT) were determined by Real-time PCR, protein levels of PPARγ, phospho (p) - PPARγ, extracellular signal regulated kinase (erk) 1/2 and p-erk1/2 in VAT were determined by western blot. GTPs treatment attenuated the VAT accumulation, hypoadiponectinemia and the decreased mRNA level of adiponectin in VAT induced by HF. Decreased expression and increased phosphorylation of PPARγ (the master regulator of adiponectin), and increased activation of erk1/2 were observed in HF group, and these effects could be alleviated by GTPs treatment. To explore the underlying mechanism, VAT was cultured in DMEM with high glucose to mimic the hyperglycemia condition in vitro. Similar to the results of in vivo study, decreased adiponectin levels, decreased expression and increased phosphorylation of PPARγ, and elevated erk1/2 phosphorylation in cultured VAT were observed. These effects could be ameliorated by co-treatment with GTPs or PD98059 (a selective inhibitor of erk1/2). Conclusion GTPs reduced fat deposit, ameliorated hypoadiponectinemia in HF-fed rats, and relieved high glucose-induced adiponectin decrease in VAT in vitro. The signaling pathway analysis indicated that PPARγ regulation mediated via erk1/2 pathway was involved.

Introduction
With a tremendous increase in China, the prevalence of obesity has been rising all over the world in the last few decades [1]. Obesity is a vital risk factor for a number of chronic diseases, such as diabetes, vascular diseases, and cancers. Therefore, prevention of obesity becomes crucial in public health. The medical and surgical methods have been used to treat obesity, however, the adverse effects of these treatments are obvious [2,3]. Usage of dietary agents to prevent or treat obesity could be a safer solution to the problem. Epidemiological and laboratory studies showed that green tea possesses significant anti-obesity, anti-diabetes, and cardio-protective properties [4,5,6]. Epigallocatechin 3-gallate (EGCG), the major component of catechines in green tea, was demonstrated to reduce body weight gain in animal models of obesity [7,8]. Human intervention studies showed that catechins, the bioactive ingredients of green tea polyphenols (GTPs), decreased body weight and affected markers of obesity in healthy, obese and diabetic conditions [9][10][11]. These data indicated that tea polyphenols are potentially powerful agents in the prevention of obesity. It was suggested that tea decreases body weight gain by reducing lipid and carbohydrate absorption, increasing lipid and carbohydrate utilization, and lessening the lipogenesis, but the mechanism of tea's anti-obesity effects is still inconclusive.
Adiponectin (also referred to as GBP-28, apM1, AdipoQ and Acrp30) is an adipocytokine exclusively secreted by adipose tissue into the blood stream. Adiponectin counts for approximately 0.01% of the total plasma proteins. Hypoadiponectinemia, which refers to a low circulating level of adiponectin, was documented in obesity and its related diseases including insulin resistance, hyperglycemia and cardiovascular diseases [12][13][14]. Accumulating evidence showed that hypoadiponectinemia played a key role in the pathogenesis of obesity and related diseases [15][16][17]. Furthermore, adiponectin administration to obese or diabetic mice can reduce body weight and blood glucose levels while enhancing insulin sensitivity [18][19][20]. Based on these data, adiponectin was conceived to be a novel therapy target for obese and insulin resistance [21]. Elevation of adiponectin level by EGCG was documented in non-obese spontaneous diabetic rats and spontaneous hypertension rats [22,23]. Based on the above research, we hypothesized that GTPs might regulate the adiponectin levels in HF fed rats, through which GTPs exert their preventive effects on obesity and related diseases.
Adiponectin expression is regulated by factors such as peroxisome proliferator-activated receptor (PPAR) c, CCAAT-enhancer-binding protein (C/EBP) a, Kruppel-like factor 7 (KLF7), and sterol regulatory element binding protein-1c (SREBP-1c), Among these factors, PPARc is recognized as the master regulator of adiponectin gene transcription. PPARc binds directly to a functional PPAR-responsive element (PPRE) in adiponectin promoter, and increases the transcription of adiponectin gene [24]. Thiazolidinediones (TZDs), agonists of PPARc, stimulated gene expression and increased the plasma level of adiponectin in obese mice and obese people with insulin resistance [25]. Dominant negative mutant in PPARc gene inhibited circulating adiponectin by 5 times [26]. Although the results were inconsistent, PPARc expression was reported to change in obese or diabetic conditions. Park et al. and Inoue et al. reported PPARc expression increased in skeletal muscle, liver or adipose tissue of obesity or diabetic subjects [27,28]. Another study showed that long term over-feeding induced obesity and decreased PPARc expressions in skeletal muscle and VAT [29]. The mechanism of these contradictory results was not fully understood. Evidence showed erk1/2 activation was involved in regulation of PPARc expressions in different tissues [30,31]. A MAP kinase site, which is confirmed to be phosphorylated by MAPKs, was identified on the NH2-terminal domain of PPARc, and phosphorylation on the site would repress the transcription activity of PPARc then decrease the adiponectin level [32]. Increased phosphorylation of PPARc was observed in obesity and insulin-resistant conditions, and the involvement of increased activation of erk1/2 was proposed [33]. Regulating effects of GTPs on the expression of PPARc were reported though the results were not consistent [34]. Additionally, our previous study demonstrated GTPs would affect the MAPK signal pathways [35]. Based on these facts, we hypothesized that GTPs may regulate adiponectin levels via PPARc by modulating erk1/2 activations.
In this study, male Wistar rats were fed on HF diet, and the effects of GTPs on body weight gain and adiponectin levels were observed. The signaling pathway mechanism was explored along with the regulatory roles of PPARc and erk1/2.

Ethics Statement
This study was carried out in strict accordance with the guidelines and authorization for the use of laboratory animals. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Huazhong University of Science and Technology (Permit number: S249). All efforts were made to minimize suffering.
Anti-PPARc antibody was purchased from Abcam (Cambridge, MA, USA); GTPs (purity .98%) were purchased from Fuzhou Rimian Inc. (Fuzhou, Fujian, China); TRIZOL was from Invitrogen Inc. (Carlsbad, CA, USA) and Real time quantitative PCR kit was purchased from TAKARA Bio Inc. (Otsu, Shiga, Japan); ELISA kits for adiponectin and insulin were purchased from R&D Systems. (MN, USA). All other chemicals were of the highest grade commercially available.

Animals
After one week's acclimation, thirty male Wistar rats, weighting 40-60 g, were randomly divided into 5 groups. The control group was fed on standard chow; the other 4 groups were fed with modified HF chow containing 60% (w/w) standard chow, 12% lard, 12% sugar, 6% peanuts powder, 8% yolk powder, and 1% milk powder. Since the 4th week, 3 of the 4 HF groups started to drink water containing different concentrations of GTPs (0.8, 1.6, 3.2 g/L). At the end of the 26 th week, all animals were sacrificed, tissues were snap frozen with liquid nitrogen then stored at 280uC freezer. Blood biochemical indexes, adiponectin levels and insulin levels were tested. This study was carried out in strict accordance with the guidelines and authorization for the use of laboratory animals. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Huazhong University of Science and Technology. All efforts were made to minimize suffering.

VATs Culture
Male Wistar rats were sacrificed and VATs were collected under asepsis condition. One hundred fifty mg VAT was maintained at 37uC in 5% CO2. VATs cultured in DMEM with high glucose were treated without GTPs, with GTPs (4 mg/ml) for 48 hrs, or treated with PD98059 for 1 hour, respectively. VATs incubated with 5.5 mmol/L glucose in medium were used as control.

Quantitative Real-time PCR
Following the manufacturer's instructions, total RNA was extracted from fat tissue using TRIZOL then quantified by UV spectrophotometry. Samples with the A260/280 ratio lies between 1.8,2.0 were used. Reverse transcription reaction (RT) was performed with 1 mg total RNA from each sample using random primers. Real time PCR analysis was carried out using qPCR SYBR Green mix with the following parameters: 1 cycle, 95uC, 5 s; 40 cycles, 95uC 10 s; 57uC, 30 s. Changes of gene expression were determined by the comparative Ct method with GADPH as reference. The primers used in the PCR were as follows: GADPH:

Enzyme-linked Immunosorbent Assay
To assess adiponectin secretion and circulating insulin, the blood of the rats was collected and serum was separated by centrifugation, and the culture supernatant of fat explants was harvested and centrifuged to remove impurities. Adiponectin and insulin concentrations were measured using an enzyme-linked immunosorbent assay according to the manufacturer's instruction. Figure 1. GTPs reduced body weight gain and VAT coefficient induced by HF diet without affecting the energy intake. GTPs treatment did not affect the energy intake in compare with the HF group, although the food intake of the control group is higher than other groups(A), no significant difference was observed in energy intake (B). Difference in body weight started from the 9 th week (C). The body weight was obviously higher in the HF group in compare with all other groups (D); VAT coefficient of the HF group was obviously higher than the control group, and GTPs treatment alleviated the effect (E). (* P,0.05 vs. the control; # P,0.05 vs. the HF group). Data is expressed as the mean 6 SEM (N = 6). doi:10.1371/journal.pone.0053796.g001 Measurements were performed in six replicates. Results were presented as ng/mL.

Statistical Analysis
All quantitative data are presented as Mean 6 S.E. Data were compared by ANOVA-SNK or Dunnett's T3 test. Differences were considered significant when P,0.05.

GTPs Reduced Body Weight Gain and VAT Coefficient without Affecting the Food Intake
As shown in fig. 1, GTPs treatment did not affect the energy intake in comparison to the HF group, although the food intake of the control group was higher than other groups, no significant difference was observed in energy intake due to the different energy densities of normal and HF diet (A, B). Difference in body weight gain started from the 9 th week (C). At the end of the experiment, the body weight and VAT coefficient were obviously higher in the HF group; the effects were ameliorated by GTPs treatment (D,E). GTPs treatment also reduced the blood glucose and HOMA-IR index and reversed the lipid profile change in HF fed rats (Table 1). However, GTPs treatment did not affect the circulating insulin level compared to the HF group.

GTPs Relieved the Down-expression of Adiponectin in VAT and Serum Induced by HF Diet
To assess the adiponectin expression and secretion, adiponectin mRNA in VAT was tested by qRT-PCR, and serum adiponectin was tested by ELISA. Fig. 2 demonstrated that HF fed rats exhibited lower expressions of adiponectin at transcriptional and phenotypic levels, while treatment of GTPs alleviated the adiponectin-reducing effect of HF diet.

GTPs Attenuated the Down-expression of PPARc, the Increased Phosphorylation of PPARc, and the Increased Phosphorylation of erk1/2 in VAT Induced by HF Diet
Researches demonstrated that adiponectin is a downstream target gene of PPARc [13] while p-PPARc preserves an inhibitory effect on adiponectin expression [20]. The mRNA of PPARc and protein expression of PPARc and p-PPARc were tested by qRT-PCR and Western Blot, respectively. As shown in Fig. 3, both the mRNA expression (A) and protein expression (B) of PPARc decreased in HF group while phosphorylation of PPARc (C) increased in HF fed group, and these changes were attenuated by GTPs treatment. Increased phosphorylation of erk1/2 induced by HF diet was also ameliorated by GTPs treatment (D).

Discussion
The present work demonstrated that GTPs alleviated VAT deposit and hypoadiponectinemia in HF fed rats. Meanwhile, GTPs up-regulated the expression while down-regulating the phosphorylation of PPARc, the principal regulator of adiponectin. GTPs also inhibited the over-activation of erk1/2 induced by HF diet. Similar results were observed in high glucose incubated VAT, co-treated with GTPs in vitro. Like being treated with GTPs, selective inhibition of erk1/2 alleviated the down-expression of adiponectin, down-regulated phosphorylation of PPARc, and upregulated the expression of PPARc induced by high glucose incubation.
Adiponectin was demonstrated to be adversely associated with obesity, insulin resistance, cardiovascular diseases, and obesity related fatty liver disease [37,38]. The production of adiponectin was reported to be related to visceral fat deposits [39]. Hypoadiponectinemia was observed in obese humans [40] and obese animal models in the present study, while increased adiponectin levels was observed after weight loss [41]. Genetic studies showed that adiponectin polymorphism, SNPs 45T to G and 276G to T are related to obesity in humans [42] and the G/G genotype for SNP276 was associated with lower serum adiponectin levels and waist-to-hip ratio [43], novel genetic determinents of adiponectin levels were identified in 2012 and the identified loci were proved to impact upon metabolic diseases [44]. Furthermore, intravenous or intra-cerebro-ventricular administration of adiponectin decreased body weight [2,45]. Diet composition and exercise, which are closely related to body weight, were showed to affect plasma adiponectin levels. Reports demonstrated that HF diet decreased adiponectin levels [46,47], which is consistent with the present study. While low fat, high carbohydrate diet [48], diets low in glycemic load and high in fiber [49], and food restriction [50,51] increased adiponectin levels. Exercise was demonstrated to increase adiponectin levels in humans and animals [52,53]. These reports suggested that food composition or exercise affect body weight via regulating adiponectin. Therefore, means to increase adiponectin level was conceived to be a novel therapy strategy for obesity and related diseases [2]. Similar to adiponectin, GTPs consumption was reported be associated with obesity, metabolic syndrome, type 2 diabetes and cardiovascular diseases [2]. In this study, GTPs treatment alleviated VATs increase and blood glucose elevation, and improved the insulin sensitivity and lipid profile in the HF fed rats. At the same time, GTPs treatment attenuated the decrease of adiponectin induced by HF or high glucose, which was also obeserved in another research using tea extracts [54]. From this point, regulation of adiponectin should be related to the mechanism by which GTPs exert anti-obesity, antidiabetic and cardiovascular protective effects. However, further studies to investigate the effects of GTPs on adiponectin knockout mice would help consolidating the conclusion.
Gene expression of adiponectin is mainly regulated by nuclear transcriptor named PPARc. PPARc binds with PPRE element in the adiponectin gene and stimulates the transcription [13]. Research demonstrated PPARc agonists would increase the circulating adiponectin in a metabolic syndrome rat model [55], and an epidemiological study proved that PPARc gene polymorphism would affect the serum adiponectin levels [56]. PPARc expression reduction was observed in obesity subjects [57,58]. In our experiments, decreased mRNA and protein expressions of PPARc and adiponectin were observed in HF fed rats and high glucose incubated VATs, and these effects could be attenuated by GTPs treatment. The transcription activity of PPARc was demonstrated to be affected by several factors, including phosphorylation or sumoylation of the receptor [59,60] and recruitment of different cofactors [61], among which phosphorylation of PPARc is investigated most. Phosphoryltion of PPARc resulted in decreased PPARc activation followed by downregulation of adiponectin gene [2]. Genetic techniques of inhibiting PPARc's phosphorylation could improve insulin resistance and increase adiponectin level [62]. Along with the decrease of adiponectin expression, increased phosphorylation of PPARc was observed in vitro and in vivo in the present study, and all these effects could be attenuated by GTPs treatment. Meanwhile, phosphorylation of PPAR gamma would prime PPARc for polyubiquitination and proteasomal degradation, increase the sumoylation of K77/K107 in a lysine motif IKVE directly adjacent to S82/112, and then synergistically repress PPARc transactivation [63]. Activation of erk1/2, which was documented in adipocytes of obese rodents and humans [64], was confirmed to induce phosphorylation of PPARc [65]. In the present study, HF diet and high glucose incubation elevated erk1/2 activation and phosphorylation of PPARc, which was consistent with the study conducted by Hosooka et al [66]. Treatment with GTPs inhibited the activation of erk1/2, and alleviated the decreased PPARc expression and increased PPARc phosphorylation induced by HF diet in vivo or by high glucose incubation in vitro. Selective inhibition of erk1/2 by PD98059 exerts same effects as GTPs treatment. These results suggested that GTPs increased PPARc expression and inhibited PPARc phosphorylation by downregulating erk1/2 in HF fed rats or under high glucose condition. However, the regulatory roles of erk1/2 activation played on PPARc expression remain uncertain. Erk1/2 might directly downregulate the PPARc expression, as well as reduce PPARc expression by triggering the phosphorylation of PPARc.

Conclusion
GTPs prevent HF-induced obesity by up-regulating adiponectin levels. The underlying mechanisms may include the inhibition of erk1/2 activation, alleviation of PPARc phosphorylation, and increase of the PPARc expression. Further research to track the PPARc and adiponectin level along with the pathogenesis of obesity would lead to better understanding of the mechanism.


Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity

Multiple natural killer (NK) cell-based anticancer therapies are currently under development. Here, we compare the efficiency of genetically modified NK-92 cells expressing chimeric antigen receptors (CARs) at killing NK cell-resistant B-lymphoid leukemia cells to the antibody-dependent cell-mediated cytotoxicity (ADCC) of NK-92 cells expressing a high affinity variant of the IgG Fc receptor (FcγRIII). First, we compared in vitro the abilities of NK-92 cells expressing CD20-targeting CARs to kill primary chronic lymphocytic leukemia (CLL) cells derived from 9 patients with active, untreated disease to the cytotoxicity of NK-92 cells expressing FcγRIII combined with either of the anti-CD20 monoclonal antibodies (mAbs) rituximab or ofatumumab. We found that CAR-expressing NK-92 cells effectively kill NK cell-resistant primary CLL cells and that such a cytotoxic response is significantly stronger than that resulting from ADCC. For studying CAR-expressing NK cell-based immunotherapy in vivo, we established xenograft mouse models of residual leukemia using the human BCR-ABL1+ cell lines SUP-B15 (CD19+CD20−) and TMD-5 (CD19+CD20+), two acute lymphoblastic leukemia (ALL) lines that are resistant to parental NK-92 cells. Intravenous injection of NK-92 cells expressing CD19-targeting CARs eliminated SUP-B15 cells, whereas they had no such effect on TMD-5 cells. However, the intrafemoral injection of NK-92 cells expressing CD19-targeting CAR resulted in the depletion of TMD-5 cells from the bone marrow environment. Comparative studies in which NK-92 cells expressing either CD19- or CD20-targeting CARs were directly injected into subcutaneous CD19+CD20+ Daudi lymphoma xenografts revealed that CD20-targeting CAR is superior to its CD19-specific counterpart in controlling local tumor growth. In summary, we show here that CAR-expressing NK-92 cells can be functionally superior to ADCC (as mediated by anti-CD20 mAbs) in the elimination of primary CLL cells. Moreover, we provide data demonstrating that the systemic administration of CAR-expressing NK-92 cells can control lymphoblastic leukemia in immunocompromised mice. Our results also suggest that the direct injection of CAR-expressing NK-92 cells to neoplastic lesions could be an effective treatment modality against lymphoma.

Introduction
Anticancer treatments based on monoclonal antibodies (mAbs) administered in combination with chemotherapy have significantly improved clinical outcome in patients with non-Hodgkin lymphoma (NHL). Nevertheless, about 40% of all NHL patients ultimately relapse 1,2 and subsequent stem cell transplantation can rescue only a small percentage of these individuals. 3 Thus, cellbased approaches are being increasingly considered as a therapeutic strategy to overcome the resistance to chemotherapy of leukemic cells and attack the malignant stem cell clone through an alternative cytotoxic modality. Recent reports on the therapeutic use of chimeric antigen receptor (CAR)-expressing autologous T lymphocytes to treat chemotherapy-resistant lymphoid Multiple natural killer (NK) cell-based anticancer therapies are currently under development. here, we compare the efficiency of genetically modified NK-92 cells expressing chimeric antigen receptors (cars) at killing NK cell-resistant B-lymphoid leukemia cells to the antibody-dependent cell-mediated cytotoxicity (aDcc) of NK-92 cells expressing a high affinity variant of the IgG Fc receptor (FcγrIII). First, we compared in vitro the abilities of NK-92 cells expressing cD20-targeting cars to kill primary chronic lymphocytic leukemia (cLL) cells derived from 9 patients with active, untreated disease to the cytotoxicity of NK-92 cells expressing FcγrIII combined with either of the anti-cD20 monoclonal antibodies (mabs) rituximab or ofatumumab. We found that car-expressing NK-92 cells effectively kill NK cell-resistant primary cLL cells and that such a cytotoxic response is significantly stronger than that resulting from aDcc. For studying car-expressing NK cell-based immunotherapy in vivo, we established xenograft mouse models of residual leukemia using the human Bcr-aBL1 + cell lines sUP-B15 (cD19 + cD20 − ) and TMD-5 (cD19 + cD20 + ), two acute lymphoblastic leukemia (aLL) lines that are resistant to parental NK-92 cells. Intravenous injection of NK-92 cells expressing cD19-targeting cars eliminated sUP-B15 cells, whereas they had no such effect on TMD-5 cells. however, the intrafemoral injection of NK-92 cells expressing cD19-targeting car resulted in the depletion of TMD-5 cells from the bone marrow environment. comparative studies in which NK-92 cells expressing either cD19-or cD20-targeting cars were directly injected into subcutaneous cD19 + cD20 + Daudi lymphoma xenografts revealed that cD20-targeting car is superior to its cD19-specific counterpart in controlling local tumor growth. In summary, we show here that car-expressing NK-92 cells can be functionally superior to aDcc (as mediated by anti-cD20 mabs) in the elimination of primary cLL cells. Moreover, we provide data demonstrating that the systemic administration of car-expressing NK-92 cells can control lymphoblastic leukemia in immunocompromised mice. Our results also suggest that the direct injection of car-expressing NK-92 cells to neoplastic lesions could be an effective treatment modality against lymphoma. malignancies substantiate the potential clinical benefits of cellular therapy. 4,5 As an alternative to T cells, natural killer (NK) cells expressing CARs directed against CD19 or CD20 (2 markers of the B-cell lineage) can also be used as cytotoxic effector cells for cell-based immunotherapy. However, current NK cellbased therapies are constrained by the necessity to isolate sufficient numbers of NK cells from donors as well as by the need to achieve acceptable transfection efficiencies. 6,7 The human NK cell line NK-92 8,9 presents an attractive alternative to donor NK cells, as it can be propagated and expanded in vitro. 10 Phase I clinical trials testing NK-92 cells have been successfully completed, confirming their clinical safety. 11,12 Furthermore, NK-92 cells are amenable to transfection with vectors coding for proteins of interest. 7 Preclinical studies performed so far have explored the genetic engineering of NK-92 cells with CARs targeting multiple tumor-associated antigens, including v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2, also known as HER-2/neu), 13 CD19, 14 CD20, 15 ganglioside GD2, 16 epithelial cell adhesion molecule (EPCAM), 17 and Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C). 18 On average, a 50% transduction has been achieved by using fresh NK-92 cells and a lentiviral construct, and the percent purity of transduced cells could be increased to 100% upon cell sorting. 7 Therefore, NK-92 cells can provide an "off the shelf," CAR-customized NK-cell product for anticancer immunotherapy.
NK cells, by virtue of expressing the IgG Fc receptor FcγRIII are also major effectors of antibody-dependent cell-mediated cytotoxicity (ADCC). 19,20 Although not all monoclonal antibodies kill target cells through ADCC, in some instances this is their primary killing mechanism. 19 In support of this notion, it has previously been shown that patients whose lymphocytes express a high affinity FcγRIII polymorphic variant achieve a better outcome in response to mAbs. 21 Unfortunately, only about 10% of the population actually harbors the allele coding for the high affinity FcγRIII variant (V/V), with the majority of individuals expressing the intermediate (F/V) or low affinity (F/F) variants of the receptor. 22 Hence, the cytotoxic effects of some mAbs may be augmented by simultaneously infusing NK cells selected for expression of high FcγRIII, as previously demonstrated by in vitro studies. 23 The objective of the study presented herein was to compare the cytotoxicity of NK-92 cells expressing CD20-targeting CARs to that of ADCC, as mediated by the anti-CD20 mAbs drugs rituximab and ofatumumab, against a panel of primary NK cellresistant chronic lymphocytic leukemia (CLL) cells. We further examined whether NK-92 cells expressing CD19-or CD20targeting CARs exert antitumor effects in xenograft models of human B-lymphoblastic leukemia and lymphoma.

Results
The cytotoxic activity of NK-92 cells expressing CD20targeting CAR against primary CLL cells is superior to ADCC induced by anti-CD20 monoclonal antibodies A number of mAbs rely on NK cells as cytotoxic effectors to mediate ADCC. 19,20 Here, we compared the abilities of two FDA-approved anti-CD20 mAbs, namely, rituximab and ofatumumab, to elicit ADCC with the cytotoxicity of NK-92 cells transduced with a lentiviral construct for the expression of CD20targeting CAR. Primary CLL cells from a total of 9 patients with active, untreated disease were tested as targets (Fig. 1). The mean cytotoxicity of NK-92 cells expressing CD20-targeting CAR (αCD20-CAR) was significantly greater than ADCC as mediated by either rituximab or ofatumumab (40.2% ± 2.6 for αCD20-CAR NK-92 cells as compared with 25.1% ± 2.1 and 30.5% ± 3.0 for rituximab and ofatumumab, respectively; p = 0.001 and p = 0.044, respectively).

Systemic administration of NK-92 cells expressing CD19targeting CAR controls the growth of metastatic SUP-B15 ALL cells transplanted in immunocompromised mice
Having shown that CAR-expressing NK-92 cells can effectively kill NK cell-resistant primary CLL cells in vitro, we developed a xenograft mouse model to test whether the intravenous infusion of NK-92 cells engineered to express CD19-specific CAR (αCD19-CAR) can eliminate human leukemia in immunocompromised NOD scid gamma (NSG) mice. SUP-B15 is an aggressive Philadelphia chromosome positive (Ph + ) CD19 + CD20 − B-cell acute lymphoblastic leukemia (ALL) line that has been previously shown to be resistant to killing by parental NK-92 cells, yet sensitive to killing by NK-92 cells expressing αCD19-CAR in vitro. 7 When injected intravenously with luciferase-(Luc-) tagged SUP-B15 cells, NSG mice succumbed within 20 to 40 d, depending on the size of the inoculum. The distribution of leukemic cells in these mice faithfully recapitulated the distribution of ALL cells observed in humans, including infiltrations into the bone marrow and the central nervous system (cranium and spine; Figure 2A-C). Mice developed signs of illness (hunched posture, scruffiness) and a reduction in mobility that eventually turned into a complete paralysis of the lower body and hind limbs. Cytofluorometric analyses at the time of sacrifice showed extensive infiltration of CD19 + CD45 + SUP-B15 blast cells in the bone marrow (femur) and spleen (86.7% ± 3.8 and 50.7% ± 19.5, respectively), as well as in the peripheral blood (14.7% ± 7.7).
Parental NK-92 cells or NK-92 cells engineered to express αCD19-CAR were injected intravenously (1 × 10 7 cells/dose) through the tail vein on 5 consecutive days, starting 24 h after the intravenous inoculation of 2 × 10 5 Luc-expressing SUP-B15 cells. Seventeen days after tumor cell injection, bioluminescence assessments showed that parental NK-92 cells had no effect on tumor burden, in sharp contrast to NK-92 cells expressing αCD19-CAR, which significantly reduced, but did not completely eliminate, tumor burden (3.12 × 10 8 ± 0.33 photons/sec vs. 5.46 × 10 8 ± 1.16 photons/sec in PBS-treated control mice, p = 0.02; Figure 2D-E). Since cellular therapy is surmised to be more efficacious in biological settings with reduced tumor burden, we titrated the limiting number of tumor-initiating cells by injecting decreasing numbers of Luc-expressing SUP-B15 cells (1 × 10 4 , 1 × 10 3 and 1 × 10 2 cells) into mice, determining that 1 × 10 3 cells was the lowest dose sufficient to induce systemic leukemia. Mice injected with 1 × 10 3 Luc-expressing SUP-B15 cells were subsequently given intravenous injections of 1 × 10 7 NK-92 cells on days 4, 5, and 6. As shown in Figure 2F and G, after 6 weeks (42 d), the tumor burden in mice treated with NK-92 cells expressing αCD19-CAR had almost completely disappeared and no malignant cells were detected by flow cytometry in the femur, spleen, or peripheral blood when mice were sacrificed at 4 mo (data not shown). On the contrary, mice injected with 1 × 10 3 Lucexpressing SUP-B15 cells and treated with parental NK-92 cells exhibited progressive leukemia similar to control mice ( Fig. 2F and G).
Therapeutic efficacy of CAR-expressing NK-92 cells against slow-growing TMD-5 ALL xenografts in immunocompromised mice In addition to rapidly-growing SUP-B15 leukemia cells, we tested a less aggressive, slow-growing Ph + CD19 + CD20 + B-cell ALL cell line, namely TMD-5 cells. Similar to SUP-B15 cells, TMD-5 cells are resistant to parental NK-92 cells in cytotoxicity assays in vitro. 7 When administered intravenously into sub-lethally irradiated NSG mice, Lucexpressing TMD-5 cells distributed in a manner similar to SUP-B15 cells, exhibiting a robust accumulation in the bone marrow and central nervous system (Fig. 3A). The relatively slow progression of the disease caused by TMD-5 cells and their distribution to the bone marrow, spleen and liver closely resembled a setting of residual leukemia.
Cytofluorometric analyses of various murine tissues confirmed that TMD-5 cells infiltrate the bone marrow and, after an initial period of slow growth, reach > 90% of the total bone marrow cell number by week 20 (data not shown). Approximately 15 weeks after the inoculation of TMD-5 cells, mice began to show signs of progressive disease including hunched posture, scruffy fur, reduced mobility, and splenomegaly. Death typically occurred around 6 months after the injection of cancer cells. At the time of sacrifice (6 mo), CD19 + /CD45 + TMD-5 cells represented > 90% of the total cell number in the bone marrow and spleen (94.2% ± 1.7 and 92.0% ± 1.6, respectively), and 50.6% ± 5.1 of the circulating cells. No hind limb paralysis was observed.
Sub-lethally irradiated NSG mice were injected intravenously with 5 × 10 6 Luc-expressing TMD-5 cells and subsequently treated with intravenous injections of parental NK-92 cells or NK-92 cells expressing either αCD19-CAR or αCD20-CAR (1 × 10 7 cells/dose) at days 7, 9, and 11 post-inoculation. Bioluminescence assessments showed that all immunotherapeutic regimens failed to control disease burden (Fig. 3B). Considering that TMD-5 cells are predominantly found in the bone marrow compartment early in the course of disease and, as such, represent a model of minimal residual leukemia, we sought to confirm whether the lack of antileukemic effects observed after the intravenous administration of NK-92 cells could be causally related to issues with the homing of NK-92 cells to disease sites in the bone marrow. To test this hypothesis, we treated NSG mice that had received 5 × 10 6 Luc-expressing TMD-5 cells with NK-92 cells expressing αCD19-CAR, applied as a single intrafemoral injection 100 d after cancer-cell inoculation. Specifically, 3 × 10 6 NK-92 cells expressing αCD19-CAR were injected into the right femur, while the left femur was injected with PBS (as a control). CAR-expressing NK-92 cells mediated a robust antitumor effect and leukemia cells were undetectable in the femur receiving NK-92 cells expressing αCD19-CAR in 48 h after treatment, whereas no marked effect could be documented in the contralateral femur (injected with PBS; Figure 3C). At sacrifice, i.e. 12 weeks after the intrafemoral injection of NK-92 cells, leukemia had recurred in the bone marrow (97% CD19 + CD45 + Luc-expressing TMD-5 cells), but no disease was detected in the blood or spleen. Furthermore, we documented an expansion of CD56 + /GFP + NK-92 cells (CAR-expressing NK-92 cells) in vivo, especially in the peripheral blood (25% of cells) and in spleen (5%), whereas no NK-92 cells could be detected in the bone marrow (data not shown).

Intratumoral injection of CAR-expressing NK-92 cells controls the local growth of lymphoma
To further investigate the local antitumor effects mediated by CAR-modified NK-92 cells, we induced the formation of a solid lymphoma tumor by subcutaneously injecting 2.5 × 10 5 Luc-expressing CD19 + CD20 + Daudi NKR cells into NOD/SCID mice, followed by the intratumoral injection of parental NK-92 cells or NK-92 cells expressing either αCD19-CAR or αCD20-CAR (5 × 10 6 per dose) on days 4, 5, and 6 after cancer cell inoculation. As shown in Figure 4, NK-92 cells expressing αCD20-CAR reduced tumor growth as compared with the PBS-treated control cohort, as demonstrated by an increase in bioluminescence of 1.79 ± 0.77 fold vs. 7.58 ± 1.19 fold, respectively (p = 0.001), between days 3 and 7 post-inoculation. In contrast, parental NK-92 did not affect tumor growth, nor did NK-92 cells expressing αCD19-CAR. Furthermore, at sacrifice (25 d), in the three mice of the cohort treated with NK-92 cells expressing αCD19CAR where the increase in bioluminescence was < 1.0 the size of lymphomas had remained stable over the Figure 1. cytotoxic potential of aDcc vs. car-expressing NK-92. antibodydependent cell-mediated cytotoxicity (aDcc) against primary chronic lymphocytic leukemia (cLL) cells (n = 9) as triggered by the anti-cD20 antibodies rituximab (gray, full), and ofatumumab (white, full) as compared with cLL cell killing mediated by NK-92 cells engineered to express a cD20-specific chimeric antigen receptor (αcD20-car) (black, full). The cytotoxic response to parental NK-92 cells alone (checkered, control for car-dependent cytotoxicity) and parental NK-92 cells with mabs (stripes, control for aDcc) is presented for each sample. In all experiments, the effector to target cell ratio was 10:1.
2 weeks period between imaging and sacrifice (0.10 ± 0.06 g, compared with 0.75 ± 0.10 g average tumor weight at sacrifice in control mice).

Discussion
Despite recent advances in chemotherapy and stem cell transplantation, a significant proportion of patients with lymphoid leukemia succumb to their disease. The elimination of the clonogenic cancer stem cell requires a multi-modality treatment approach involving cooperative components that should not counteract each other. Cellular therapy is considered one of those treatments and the infusion of autologous T lymphocytes expanded in culture and engineered to express CD19-targeting CARs has recently been shown to control advanced B-lymphoid malignancies. 4,5 Much of the focus of cellular immunotherapy so far has been on bulk peripheral T cells, EBV-specific T cells, or lymphoid progenitor cells (reviewed in ref. 24). However, autologous cytotoxic cells are frequently dysfunctional due to prior chemotherapy and/or exposure to the immunosuppressive tumor microenvironment. 25 Additionally, the absent or aberrant expression of MHC molecules by malignant cells possibly precludes their recognition by autologous MHC-restricted T cells. Furthermore, allogeneic T lymphocytes can cause acute graft-vs.-host disease (GvHD), a pathological condition that can be debilitating and, in extreme cases, fatal. 26 Conversely, NK cells do not induce GvHD and necessitate only the presence of "foreign" epitopes, often mutated MHC antigens, to become fully activated (reviewed in ref. 27). NK cells have been found to be suitable effectors for CARdependent immunotherapy, with the caveat that their isolation and expansion from the peripheral blood is labor intensive and costly (reviewed in Refs. 6,28). There is also a significant donorto-donor variability with respect to NK cells yield and cytotoxic activity. 29 As an immortalized NK cell line that can be easily and predictably expanded in culture while maintaining a broad anticancer activity, NK-92 cells provide a potential solution to these issues. 8, 9 Phase I clinical trials testing NK-92 cells have been completed establishing an acceptable safety profile. 11,12 Importantly, NK-92 cells are amenable to be genetically engineered by various methods. 7 Six variants of NK-92 cells expressing unique CARs have indeed been reported to date. [13][14][15][16][17][18] Furthermore, since the parental NK-92 cell line does not express endogenous Fc receptors, a NK-92 cell derivative expressing high affinity variant of FcγRIII has been developed. 30 This variant has also been employed for quantifying the potential of individual mAbs to trigger ADCC. 20 The principal aim of this project was to compare the ADCC triggered by 2 FDA-approved anti-CD20 mAbs against primary CLL cells to the anticancer cytotoxicity achieved by NK-92 cells engineered to express a CD20-specific CAR. Our findings indicate that NK-92 cells expressing αCD20-CAR exert a significantly higher cytotoxicity than the anti-CD20 antibodies rituximab or ofatumumab through ADCC. Although these results suggests that CAR-based immunotherapies may be intrinsically superior to mAb-based approaches, one should bear in mind that mAbs may kill their target through multiple mechanisms, including ADCC, complement-mediated cytotoxicity, and the interference with key signaling pathways. Hence, the actual antineoplastic activity of mAbs in vivo originates from a compilation of these distinct effects. Additionally, the FcγRIII employed in our assays does not possess an intracellular activation domain, and is therefore dependent on endogenous proteins for the transduction of an activatory signal, unlike CAR molecules, which contain the CD3ζ chain of the T-cell receptor (TCR) signaling complex. It should also be noted that FcR and TCR preferentially signal through different tyrosine kinases (SYK/LYN and ZAP70, respectively), which may affect the strength of the transduced signal.
Next, we assessed the antineoplastic potential of CAR-expressing NK cells in xenograft models of human BCR-ABL1 + B-lymphoblastic leukemia in NSG mice, using the B-ALL cell lines SUP-B15 (CD19 + CD20 − ) and TMD-5 (CD19 + CD20 + ). We showed that the intravenous delivery of NK-92 cells expressing a CD19-specific CAR can effectively control the growth of aggressive SUP-B15 cells, but not that of slow-growing TMD-5 cells. The eradication of TMD-5 cells from the bone marrow could only be achieved when the NK-92 cells expressing αCD19-CAR were injected directly into leukemia-infiltrated femurs. In this context, the clearance of leukemic cells from the bone marrow was relatively rapid (within 48 h), suggesting that TMD-5 cells are sensitive to the cytotoxic potential of NK-92 cells in vivo and that NK-92 cells are capable of eliminating a large number of targets upon engagement. It also implies that the lack of response to the intravenous delivery of NK-92 cells is caused by factors that are not directly related to their cytolytic functions. For example, the contact between NK cells and slow-growing TMD-5 cells could alter signaling pathways in NK cells that are critical for their sustained activation. 31 TMD-5 cells may also secrete a different spectrum of chemokines than SUP-B15 cells, such that TMD-5 cells may be less efficient at attracting NK-92 cells to the bone marrow. The recurrence of TMD-5 cells in the bone marrow several weeks after apparent clearance could result from either the incomplete elimination of tumor cells by CARexpressing NK-92 cells or, alternatively, from the re-colonization of the bone marrow by tumor cells coming from other parts of the body. This finding also suggests that NK-92 cells are not capable of long-term engraftment and proliferation in the bone marrow compartment.
We also sought to determine whether NK-92 cells expressing αCD19-CAR or αCD20-CAR exhibit preferential anticancer cytotoxicity. To this aim, we injected NK-92 cells expressing either the CD19-or CD20-targeting CAR into a subcutaneous Daudi lymphoma that expresses both CD19 and CD20. In this system, only NK-92 cells bearing the CD20-targeting CAR were effective, although it is unclear to what extent the relative density of CD19 and CD20 molecules on Daudi cells could have biased these results.
The superiority of CAR-expressing NK-92 cells over ADCC as triggered by anti-CD20 mAbs could be relevant for patients affected by multiple types of hematological cancers. Although we only tested primary tumor cells from patients with CLL, similar data were reported by Tassev et al., who found that the antineoplastic potential of NK-92 cells engineered to express CARs is superior to that of ADCC when the target is an EBV-transformed B-cell tumor. 18 . Importantly, we found that CAR-expressing NK-92 cells also controlled the growth of some neoplastic B-lymphoid xenografts in immunocompromised mice when given systemically or locally. Altogether, our findings support the initiation of clinical assays testing the antineoplastic potential of CAR-expressing NK-92 in patients with hematological malignancies.

Materials and Methods
Primary effector and target cells Primary CLL samples from 9 patients with untreated CLL diagnosed according to the National Cancer Institute-Working Group criteria were available for testing. All patients had stage 0 or I CLL (Rai staging system). Mononuclear cells (MNCs) from all samples were obtained by density gradient centrifugation using Ficoll-Hypaque Plus (Amersham Biosciences).

Antibodies and cytofluorometry
The anti-CD20 mAb rituximab and the anti-HER2 mAb trastuzumab (also known as Herceptin) were obtained from Genentech/Roche. The anti-CD20 mAb ofatumumab was obtained from GlaxoSmithKline. For the detection of human cells from xenografts by fluorescence cytometry, phycoerythrin (PE)-conjugated anti-CD56, PE-conjugated anti-CD45, and fluorescein isothyocyanate (FITC)-conjugated anti-CD19 mAbs were purchased from BD Biosciences PharMingen. Cytofluorometric analyses were performed on a Dako Cyan TM flow cytometer (Beckman-Coulter, Inc) and analyzed using the Summit software (Beckman-Coulter, Inc).

Recombinant lentiviral vectors and lentivirus production
The Luc-expressing reporter construct used in this study was provided by Dr M Rosenblatt (Tufts University School of Medicine). This construct encodes firefly luciferase (Luc), the mCherry fluorescent protein and a puromycin resistance determinant separated by 2A "self-cleaving" peptides, all cloned as a polycistronic cDNA into a pFUW lentiviral vector. The CAR constructs used in this study consist of single chain variable fragments (scFvs) from a murine antibody specific for human CD19 or CD20 linked to the CD3ζ chain of the TCR complex (first-generation CAR). The cDNAs coding for CD19-and CD20-specific CARs were subcloned from the retroviral vector pLXSN 14,15 into the lentiviral vector pCL20c-IRES-GFP (provided by Dr R Childs, NHLBI). The CAR-coding constructs were transfected into HEK-293T packaging cells alongside helper plasmids, using the Fugene TM lipofection system (Roche). Culture supernatants were collected after 48 h, filtered (with 0.22 µm filters) and stored at -80°C. The supernatants consistently had a titer of > 10 7 infectious units/mL.

Lentiviral transduction
Daudi, SUP-B15 and TMD-5 cells were transduced with pFUW-Luc lentivirus and selected with puromycin (Sigma-Aldrich). Daudi cells, which are normally sensitive to the cytotoxic activity of NK and NK-92 cells, lost such sensitivity upon transfection with this construct, and were subsequently referred as Daudi NKR (NK resistant).
NK-92 cells were transduced with pCL20c lentiviral particles as previously described, 7 using 2 × 10 6 cells in 6-well plates mixed with the corresponding lentiviral supernatant and at a multiplicity of infection (MOI) of ~5. Since polybrene is toxic to NK-92 cells, only 15 µg/mL protamine sulfate (Sigma-Aldrich) was used. Transduced cells were expanded in Myelocult ® medium supplemented with 1000 UI/mL Proleukin and GFP-expressing (i.e. transduced) NK-92 cells were further enriched by cell sorting to achieve > 95% purity (MoFlo, DakoCytomation). Transgene expression was confirmed by flow cytometry, measuring either mCherry or GFP expression directly, or CAR expression by using biotin-conjugated anti-scFv antibody (Jackson Immunoresearch) and allophycocyanin (APC)-conjugated streptavidin (BD Biosciences PharMingen). Of note, we always observed a strict correlation between GFP and CAR expression.
Cytotoxicity assay Effector and target cells were co-cultured for 3 h and cytotoxicity was measured by flow cytometry, as previously published. 7 ADCC assays were performed as previously described. 20 Briefly, CLL cells were incubated with rituximab, ofatumumab, or trastuzumab (as a negative control) for 30 min, followed by the addition of effector cells (parental NK-92 cells or NK-92Fc cells, which expressing high affinity FcγRIII), at an effector to target cell ratio of 10:1. Heat-inactivated FBS was utilized throughout the assays to exclude any contribution of complement-mediated cytotoxicity. For NK-mediated cytolysis, CLL target cells were incubated with parental NK-92 cells or NK-92 cells expressing CD20-specific CAR as above, in the absence of mAbs. Samples were analyzed immediately by flow cytometry. ADCC was calculated by subtracting the percentage of cytotoxicity obtained with mAbs plus parental NK-92 cells (which do not express FcγRIII) from that obtained with mAbs plus NK-92fc cells. The percentage of cytotoxicity obtained with mAbs or effector cells alone was consistently lower than that detected with mAbs plus parental NK-92 cells. Trastuzumab consistently failed to induce ADCC. CAR-dependent killing was calculated by subtracting the percentage of target cell killing obtained with parental NK-92 cells from that obtained with CAR-expressing NK-92 cells.
Xenotransplantation experiments NOD.CB17-Prkdc scid /J (NOD/SCID) and NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NOD scid gamma, or NSG) mice were purchased from the Jackson Laboratory and were housed under sterile conditions in the animal facility at Tufts University School of Medicine. This study was approved by the Tufts Institutional Animal Care and Use Committee. For the Ph + B-acute lymphoblastic leukemia model, 6-to 10-week old female NSG mice were sub-lethally irradiated with 350 cGy 24 h before the intravenous injection of 2 × 10 5 or 1 × 10 3 Luc-expressing SUP-B15 cells, or 5 × 10 6 Lucexpressing TMD-5 cells (in PBS). Mice were then treated with 3 to 5 intravenous injections of 1 × 10 7 parental NK-92 cells, or NK-92 cells expressing CD19-or CD20-targeting CARs, every day or every 2 d as indicated. Intrafemoral injections were performed using 3 × 10 6 NK-92 cells expressing CD19-specififc CAR in PBS, or PBS alone (control), as previously described. 33 To test the local effect of CAR-expressing NK-92 cells, nonirradiated 6-to 10-week old female NOD/SCID mice were injected subcutaneously with 2.5 × 10 5 Luc-expressing Daudi NKR cells in PBS. When the tumor had grown to about 0.5 cm 3 , mice were treated on days 4, 5 and 6 with intratumoral injections of 5 × 10 6 parental NK-92 cells or NK-92 cells expressing CD19-or CD20-targeting CARs, in PBS.
In vivo imaging For the detection of tumor burden in vivo, D-luciferin (Caliper Life Sciences) was injected intraperitoneally (75 mg/Kg in PBS) 10 min prior to imaging. Mice were anesthetized using 2.5% isofluorane and imaging was performed using a Xenogen IVIS 200 Biophotonic Imager (PerkinElmer) at the Tufts small animal imaging facility. Bioluminescence quantification was performed using the Living Image ® Software (PerkinElmer), and individual values were recorded as the average of measurements from the 4 sides of the whole body.

Statistical analysis
The SPSS 11.5 software (SPSS) was used to calculate statistical significance using a one-sided Student t-test (independent samples, unpaired). For small sample sizes we also used a Wilcoxon rank-sum test. All p values < 0.05 were considered statistically significant. Data are presented as means ± SEM, unless otherwise noted.

Disclosure of Potential Conflicts of Interest
HK is the Founder and employee of Conkwest Inc. The other authors do not declare any competing financial interest.


text:
Evaluation of Multiplex PCR with Enhanced Spore Germination for Detection of Clostridium difficile from Stool Samples of the Hospitalized Patients

Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37°C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.

Introduction
Clostridium difficile is a motile, rod-shaped, Gram-positive bacterium, which is known to be a leading cause of antibioticassociated diarrhea, especially nosocomial infections [1]. Though C. difficile is not a major component of natural gut flora, treatment with broad-spectrum antibiotics impedes the growth of other bacterial species and allows C. difficile to colonize. Following the colonization, an enterotoxin, TcdA, which is found in ∼70% of C. difficile strains, and a cytotoxin, TcdB, which is found in all C. difficile strains, can be produced, thereby disrupting tight junctions of the intestinal epithelial cells resulting in inflammation and increased permeability of the intestine [2]. Approximately less than 10% of clinical C. difficile isolates possess binary toxins (cdtA/B), which have been associated with increased severity of the symptoms [3]. The pathogenic role of cdtA/B has been suggested to trigger microtubule protrusion, thereby increasing the adherence of C. difficile to the gut epithelium [4]. C. difficile infection (CDI) results in a wide range of symptoms including fever, abdominal pains, mild diarrhea, and pseudomembranous colitis. Although CDI can be treated with certain antibiotics, the emergence of hypervirulent strains that are resistant to current chemotherapy and are able to produce high titers of toxins poses a challenge to the treatment of CDI worldwide [5].
To date, there are several diagnostic assays for the detection of C. difficile, each of which reveals the advantages and disadvantages and discrepancies in the performance existing in the literature [6,7]. Conventional diagnostic methods, including toxigenic bacterial cell culture and tissue cell culture cytotoxicity neutralization assays, have been considered as the reference standard [8]. These assays require technical expertise and several days to obtain results: therefore, they are not appropriate for the clinical setting, where an accurate and rapid diagnosis is needed. An enzyme immunoassay (EIA) for TcdB alone or both TcdA and TcdB has been widely used in most laboratories because it is relatively simple, rapid, and commercially available [9]. However, it has been revealed that the sensitivity of EIA is as low as 23% and the specificity as low as 75% [10]. Therefore, in practice, a symptomatic patient with the EIA-negative result is usually tested by another assay with higher sensitivity. Many laboratories have reported the combination of assays to increase the sensitivity and specificity of the detection [11][12][13]. An example include a 2-step algorithm, in which the first step is to perform an EIA for glutamate dehydrogenase (GDH) and the second step is to test the GDH-positives with an EIA for toxins. The EIA for GDH step yields a highly sensitive result for C. difficile, but is not specific for toxigenic isolates; therefore, the GDH-positive results must be confirmed with a subsequent specific test such as EIA for toxins [13]. Although these algorithms improve the diagnostic performance, it has been shown that the levels of sensitivity of the EIA for GDH in this two-step algorithm vary depending on the C. difficile ribotypes [14]. Moreover, the two-or more step assays are cost-ineffective [15]. Recently, nucleic acid amplification tests (NAATs) have been developed as a single assay with the same day results for CDI. These assays aim to detect the toxin gene(s) and have been proven to be more superior than other methods, except the toxigenic bacterial cell culture, as they yield the high sensitivity and high negative predictive value [16]. Currently, there are a number of FDA-approved commercially available NAATs including (i) the Xpert C. difficile, (ii) Xpert C. difficile/Epi assays that detect tcdB by real-time PCR, and (iii) the Illumigene C. difficile assay that detects tcdA by loop-mediated isothermal amplification [17].
Although the NAATs have gained popularity for CDI diagnosis, the common drawbacks of this type of assays to detect pathogens directly from stool samples are the presence of PCR inhibitors, contamination of DNA from host and other microorganisms, and low quality and yield of bacterial DNA that is extracted from spores in stool samples from suspected patients. Thus, the objective of this study was to evaluate the multiplex PCR with enhanced spore germination for the detection of C. difficile directly from stool samples of hospitalized patients. The combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.

Specimen Collection and Acquisition.
A total of 238 fecal specimens from inpatients that aged more than 15 years and developed diarrhea during hospitalization at Ramathibodi hospital, a 1,000-bed tertiary health care university Hospital, were collected from May 2010 to January 2011. The samples were subjected to the routine EIA test using VIDAS C. difficile Toxin A&B qualitative assay (BioMérieux, Marcy l'Etoile, France) according to the manufacturer's recommendations. The samples were also subjected to C. difficile selective culture by plating onto cycloserine cefoxitin fructose agar (CCFA) and incubated anaerobically at 37 ∘ C for up to 5 days. All samples were then subsequently stored at −80 ∘ C before use. The use of human materials has been approved by the research ethics committee of the Faculty of Medicine at Ramathibodi Hospital, Mahidol University, Thailand.

Bacterial Cell
Culture. Clostridium strains were grown anaerobically in BHIS medium, brain heart infusion broth at 37 ∘ C (Oxoid, Basingstoke, UK), supplemented with 5% yeast extract, 0.1% sodium thioglycolate (TCI, Tokyo, Japan), and 0.1% L-cysteine (TCI). Before sterilization, anaerobic conditions were created by boiling the medium for 10 min and, during cooling, flushing the medium with nitrogen gas. All other bacteria were cultivated at 37 ∘ C in tryptone soy broth (Oxoid).

Multiplex PCR for the Detection of C. difficile Toxin
Genes. A multiplex PCR was developed for the detection of toxin genes including tcdA, tcdB, cdtA, and cdtB as well as 16S rDNA as a internal control. A total volume of 20 L PCR reaction consisted of 1 × PCR buffer (10 mM Tris-HCl, 50 mM KCl, and pH 8.3), 5 mM MgCl 2 , 250 M dNTP, 1 × enhancer (0.5 M betaine, 1% DMSO), 1 U of Taq DNA polymerase (New England Biolabs, MA, USA), and 5 pairs of primers at indicated concentrations (Table 1). Thermal cycling parameters included (i) an initial denaturation at 92.5 ∘ C for 2 min; (ii) 30 cycles of denaturation at 92.5 ∘ C for 20 s, annealing at 60 ∘ C for 65 s, and extension at 68 ∘ C for 70 s; and (iii) a final extension at 68 ∘ C for 5 min. PCR products were resolved by electrophoresis on a 2% agarose gel stained with ethidium bromide.

Preparation of C. difficile Spores.
Spores from C. difficile strain R20291 were produced in a sporulation medium as described previously [18]. Briefly, a culture of C. difficile was spread onto BHIS agar supplemented with 0.1% taurocholate (BHIS/TA). The plates were then incubated at 37 ∘ C under anaerobic conditions for 5 days. Spores were scraped off the plates and resuspended in deionized water. The samples were then washed ten times with water. The spores were checked for purity and enumerated using phase-contrast microscopy and light microscopy after staining with malachite green and

Results and Discussion
Toxigenic C. difficile strains are recognized as the main cause of nosocomial diarrhea [1][2][3]. Therefore, a rapid and costeffective method to detect C. difficile directly from stool samples facilitates patient management to control CDI. The aim of this work was to design an optimized multiplex PCR for the detection of toxigenic C. difficile from stool samples of hospitalized patients and to evaluate the combination of various sample processing conditions and multiplex PCR on such detection.

Multiplex PCR for the Detection of Toxigenic C. difficile.
In the past years, there have been an increasing number of C. difficile genome and toxin gene sequences deposited in the GenBank database, enabling us to design more specific primers. The 5-plex PCR primers were developed for the detection of the four C. difficile toxin genes including tcdA, tcdB, cdtA, and cdtB, together with 16S rDNA as an internal PCR control (Figure 1). The primer set was chosen to amplify products with distinguishable sizes on agarose gel electrophoresis. The individual primer pairs for the amplification of the regions in the tcdA, tcdB, cdtA and cdtB genes were tested in single-plex PCRs (Figure 1; lanes 1-4, respectively). The multiplex PCR with a combination of all five primer pairs was optimized (Figure 1; lane 5) and was tested with different PCR C. difficile ribotypes (Figure 2). Our results are consistent with the previously reported data [19].

Sensitivity and Specificity
Test. The sensitivity and specificity of the developed multiplex PCR for the detection of C. difficile were evaluated. The detection limit as measured with genomic DNA from toxigenic reference strain C. difficile R20291 was 0.1 pg or ∼22 genomic copy number per reaction.
To further evaluate the primer specificities for C. difficile, ND stands for "not determined" because there were too many contaminated bacterial species, rendering it impossible to distinguish C. difficile colonies on the plates. −/+ indicates that typical C. difficile colonies could not be observed in at least one of the three replicates.

Enrichment Multiplex PCR for Detection of Toxigenic C.
difficile. The multiplex PCR assays for detection of toxigenic C. difficile directly from fecal specimens have been previously reported [20][21][22]. However, the detection limit has been shown to be as low as 5 × 10 4 cfu per 1 g of feces [22]. The sensitivity of the PCR is usually affected by inhibitors found in stool samples including bile salts, complex polysaccharides, proteinases, a high concentration of background flora, and a low concentration and uneven distribution of target microorganism [23], rendering it not suitable for PCR detection of the pathogen from direct stool samples. In this study, different combinations of pretreatment conditions, enrichment conditions, and enrichment times prior to both multiplex PCR and conventional bacterial cell culture methods were evaluated to determine the possibility of detecting 100 C. difficile spores per 1 g of feces (Table 2). This low spore concentration was not detected by nonenrichment multiplex PCR and culture methods. Regarding the pretreatment step, the C. difficile colonies were observed on CCFA after 1 h of enrichment without alcohol shock, compared to 3 h with the alcohol shock. Under nonselective plating, most conditions yielded uninterpretable results due to the high levels of contamination of other microorganisms. Positive PCR results were obtained under the conditions of the 1 h enrichment time, regardless of pretreatment or enrichment broth. Therefore, the condition of no alcohol shock and enrichment with BHIS/TA for 1 h was identified as the optimal combination with practical working hours in laboratories and was appropriate for both bacterial cell culture and multiplex PCR diagnosis. Furthermore, the sensitivity of the developed multiplex PCR under the described conditions was also tested with fecal samples spiked with different numbers of spores. We found that the multiplex PCR was able to detect as low as 5 spores/g feces. Therefore, the enrichment broth could enhance spore germination and transformation into vegetative cells, allowing them to grow to PCR-detectable levels.

Detection of Toxigenic C. difficile in Clinical Stool Samples.
To examine the efficacy of the assay described in this study, 238 stool samples of patients suspected of CDI were subjected to multiple PCR with the enhanced spore germination as above. Routine EIA and bacterial cell culture assays were also performed. All samples were tested positive for the 16S rDNA control gene. Comparison of different diagnostic assays is shown in Table 3. Sixteen cases were tested positive while 166 cases gave rise to negative results in all three assays. Eighteen samples were EIA positive, but both PCR and culture negative, and were, therefore, considered to be negative due to the low level of EIA specificity that requires the second assay to confirm the EIA results. Twenty-four cases were shown to be PCR positive with either EIA positive or culture positive. These results represented another twostep diagnostic process that increased the reliability of the PCR outcomes. Twenty-four samples were PCR positive, but both EIA and culture turned out negative. Although anaerobic culture was considered the most sensitive assay for the detection of C. difficile, it is also likely that the low spore counts in fecal samples and different rates of spore viability would account for such results. However, another confirmatory assay should be performed to support the PCR results.
Of 54 PCR-positive samples, 7 were  [24,25], this is the first time that the + + / + isolates were detected. It is also noteworthy that PCR detection of C. difficile toxin genes might only detect DNA without the presence of toxin production; therefore, the PCR-positive cases should be carefully interpreted.
Although the enrichment multiplex PCR in this study showed superiority over the other two diagnostic methods, the limitations of our experimental setting include the lack of another confirmatory diagnostic test such as cell cytotoxicity; so the false positives or negatives in cases of discordant test results could not be identified. However, analytical sensitivity with the reference C. difficile strain and spore-inoculated fecal samples and specificity of the multiplex PCR assay with various C. difficile ribotypes, other Clostridium spp., and other bacteria species revealed high sensitivity with no false positive or false negative.

Conclusions
This work revealed the comprehensive evaluation of sample processing prior to the multiplex PCR diagnosis of C. difficile directly from clinical stool samples. The results demonstrated that the enrichment of nonselective medium for 1 h prior to both PCR or bacterial cell culture assays yielded the most optimal condition. The performance of the enrichment multiplex PCR was proven to be more superior to that of the routine EIA. This rapid and cost-effective diagnostic assay provides an alternative approach for the detection of C. difficile, thereby improving the CDI management. However, a large-scale clinical testing is warranted to further validate this assay.