Oncolytic Immunotherapy for Treatment of Cancer.

Immunotherapy entails the treatment of disease by modulation of the immune system. As detailed in the previous chapters, the different modes of achieving immune modulation are many, including the use of small/large molecules, cellular therapy, and radiation. Oncolytic viruses that can specifically attack, replicate within, and destroy tumors represent one of the most promising classes of agents for cancer immunotherapy (recently termed as oncolytic immunotherapy). The notion of oncolytic immunotherapy is considered as the way in which virus-induced tumor cell death (known as immunogenic cancer cell death (ICD)) allows the immune system to recognize tumor cells and provide long-lasting antitumor immunity. Both immune responses toward the virus and ICD together contribute toward successful antitumor efficacy. What is now becoming increasingly clear is that monotherapies, through any of the modalities detailed in this book, are neither sufficient in eradicating tumors nor in providing long-lasting antitumor immune responses and that combination therapies may deliver enhanced efficacy. After the rise of the genetic engineering era, it has been possible to engineer viruses to harbor combination-like characteristics to enhance their potency in cancer immunotherapy. This chapter provides a historical background on oncolytic virotherapy and its future application in cancer immunotherapy, especially as a combination therapy with other treatment modalities.

First Report of Stem Rot of Konjac Caused by Phytophthora nicotianae in China.

Konjac (Amorphophallus) is an important economic crop widely used in health products and biomaterials in Asia (2). A serious foliage disease of Konjac was observed in Fuyuan County, Yunnan Province, China, in July 2012. The symptoms began with leaf color changes from light green to yellow, followed by discoloration on the stem base, plant wilting, bulb rotting, and ultimately plant death. Symptomatic tissues were cut into small pieces, surface-sterilized, and cultured on 20% V8 juice agar at 28°C. Five days after incubation, white fluffy colonies with the typical sporangium of Phytophthora sp. were observed from root and stem pieces. Isolates were identified as P. nicotianae based on morphological characteristics and DNA analysis. The growth rate of the colonies was 16 mm/d at 28°C. Sporangia were pyriform, ovoid to spherical, and papillate, and the dimensions of the 80 sporangia measured ranged from 23.7 to 60.4 × 19.4 to 45.7 µm (avg. 42.4 × 31.5 µm) with length-to-breadth ratios of 1.19 to 1.44 (avg. 1.34). The chlamydospores were spherical with a smooth surface, and their dimensions ranged from 20.3 to 47.3 × 18.9 to 45.9 µm (avg. 32.7 × 30.4 µm) (3). DNA was extracted from one colony containing spores and hyphae of the isolated P. nicotianae, and the nuclear ribosomal DNA internal transcribed spacer (ITS) region was amplified with primers ITS6 and ITS4 (4). The obtained 854-bp amplicon was purified and sequenced. NCBI BLAST retrieved a 100% identity with P. nicotianae (GenBank Accession No. KJ506732). A pathogenicity test of the isolated P. nicotianae was conducted in a greenhouse. After 7 days in a humidity-controlled greenhouse, all 10 inoculated plants showed similar symptoms as observed initially in the field, while control plants were symptomless. P. nicotianae was re-isolated from the inoculated stems, thus successfully completing Koch's postulates (1). To our knowledge, this is the first report of P. nicotianae as a pathogen of Konjac in China. References: (1) B. Alvarez-Rodriguez et al. Plant Dis. 97:1257, 2013. (2) H. Ban, et al. Plant Cell Rep. 28:1847, 2009. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St. Paul, MN, 1996. (4) J. M. French et al. Plant Dis. 95:1028, 2011.

[Establishment of a nomogram predicting risk factors of postoperative perineal wound complications after abdominoperineal resection for rectal cancer].

Objective: To investigate the risk factors of perineal incision complications after abdominoperineal resection (APR) for rectal cancer, and to establish a nomogram model to predict the complications of perineal incision. Methods: A case-control study was conducted to retrospectively collect the medical records of 213 patients with colorectal cancer who underwent APR at the First Affiliated Hospital of Nanjing Medical University from January 2010 to December 2016. The complications of perineal incision after APR were classified according to the modified Clavien-Dindo classification of surgical complications (Version 2019), and the complications of grade II and above were defined as "clinically significant complications" .Twenty-two factors related to complication of perineal incision, such as gender, age, surgical procedure, surgical approach, perineal repair, placement of drainage tube, skin position of drainage tube, operation time, intraoperative blood loss, preoperative radiotherapy and chemotherapy, intraoperative local perfusion chemotherapy, tumor classification, pathological grade, tumor T stage, tumor TNM stage and so on, were analyzed by chi-square test for univariate risk factor of complication in all variables, and variables with P<0.2 in univariate analysis were further included in multivariate analysis. Logistic regression analysis was used to screen out independent risk factors. R software (R 3.3.2) was introduced. The rms software package was used to construct a nomogram prediction model. The C-index was calculated (higher meaning better consistency with actual risk) to evaluate the discriminant degree of the model. The Bootstrap method was used to repeat the sampling for internal verification. A total of 42 patients with colorectal cancer who underwent APR from January 2017 to December 2017 at the First Affiliated Hospital of Nanjing Medical University were externally validated, and the corrected C-index was calculated. The model conformity was determined by comparing the C-index calibration difference between the predicted and actual risks. Results: Of the 213 patients with colorectal cancer, 131 were male and 82 were female, with mean age of (59.6±11.6) years. The incidence of postoperative perineal incision complications was 20.2% (43/213), including 27 cases of Clavien-Dindo II and above complications. Univariate analysis showed that the Eastern Cancer Cooperative Group (ECOG) score, preoperative albumin, skin position of drainage tube, intraoperative blood loss, preoperative radiotherapy and chemotherapy were associated with complications of postoperative perineal incision (All P<0.05) . Multivariate analysis showed that preoperative albumin levels ≤38 g/L (OR=105.261, 95% CI: 7.781 to 1423.998, P<0.001), perinead drainage (OR=11.493, 95% CI: 1.379 to 95.767, P=0.024), intraoperative blood loss >110 ml (OR=6.476, 95% CI: 1.505 to 27.863, P=0.012) and preoperative radiotherapy and chemotherapy (OR=7.479, 95% CI: 1.887 to 29.640, P=0.004) were postoperative clinically significant independent risk factors for perineal incision complications. The nomogram model was established. Preoperative albumin level <38 g/L was for 100 points, the preoperative chemoradiotherapy was for 52.5 points, the intraoperative blood loss >110 ml was for 28.5 points, and the perineal drainage was for 17.5 points. Adding all the points was the total score, and the complication rate corresponding to the total score was the predicted rate of the model. The model had a C-index of 0.863. After internal verification, the C-index dropped by 0.005. External verification showed a C-index of 0.841. Conclusions: Preoperative nutritional status, skin position of drainage tube, intraoperative blood loss and preoperative radiotherapy and chemotherapy may affect the occurrence of perineal wound complications after APR for rectal cancer. The nomogram model constructed in this study is helpful for predicting the probability of clinically significant complications after APR.

Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

Identification of the transcriptional regulatory sequences of human kallikrein 2 and their use in the construction of calydon virus 764, an attenuated replication competent adenovirus for prostate cancer therapy.

Human glandular kallikrein (hK2) and prostate-specific antigen (PSA) are related members of the human kallikrein gene family. The genes for hK2 and PSA are expressed predominately in the prostate, are transcriptionally up-regulated by androgens, and share 78% homology. Previously, one functional androgen response element was identified within the proximal promoter (-324 to +33 relative to the cap site) of the hK2 gene. To detect additional upstream regulatory elements, the 12.3 kbp between the PSA gene and 5' to the hK2 gene were amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed an androgen-dependent enhancer, located between -3.4 and -5.2 kb upstream of the transcription start site of the hK2 gene. This hK2 enhancer increased luciferase expression 100-fold in the presence of the testosterone analogue R1881. The hK2 enhancer contains an androgen response element that lost activity when mutated. The hK2 enhancer/promoter demonstrated activity in PSA(+) LNCaP cells whereas the enhancer/promoter was inactive in PSA(-) 293, A549, HBL100, HUH-7, LoVo, MCF-7, OVCAR-3, and PC-3 cells. Insertion of the hK2 enhancer/promoter into adenovirus to drive the E1A genes of adenovirus type 5 (Ad5) created an attenuated replication competent adenovirus variant Calydon virus (CV) 763, which replicates similarly to wild-type adenovirus in prostate tumor cells but is attenuated in nonprostate tumor cells. In addition, CV764, an adenovirus variant containing the previously cloned prostate-specific enhancer (to drive the Ad5 E1A genes) and the hK2 enhancer/promoter (to drive the Ad5 E1B genes) was constructed. CV764 is significantly attenuated and has a high therapeutic index with a cell specificity of 10,000:1 for PSA(+) LNCaP cells, compared to ovarian cancer OVCAR-3 cells and SK-OV-3 cells and PA-1 cells. CV764 is also highly attenuated in primary human microvascular endothelial cells.

The addition of adenovirus type 5 region E3 enables calydon virus 787 to eliminate distant prostate tumor xenografts.

CV787, a novel highly prostate-specific replication-competent adenovirus with improved efficacy, was constructed. CV787 contains the prostate-specific rat probasin promoter, driving the adenovirus type 5 (Ad5) E1A gene, and the human prostate-specific enhancer/promoter, driving the E1B gene. To improve efficacy, we constructed CV787 such that it also contains the entire Ad5 E3 region. CV787 replicates in prostate-specific antigen (PSA)+ cells as well as wild-type adenovirus, but in PSA- cells, CV787 replicates 10(4)-10(5) times less efficiently. CV787 destroys PSA+ prostate cancer cells 10,000 times more efficiently than PSA- cells. Incorporation of the Ad5 E3 region significantly improves the target cell killing ability or efficacy of CV787. In nu/nu mice carrying s.c. LNCaP xenografts, a single i.v. tail vein injection of CV787 eliminates 300-mm3 tumors within 4 weeks. CV787 could be a powerful therapeutic for human metastatic prostate cancer.

A hepatocellular carcinoma-specific adenovirus variant, CV890, eliminates distant human liver tumors in combination with doxorubicin.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world. Tumor resection remains the only curative treatment but is often not possible because of advanced stage and frequently unsuccessful because of intrahepatic or distant tumor recurrence. alpha-Fetoprotein (AFP), a tumor marker currently used for the diagnosis and management of HCC, is an oncofetal protein expressed in a majority of HCCs but rarely in normal hepatocytes. Because AFP gene expression is tightly regulated at the level of transcription, AFP transcriptional regulatory elements (TRE) are excellent candidates for generating HCC-specific oncolytic adenoviruses. We devised a new strategy for the AFP TRE to control an artificial E1A-IRES-E1B bicistronic cassette in an adenovirus 5 vector (Ad5) and constructed an HCC-specific oncolytic virus, CV890. In vitro, CV890 expression of the E1A and E1B genes, virus replication, and cytopathic effects were examined by Northern blot, Western blot, virus yield assay, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in AFP-producing cell lines (HepG2, Huh7, Hep3B, PLC/PRF/5, and SNU449), non-AFP-producing cell lines (Sk-Hep-1, Chang liver cell, LNCaP, HBL-100, PA-1, UM-UC-3, SW 780, Colo 201, and U118 MG), and non-AFP-producing human primary cells (lung fibroblast, bladder smooth muscle, and mammary epithelial). CV890 efficiently replicates in and destroys AFP-producing HCC cells as well as wild-type Ad5, but replication is highly attenuated in non-AFP-producing HCC cells or non-HCC cells. CV890 produced 5,000-100,000-fold less virus than wild-type Ad5 in non-AFP-producing cells. CV890 was attenuated 100-fold more than CV732, a virus containing the AFP TRE driving the E1A gene alone, in non-AFP-producing cells. These studies demonstrated that expression of both E1A and E1B genes under the control of a bicistronic AFP-E1A-IRES-E1B cassette yielded improvements in virus specificity equivalent to driving the E1A and E1B genes with two independent TREs yet requires only one TRE thereby conserving genomic space within the virus. Significantly, CV890 produced nearly the same yield of virus in cells that produced AFP over a 75-fold range, from a low of 60 ng AFP/10(6) cells/10 days to as high as 4585 ng AFP/10(6) cells/10 days. In vivo, antitumor efficacy of CV890 was examined in BALB/c-nu/nu mice containing large s.c. HepG2 or Hep3B tumor xenografts. Tumor volume of distant xenografts dropped below baseline 4 weeks after a single i.v. injection. Combination of CV890 with doxorubicin demonstrated synergistic antitumor efficacy, yielding complete elimination of distant Hep3B tumors 4 weeks after a single i.v. administration of both compounds. Our results support the clinical development of CV890 as an antineoplastic agent for the treatment of localized or metastatic HCC.

Antitumor synergy of CV787, a prostate cancer-specific adenovirus, and paclitaxel and docetaxel.

CV787, a PSA+ prostate cell-specific adenovirus variant, is currently in Phase I/II clinical trials for the treatment of prostate cancer. We have previously demonstrated that a single administration of CV787 at 1 x 10(11) particle/animal could eliminate established tumors within 6 weeks in nude mouse xenografts (Yu et al., Cancer Res., 59: 4200-4203, 1999). We now demonstrate that CV787-mediated replication-dependent cytotoxicity is synergistic with the chemotherapeutic agents paclitaxel (Taxol) or docetaxel (Taxotere) both in vitro and in vivo. In vitro, cells were pretreated with CV787 24 h before taxane, pretreated with taxane 24 h before CV787, or treated with both agents simultaneously. Cell viability was determined at various time points by 3-[4,5-dimethylthiazole-2-4]-2,5-diphenyl-2H-tetrazolium bromide assay, and virus yield was examined by plaque assay. Addition of taxane to CV787 resulted in a synergistic increase of cytotoxicity toward the human prostate cancer cell line LNCaP, regardless of the timing of administration. There was no reduction in virus replication or specificity of CV787-based cytopathogenicity for prostate cancer cells (approximately 10,000 to 1) with the taxanes. p53 expression was significantly elevated in the cells treated with CV787 and taxane. In vivo, using the PSA+ LNCaP xenograft model of prostate cancer, a single i.v. dose of 1 x 10(8) particles CV787 and docetaxel in combination eliminates large preexistent distant tumors. Toxicity studies do not show a synergistic increase of toxicity of CV787 and taxane. These experiments demonstrate a synergistic antitumor efficacy for CV787 when combined with taxane and demonstrate an in vivo single-dose curative therapeutic index for CV787 of over 1000:1.

CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity.

Radiation is an effective means of treating localized prostate cancer. However, up to 40% of men with certain risk factors will develop biochemical failure 5 years after radiotherapy. CV706, a prostate cell-specific adenovirus variant, is currently in clinical trials for the treatment of recurrent organ-confined prostate cancer. We demonstrated previously that a single administration of CV706 at 5 x 10(8) particles/mm3 of tumor eliminated established tumors within 6 weeks in nude mouse xenografts (Rodriguez et al., Cancer Res. 57: 2559-2563, 1997). We now demonstrate that CV706-mediated cytotoxicity is synergistic with radiation. In vitro, addition of radiation to CV706 resulted in a synergistic increase of cytotoxicity toward the human prostate cancer cell line LNCaP and a significant increase of virus burst size, with no reduction in specificity of CV706-based cytopathogenicity for prostate cancer cells. In vivo, prostate-specific antigen (+) LNCaP xenografts of human prostate cancer were treated with CV706 (1 x 10(7) particles/mm3 of tumor), 10 Gy of single fraction local tumor radiation, or both. Tumor volumes of the group treated with CV706 or radiation was 97% or 120% of baseline 6 weeks after treatment. However, when the same dose of CV706 was followed 24 h later with the same dose of radiation, the tumor volume dropped to 4% of baseline at this time point and produced antitumor activity that was 6.7-fold greater than a predicted additive effect of CV706 and radiation. Histological analyses of tumors revealed that, compared with CV706 or radiation alone, combination treatment with two agents increased necrosis by 180% and 690%, apoptosis by 330% and 880%, and decreased blood vessel number by 1290% and 600%, respectively. Importantly, no increase in toxicity was observed after combined treatment when compared with CV706 or radiation alone. These data demonstrate that CV706 enhances the in vivo radioresponse of prostate tumors and support the clinical development of CV706 as a neoadjuvant agent with radiation for localized prostate cancer.

A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy.

CV706 is a prostate-specific antigen (PSA)-selective, replication-competent adenovirus that has been shown to selectively kill human prostate cancer xenografts in preclinical models. To study the safety and activity of intraprostatic delivery of CV706, a Phase I dose-ranging study for the treatment of patients with locally recurrent prostate cancer after radiation therapy was conducted. Twenty patients in five groups were treated with between 1 x 10(11) and 1 x 10(13) viral particles delivered by a real-time, transrectal ultrasound-guided transperineal technique using a three-dimensional plan. The primary end point was the determination of treatment-related toxicity. Secondary objectives included evaluation of the antitumor activity of CV706 and monitoring for other correlates of antineoplastic action. In this study, CV706 was found to be safe and was not associated with irreversible grade 3 or any grade 4 toxicity. No grade >1 alterations in liver function tests associated with CV706 administration were observed. Posttreatment prostatic biopsies and detection of a delayed "peak" of circulating copies of virus provided evidence of intraprostatic replication of CV706. The study defined the timing of CV706 shedding into blood and urine as well as the appearance of circulating Ad5 neutralizing antibodies. Finally, this study documents the serum PSA response of treated patients and reveals a dose response showing that all five patients who achieved a > or =50% reduction in PSA were treated with the highest two doses of CV706. This study represents the first clinical translation of a prostate-specific, replication-restricted adenovirus for the treatment of prostate cancer. Taken together, this study documents that intraprostatic delivery of CV706 can be safely administered to patients, even at high doses, and the data also suggest that CV706 possesses enough clinical activity, as reflected by changes in serum PSA, to warrant additional clinical and laboratory investigation.

Pre-existent adenovirus antibody inhibits systemic toxicity and antitumor activity of CN706 in the nude mouse LNCaP xenograft model: implications and proposals for human therapy.

Pre-existent humoral antibody to adenovirus potentially confounds human clinical trials involving intravascular administration of adenovirus. Using the LNCaP prostate cancer xenograft model in BALB/c nu/nu mice and the prostate-specific attenuated replication-competent adenovirus (ARCATM) CN706, we developed an animal model that systematically controls both the dose of intravascularly administered adenovirus and the titer of the pre-existent anti-Ad5 antibody, and then measures the virus-induced toxicity as well as antitumor activity. We prepared hyperimmune sera to adenovirus in rabbits, passively injected the purified rabbit anti-Ad5 antibody into tumor-bearing mice, and established measurable humoral anti-Ad5 antibody titers. CN706 was intravenously injected into the tail vein of animals 24 hr after passive anti-Ad5 antibody administration. In the absence of pre-existent antibody, the lethal dose (LD100) for BALB/c nu/nu mice was 2.5x10(11) CN706 particles, whereas 1x10(11) CN706 particles was not lethal. However, in the presence of a 1:80 pre-existent titer of Ad5 neutralizing antibody (NAb), intravenous injection of 5x10(11) CN706 particles was no longer lethal. In addition, pre-existent antibody also prevented antitumor activity in a dose-dependent manner: 1x 10(11) CN706 particles prevented LNCaP xenograft tumor progression, but antitumor activity was eliminated by a pre-existent 1:80 NAb titer. These results led us to propose transient removal of pre-existent adenovirus antibody by immunoapheresis. An affinity column of cloned virus capsid proteins was constructed that was able to specifically remove adenovirus antibody from human clinical serum samples. A 5-min disposable immunoassay was also developed to monitor the level of pre-existent antibody in sera before and after immunoapheresis. Clinically, this approach may enable controlled clinical studies of intravenously administered adenovirus in patients with pre-existent anti-adenovirus antibody.

Kinetics and modeling of L-6-[18F]fluoro-dopa in human positron emission tomographic studies.

Kinetics of L-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) in striatum and cerebellum were measured in 10 normal human subjects with positron emission tomography (PET) from 0 to 120 min after an intravenous bolus injection of the tracer. The time course of the arterial plasma concentrations of the tracer and its metabolites was also assayed biochemically. FDOPA compartmental models that are based on biochemical information were investigated for their consistency with the measured striatal and cerebellar tissue kinetics. A modeling approach was also developed for separating plasma FDOPA and metabolite time-activity curves from the measured total 18F time-activity curve in plasma. Results showed that a model consisting of three separate compartments for tissue FDOPA, tissue 6-[18F]fluorodopamine (FDA) and its metabolites, and tissue L-3,4-dihydroxy-6-[18F]fluoro-3-O-methylphenylalanine (3-OMFD) could describe adequately the striatal kinetics in humans. Based on this model, the FDOPA transport constant across the blood-brain barrier (BBB) (K1), the FDOPA decarboxylation rate constant (k3), and the turn-over rate constant of FDA and its metabolites (k4) could be estimated by model fitting to the tissue kinetics and were found for the normal subjects to be 0.031 +/- 0.006 ml/min/g (mean +/- SD), 0.041 +/- 0.015/min, and 0.004 +/- 0.002/min, respectively. About 50% of the FDOPA that crossed the BBB from plasma to striatum was decarboxylated. The decarboxylation constant with respect to plasma FDOPA (K3) was 0.015 +/- 0.003 ml/min/g. The BBB transport corresponded to a permeability-surface area product of 0.032 ml/min/g for FDOPA. For 3-OMFD, the BBB transport was 1.7 times faster. The effects of tissue heterogeneity on the FDOPA kinetics and on the estimated model parameters were also investigated. The usefulness and implications of these findings for interpretation of PET FDOPA studies are discussed.

Radiofluorinated L-m-tyrosines: new in-vivo probes for central dopamine biochemistry.

In this work, we introduce 6-[18F]fluoro-L-m-tyrosine (6-FMT) and compare its in-vivo kinetic and bio-chemical behaviors in monkeys and rodents with those of 4-FMT and 6-[18F]fluoro-L-3, 4-dihydroxyphenylalanine (DOPA) (FDOPA). These radiofluorinated m-tyrosine presynaptic dopaminergic probes, resistant to peripheral 3-O-methylation, offer a nonpharmacological alternative to the use of catechol-O-methyltransferase inhibitors. Like FDOPA, 4-FMT and 6-FMT are analogs that essentially follow the L-DOPA pathway of central metabolism. After i.v. administration in nonhuman primates and rodents, these new radiofluorinated m-tyrosine analogs accumulate selectively in striatal structures and allow for the detection of additional innervation sites (e.g., brain stem) rich in aromatic amino acid decarboxylase. Bio-chemical analyses in rodents and monkeys revealed the specificity of their central and peripheral metabolism. Molecular and enzymatic mechanisms involved in their retention in central brain structures are consistent with involvement of dopaminergic neurons. The high signal-to-noise ratios observed make these radiofluorinated m-tyrosine analogs outstanding candidates for probing the integrity of central dopaminergic mechanisms in humans.

Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia.

Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase (luc) gene in G. lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G. lamblia. An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia.

Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA.

Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes. One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs. Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF). A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB). Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia. A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation. Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts. It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing. This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.

Using fast sequential asymmetric fanbeam transmission CT for attenuation correction of cardiac SPECT imaging.

UNLABELLED: The objective of this study was to determine the feasibility of using a fast (short-duration) transmission computed tomogram (TCT), acquired immediately before or after the emission CT, to correct for photon attenuation in cardiac SPECT. METHODS: The asymmetric fanbeam geometry with a 99mTc line source was used to acquire TCTs after conventional cardiac emission CT imaging on a triple-head SPECT system. The TCTs were reconstructed to generate patient-specific attenuation maps, which were used with an iterative maximum likelihood algorithm to reconstruct attenuation-corrected cardiac SPECT studies. The results of attenuation correction based on TCTs as short as 1 min were compared with long-duration transmission imaging for a phantom and several human studies. RESULTS: Attenuation correction based on asymmetric fanbeam TCT significantly improves the uniformity of images of a uniform tracer distribution in a cardiac-thorax phantom configured to simulate a large patient. By using a high-activity line source and a rapid camera rotation, a suitable attenuation map for this phantom can be obtained from a 4-min TCT. A similar result is obtained for patients with thorax widths of <40 cm. CONCLUSION: A sequential imaging protocol for acquiring a fast TCT can be used for attenuation correction of cardiac SPECT imaging. The sequential TCT can be acquired without significantly extending the duration of the imaging study. This method provides a way to perform attenuation correction on existing triple-head SPECT systems without extensively modifying the system.

Methods for improving quantitation of putamen uptake constant of FDOPA in PET studies.

To estimate the striatal uptake constant of [18F]-L-6-fluorodopa (FDOPA) in humans, we studied two methods that can account for the image resolution and the background level of FDOPA. These two methods utilize information obtained from multiple ROIs varying in size around the putamen and from profiles crossing the middle of the putamen. The estimation of the uptake constant was based on a model of one-dimensional activity variation. Simulated data were used to evaluate the adequacy of these two methods. Parametric images of FDOPA uptake constants were generated using Patlak analysis for five studies in normals and were then analyzed with the two methods. Results from the simulated data indicated a good agreement between the estimated values and the true simulated values. Results from studies in normals show stable estimates of the FDOPA uptake constant that are not affected by image resolution. The two methods were not sensitive to the misplacement of ROIs and profiles.

[18F]Fluoro-beta-fluoromethylene-m-tyrosine derivatives show stereo, geometrical, and regio specificities as in vivo central dopaminergic probes in monkeys.

Stereo (D and L), geometrical (E and Z), and regiospecific (2-, 4-, and 6-[18F]fluoro) analogs of beta-fluoromethylene-m-tyrosine (FMMT) have been investigated in adult vervet monkeys (Cercopithecus aethiops sabaeus, n = 12) in vivo with positron emission tomography (PET). Brain transport through the blood-brain barrier and central aromatic amino acid decarboxylase (AAAD)-mediated decarboxylation rates were established. Results show strict structural dependency of the kinetic behavior of radiofluorinated FMMT analogs, with the E-isomer exhibiting a higher specificity over the (Z) geometrical counterpart for central dopaminergic structures. The 6-[18F]fluoro substituted L-(E)-FMMT was also favored over the 2- and 4-[18F]fluorosubstituted isomers in terms of their ability to localize in the same brain areas. The role of PET in drug development is also exemplified in this work.

The effects of measurement errors in the plasma radioactivity curve on parameter estimation in positron emission tomography.

The effects of three error sources in plasma curve measurements on parameter estimation in kinetic analysis of positron emission tomography (PET) fluorodeoxyglucose (FDG) data are investigated by computer simulation. The three error sources are: (1) measurement noise in the radioactivity concentrations of plasma samples; (2) linear interpolation between adjacent plasma sampling points of the plasma time activity curve; and (3) incorrect weights used for the least-squares regression. All three error sources are found to increase the variability of the parameter estimates, with the first one a primary error source in normal PET FDG studies. The performance of five estimation methods which account for the error sources are evaluated. When the noise variances of the plasma and the tissue measurements are not known, an iterative weighting procedure is shown to give accurate and reliable estimates.