AAV-RPGR Gene Therapy Rescues Opsin Mislocalisation in a Human Retinal Organoid Model of RPGR-Associated X-Linked Retinitis Pigmentosa.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

Post Flywheel Squat Potentiation of Vertical and Horizontal Ground Reaction Force Parameters during Jumps and Changes of Direction.

(1) Background: The aim of the study was to determine the post-activation performance enhancement (PAPE) of vertical and horizontal ground reaction force parameters during jumps and change of direction following flywheel squat exercise using two different flywheel inertias. (2) Methods: Eleven male athletes performed a countermovement jump (CMJ), standing broad jump (SBJ), and "modified 505" change of direction (COD) in a control condition and 6 minutes following three sets of six repetitions of flywheel half squats at one of two inertias (0.029 kg·m2 and 0.061 kg·m2). Peak directional ground reaction force, power, and rate of force development were calculated for each test. (3) Results: Higher inertia flywheel squats were able to acutely enhance CMJ peak vertical force (Bayes Factor (BF10) = 33.5, very strong; δ = 1.66; CI: 0.67, 2.70), whereas lower inertia flywheel squats were able to acutely enhance CMJ peak vertical power (BF10 = 3.65, moderate; δ = 0.93; CI: 0.11, 1.88). The vertical squat exercise induced no PAPE effect on resultant SBJ or horizontal COD ground reaction force parameters, nor were any differences observed between the inertias. (4) Conclusions: Researchers and practitioners should consider the kinetic and kinematic correspondence of a pre-load stimulus to the subsequent sport-specific activity (i.e., flywheel squat to CMJ).

Effect of Postactivation Potentiation After Medium vs. High Inertia Eccentric Overload Exercise on Standing Long Jump, Countermovement Jump, and Change of Direction Performance.

ABSTRACT: Beato, M, De Keijzer, KL, Leskauskas, Z, Allen, WJ, Dello Iacono, A, and McErlain-Naylor, SA. Effect of postactivation potentiation after medium vs. high inertia eccentric overload exercise on standing long jump, countermovement jump, and change of direction performance. J Strength Cond Res 35(9): 2616-2621, 2021-This study aimed to evaluate the postactivation potentiation (PAP) effects of an eccentric overload (EOL) exercise on vertical and horizontal jumps and change of direction (COD) performance. Twelve healthy physically active male subjects were involved in a crossover study. The subjects performed 3 sets of 6 repetitions of EOL half squats for maximal power using a flywheel ergometer. Postactivation potentiation using an EOL exercise was compared between a medium (M-EOL) vs. high inertia (H-EOL) experimental condition. Long jump (LJ) was recorded at 30 seconds, 3, and 6 minutes after both EOL exercises and compared with baseline values (control). The same procedure was used to assess countermovement jump (CMJ) height and peak power and 5-m COD test (COD-5m). A fully Bayesian statistical approach to provide probabilistic statements was used in this study. Long jump performance reported improvements after M-EOL and H-EOL exercise (Bayes factor [BF10] = 32.7, strong; BF10 = 9.2, moderate), respectively. Countermovement jump height (BF10 = 135.6, extreme; BF10 > 200, extreme), CMJ peak power (BF10 > 200, extreme; BF10 = 56.1, very strong), and COD-5m (BF10 = 55.7, very strong; BF10 = 16.4, strong) reported improvements after M-EOL and H-EOL exercise, respectively. Between analysis did not report meaningful differences in performance between M-EOL and H-EOL exercises. The present outcomes highlight that PAP using an EOL (M-EOL and H-EOL) improves LJ, CMJ height, CMJ peak power, and COD-5m in male athletes. The optimal time window for the PAP effect was found for both EOL conditions from 3 to 6 minutes. However, M-EOL and H-EOL produce similar PAP effect on LJ, CMJ, and COD-5m tasks.

Multisite Analysis of Lesions in the Respiratory Tract of the Rat and Nonhuman Primate (Cynomolgus Monkey) Exposed to Air, Vehicle, and Inhaled Small Molecule Compounds.

This paper presents a review of the nature, range, and incidences of background pathology findings in the respiratory tract of cynomolgus monkeys and rats. Data were collected from 81 inhalation studies and 133 non-inhalation studies evaluated at 3 geographically distinct contract research organization facilities. The inhalation studies were comprised of 44 different small molecule pharmaceuticals or chemicals which were also analyzed in order to understand the patterns of induced changes within the respiratory tract. The lung was the most frequently affected organ in both species, with increased alveolar macrophages being the most common background and test article-related finding. In the upper respiratory tract (URT), inflammatory cell infiltrates were the most common background findings in the nasal cavity in monkeys. Induced URT findings were more frequent in rats than monkeys, with squamous metaplasia in the larynx, and goblet cell hyperplasia in the nasal cavity being the most common. Overall, the data revealed a limited pattern of response to inhaled molecules in the respiratory tract, with background and test article-related findings often occurring in the same regions. It is hoped that these data will assist in the interpretation of findings in the respiratory tract induced by novel inhaled small molecule entities.

Post-activation potentiation effect of eccentric overload and traditional weightlifting exercise on jumping and sprinting performance in male athletes.

The aim of this study was to evaluate the post-activation potentiation (PAP) effects following eccentric overload (EOL) and traditional weightlifting (TW) exercise on standing long jump (SLJ), countermovement jump (CMJ), and 5 m sprint acceleration performance. Ten male athletes were involved in a randomized, crossover study. The subjects performed 3 sets of 6 repetitions of EOL or TW half squat exercise followed by SLJ, CMJ, and 5 m sprint tests at 1 min, 3 min and 7 min, in separate sessions using a randomized order. Bayes factor (BF10) was reported to show the strength of the evidence. Differences were found using EOL for SLJ distance at 3 min (BF10 = 7.24, +8%), and 7 min (BF10 = 19.5, +7%), for CMJ at 3 min (BF10 = 3.25, +9%), and 7 min (BF10 = 4.12, +10.5%). Differences were found using TW exercise for SLJ at 3 min (BF10 = 3.88, +9%), and 7 min (BF10 = 12.4, +9%), CMJ at 3 min (BF10 = 7.42, +9.5%), and 7 min (BF10 = 12.4, +12%). No meaningful differences were found between EOL and TW exercises for SLJ (BF10 = 0.33), CMJ (BF10 = 0.27), and 5 m sprint (BF10 = 0.22). In conclusion, EOL and TW exercises acutely increase SLJ and CMJ, but not 5 m sprint performance. The PAP time window was found between 3 min and 7 min using both protocols. This study did not find differences between EOL and TW exercises, and so both methodologies can be used to stimulate a PAP response.

Normal Anatomy, Histology, and Spontaneous Pathology of the Kidney, and Selected Renal Biomarker Reference Ranges in the Cynomolgus Monkey.

To further our understanding of the nonhuman primate kidney anatomy, histology, and incidences of spontaneous pathology, we retrospectively examined kidneys from a total of 505 control Cynomolgus monkeys (Macaca fascicularis; 264 male and 241 females) aged 2 to 6 years, from toxicity studies. Kidney weights, urinalysis, and kidney-related clinical biochemistry parameters were also evaluated. Although the functional anatomy of the monkey kidney is relatively similar to that of other laboratory animals and humans, a few differences and species-specific peculiarities exist. Unlike humans, the macaque kidney is unipapillate, with a relatively underdeveloped papilla, scarce long loops of Henle, and a near-equivalent cortical to medullary ratio. The most common spontaneous microscopic findings were interstitial infiltrates or interstitial nephritis and other tubular lesions, but several forms of glomerulopathy that may be interpreted as drug-induced were occasionally observed. Common incidental findings of little pathological significance included: papillary mineralization, epithelial pigment, multinucleate cells, cuboidal metaplasia of the Bowman's capsule, and urothelial inclusions. Kidney weights, and some clinical chemistry parameters, showed age- and sex-related variations. Taken together, these data will aid the toxicologic pathologist to better evaluate the nonhuman primate kidney and assess the species' suitability as a model for identifying and characterizing drug-induced injury.

Adrenal Gland Background Findings in CD-1 (Crl:CD-1(ICR)BR) Mice from 104-week Carcinogenicity Studies.

The authors performed a retrospective study to determine the incidences of spontaneous findings in the adrenal glands of control CD-1 mice. Data were collected from 2,163 mice from control dose groups in 104-week carcinogenicity studies carried out between 2000 and 2010. Adrenal gland nonproliferative lesions were more common in males than in females. In males, the most common nonproliferative lesions were cortical hypertrophy, cortical atrophy, pigment deposition/pigmentation, cysts, and extramedullary hematopoiesis. In females, the most common nonproliferative lesions were pigment deposition/pigmentation, extramedullary hematopoiesis, and cortical atrophy. Proliferative lesions were more common in females than in males. In both sexes, the most common proliferative lesions were subcapsular cell hyperplasia, focal cortical hyperplasia, and subcapsular cell tumor. Pheochromocytomas were uncommon in both sexes, with a slightly higher incidence in females, and the benign type was more frequent than the malignant type. Lymphoma was the most common metastatic tumor in both males and females, followed by histiocytic sarcoma and erythroid/myeloid leukemia. To the best knowledge of the authors, there are no recent reports on spontaneous pathological findings in the adrenal glands of CD-1 mice, and these results will facilitate the interpretation of background findings in carcinogenicity studies.

Incidences and Range of Spontaneous Lesions in the Eye of Crl:CD-1(ICR)BR Mice Used in Toxicity Studies.

The incidence and range of spontaneous pathology findings were determined in the eyes of male and female control Crl:CD-1(ICR)BR mice. Data were collected from 250, 430, 510, and 2,266 mice from control dose groups of 4-, 13-, 80- and 104-week studies, respectively, carried out between 2005 and 2013. Lesions of the eye were very rare in 4- and 13-week studies, uncommon in 80-week studies, and were of relatively higher incidence in 104-week studies. No sex predilection in the incidence of eye lesions was apparent. No neoplastic lesions were observed, and congenital lesions were very rare. The most common findings were cataracts, retinal degeneration, mineral deposits in the iris, keratitis, anterior uveitis, and mineral deposits in the corneal stroma. These lesions were observed only in animals from 80- and 104-week studies, except retinal degeneration which was observed in animals from all age-groups. There are no previous reports of mineral deposits in the iris in this strain of mice. It is hoped that reference to the incidences reported here will facilitate the differentiation of spontaneous lesions from compound-induced lesions in toxicology studies in this strain of mouse.

Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

PURPOSE: StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. METHODS: Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. RESULTS: Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. CONCLUSIONS: In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

Analysis of pre-treatment markers predictive of treatment benefit for the therapeutic cancer vaccine MVA-5T4 (TroVax).

Cancer vaccines such as MVA-5T4 (TroVax(®)) must induce an efficacious immune response to deliver therapeutic benefit. The identification of biomarkers that impact on the clinical and/or immunological efficacy of cancer vaccines is required in order to select patients who are most likely to benefit from this treatment modality. Here, we sought to identify a predictor of treatment benefit for renal cancer patients treated with MVA-5T4. Statistical modeling was undertaken using data from a phase III trial in which patients requiring first-line treatment for metastatic renal cell carcinoma were randomized 1:1 to receive MVA-5T4 or placebo alongside sunitinib, IL-2 or IFN-α. Numerous pre-treatment factors associated with inflammatory anemia (e.g., CRP, hemoglobin, hematocrit, IL-6, ferritin, platelets) demonstrated a significant relationship with tumor burden and patient survival. From these prognostic factors, the pre-treatment mean corpuscular hemoglobin concentration (MCHC) was found to be the best predictor of treatment benefit (P < 0.01) for MVA-5T4 treated patients and also correlated positively with tumor shrinkage (P < 0.001). Furthermore, MCHC levels showed a significant positive association with 5T4 antibody response (P = 0.01). The latter result was confirmed using an independent data set comprising phase II trials of MVA-5T4 in patients with colorectal, renal and prostate cancers. Retrospective analyses demonstrated that RCC patients who had very large tumor burdens and low MCHC levels received little or no benefit from treatment with MVA-5T4; however, patients with smaller tumor burdens and normal MCHC levels received substantial benefit from treatment with MVA-5T4.

Spontaneous cerebellar primitive neuroectodermal tumor in a juvenile cynomolgus monkey (Macaca fascicularis).

A neoplastic mass compressing the left cerebellar hemisphere and hindbrain was observed at trimming in a 3½-year-old male cynomolgus monkey from a control dose group. Microscopically, the neoplastic mass was nonencapsulated, invasive, and showed two morphological patterns. The predominant area consisted of densely packed undifferentiated, polygonal to spindle cells arranged in vague sheets supported by a scant fibrovascular stroma. The other area was less cellular and composed of round neoplastic cells separated by eosinophilic fibrillar material. Immunohistochemical staining for vimentin, synaptophysin, glial fibrillary acidic protein, neuron-specific enolase, neurofilament, and S-100 confirmed the presence of primitive undifferentiated neuroectodermal cells and some cells with neuronal or glial differentiation. On the basis of histopathology and immunohistochemical findings, a diagnosis of cerebellar primitive neuroectodermal tumor with neuronal and glial differentiation was made. Primitive neuroectodermal tumors are rare in animals including nonhuman primates; this is the first published report in this species.

MVA-5T4-induced immune responses are an early marker of efficacy in renal cancer patients.

Few immunotherapy compounds have demonstrated a direct link between the predicted mode of action of the product and benefit to the patient. Since cancer vaccines are thought to have a delayed therapeutic effect, identification of the active moiety may enable the development of an early marker of efficacy. Patients with renal cancer and requiring first-line treatment for metastatic disease were randomized 1:1 to receive MVA-5T4 (TroVax(®)) or placebo alongside Sunitinib, IL-2 or IFN-α in a multicentre phase III trial. Antibody responses were quantified following the 3rd and 4th vaccinations. A surrogate for 5T4 antibody response (the immune response surrogate; IRS) was constructed and then used in a survival analysis to evaluate treatment benefit. Seven hundred and thirty-three patients were randomized, and immune responses were assessed in 590 patients. A high 5T4 antibody response was associated with longer survival within the MVA-5T4-treated group. The IRS was constructed as a linear combination of pre-treatment 5T4 antibody levels, hemoglobin and hematocrit and was shown to be a significant predictor of treatment benefit in the phase III study. Importantly, the IRS was also associated with antibody response and survival in an independent dataset comprising renal, colorectal and prostate cancer patients treated with MVA-5T4 in phase I-II studies. The derivation of the IRS formed part of an exploratory, retrospective analysis; however, if confirmed in future studies, the results have important implications for the development and use of the MVA-5T4 vaccine and potentially for other similar vaccines.

Genetically modified macrophages expressing hypoxia regulated cytochrome P450 and P450 reductase for the treatment of cancer.

This study describes a combined gene and cell therapy based on the genetic modification of primary human macrophages, as a treatment for cancer. Here, we have utilised the tumour-infiltrating properties of macrophages as vehicles to deliver a gene encoding a prodrug-activating enzyme such as human cytochrome P450 2B6 (CYP2B6) inside tumours followed by killing the tumour cells with the prodrug cyclophosphamide (CPA). Macrophages were transduced with an adenoviral vector that expresses human cytochrome CYP2B6 via a synthetic hypoxia responsive promoter (OBHRE) and with human P450 reductase (P450R), via the CMV promoter. In the presence of CPA, these genetically modified macrophages showed increased cytotoxicity against various tumour cell lines compared to untransduced macrophages or macrophages transduced with CYP2B6 alone. In human ovarian carcinoma xenograft models, the median survival of mice treated with genetically modified macrophages plus CPA increased up to two-fold compared to the survival of mice treated with untransduced macrophages and CPA. Genetically modified autologous macrophages may be a feasible therapeutic option for the treatment of some solid tumours, such as ovarian cancer.

Vaccination of metastatic renal cancer patients with MVA-5T4: a randomized, double-blind, placebo-controlled phase III study.

PURPOSE: The TroVax Renal Immunotherapy Survival Trial was a randomized, placebo-controlled phase III study that investigated whether modified vaccinia Ankara encoding the tumor antigen 5T4 (MVA-5T4) prolonged survival of patients receiving first-line standard-of-care (SOC) treatment for metastatic renal cell cancer. EXPERIMENTAL DESIGN: Patients with metastatic clear cell renal cancer, prior nephrectomy, and good or intermediate prognosis were randomized 1:1 to receive up to 13 immunizations of MVA-5T4/placebo in combination with either sunitinib, interleukin-2 or interferon-α. The primary end point was overall survival. Secondary end points included progression-free survival, overall response rate, and safety. RESULTS: Seven hundred thirty-three patients were recruited (365 MVA-5T4 and 368 placebo). Treatment arms were well balanced for SOC and prognosis. No significant difference in the incidence of adverse events or serious adverse events was observed. No significant difference in overall survival was evident in the two treatment arms (median 20.1 months MVA-5T4 versus 19.2 months placebo; P = 0.55). The magnitude of the 5T4-specific antibody response induced by vaccination with MVA-5T4 was associated with enhanced patient survival. Furthermore, exploratory analyses suggested a number of pretreatment hematologic factors that could identify patients who derive significant benefit from this vaccine. CONCLUSION: MVA-5T4 in combination with SOC was well tolerated, but no difference in survival was observed in the overall study population. Exploratory analyses indicate that there may be subsets of patients who could gain significant benefit from MVA-5T4, but such results would need to be confirmed in future randomized clinical studies.

Incidences and range of spontaneous findings in control cynomolgus monkeys (Macaca fascicularis) used in toxicity studies.

The authors performed a retrospective study to determine the incidences and range of spontaneous pathology findings in control cynomolgus monkeys. Data were collected from 570 monkeys (285 animals per sex), aged twelve to thirty-six months, from sixty regulatory studies evaluated at our laboratory between 2003 and 2009. The most common finding overall was lymphoplasmacytic infiltrates observed in the following incidence: liver (60.7%), kidneys (28.8%), heart (25.8%), salivary glands (21.2%), and stomach (12.1%). Inflammation also commonly occurred in the heart, kidneys, lungs, and stomach. The most common degenerative changes were localized fatty change in the liver, myocardial degeneration, and mineralization and pigment deposits in various tissues. Parathyroid, thyroid, and pituitary cysts; ectopic thymus in the parathyroid or thyroid gland; accessory spleen within the pancreas; and adrenohepatic fusion were among the most common congenital findings. Some incidental findings bearing similarities to drug-induced lesions were also encountered in various organs. It is hoped that the results presented here and elsewhere could form the groundwork for the creation of a reliable database of incidental pathology findings in laboratory nonhuman primates.

Vaccination of renal cell cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) alone or administered in combination with interferon-alpha (IFN-alpha): a phase 2 trial.

Attenuated vaccinia virus, modified vaccinia Ankara (MVA) has been engineered to deliver the tumor antigen 5T4 (TroVax). MVA-5T4 has been evaluated in an open-label phase 2 trial in metastatic renal cell cancer patients in which the vaccine was administered alone or in combination with interferon-alpha-2b (IFN-alpha). The safety, immunologic, and clinical efficacy of MVA-5T4 with or without IFN-alpha was determined. Twenty-eight patients with metastatic renal cell cancer were treated with MVA-5T4 alone (13) or plus IFN-alpha (15). The 5T4-specific cellular and humoral responses were monitored throughout the study. Clinical responses were assessed by measuring changes in tumor burden by computed tomography or magnetic resonance imaging scan. MVA-5T4 was well tolerated with no serious adverse event attributed to vaccination. Of 23 intent-to-treat patients tested for immune responses postvaccination, 22 (96%) mounted 5T4-specific antibody and/or cellular responses. One patient treated with MVA-5T4 plus IFN-alpha showed a partial response for >7 months, whereas an additional 14 patients (7 receiving MVA-5T4 plus IFN and 7 receiving MVA-5T4 alone) showed periods of disease stabilization ranging from 1.73 to 9.60 months. Median progression free survival and overall survival for all intent-to-treat patients was 3.8 months (range: 1 to 11.47 mo) and 12.1 months (range: 1 to 27 mo), respectively. MVA-5T4 administered alone or in combination with IFN-alpha was well tolerated in all patients. Despite the high frequency of 5T4-specific immune responses, it is not possible to conclude that patients are receiving clinical benefit. The results are encouraging and warrant further investigation.

Vaccination of patients with metastatic renal cancer with modified vaccinia Ankara encoding the tumor antigen 5T4 (TroVax) given alongside interferon-alpha.

Approximately 90% of renal cell tumors overexpress the tumor antigen 5T4. The attenuated strain of vaccinia virus, modified vaccinia Ankara, has been engineered to express 5T4 (TroVax). We conducted an open-label phase 1/2 trial in which TroVax was administered alongside interferon-alpha (IFNalpha) to 11 patients with metastatic renal cell carcinoma. Antigen-specific cellular and humoral responses were monitored throughout the study, and clinical responses were assessed by measuring the changes in tumor burden by computed tomography scan (Response Evaluation Criteria In Solid Tumors). The primary objective was to assess the safety, immunogenicity, and efficacy of TroVax when given alongside IFNalpha. Treatment with TroVax plus IFNalpha was well tolerated with no serious adverse events attributed to TroVax. All 11 patients mounted 5T4-specific antibody responses and 5 (45%) mounted cellular responses. No objective tumor responses were seen, but the overall median time to progression (TTP) of 9 months (range: 2.1 to 26+ mo) was longer than expected for IFNalpha alone. For the 10 clear cell patients the TTP ranged from 3.9 to 26+ months, with a median TTP of 10.4 months. The high frequency of 5T4-specific immune responses and prolonged median TTP for clear cell patients compared with that expected for IFNalpha alone is encouraging and warrants further investigation.

Phase II trial of Modified Vaccinia Ankara (MVA) virus expressing 5T4 and high dose Interleukin-2 (IL-2) in patients with metastatic renal cell carcinoma.

BACKGROUND: Interleukin-2 (IL-2) induces durable objective responses in a small cohort of patients with metastatic renal cell carcinoma (RCC) but the antigen(s) responsible for tumor rejection are not known. 5T4 is a non-secreted membrane glycoprotein expressed on clear cell and papillary RCCs. A modified vaccinia virus Ankara (MVA) encoding 5T4 was tested in combination with high-dose IL-2 to determine the safety, objective response rate and effect on humoral and cell-mediated immunity. METHODS: 25 patients with metastatic RCC who qualified for IL-2 were eligible and received three immunizations every three weeks followed by IL-2 (600,000 IU/kg) after the second and third vaccinations. Blood was collected for analysis of humoral, effector and regulatory T cell responses. RESULTS: There were no serious vaccine-related adverse events. While no objective responses were observed, three patients (12%) were rendered disease-free after nephrectomy or resection of residual metastatic disease. Twelve patients (48%) had stable disease which was associated with improved median overall survival compared to patients with progressive disease (not reached vs. 28 months, p = 0.0261). All patients developed 5T4-specific antibody responses and 13 patients had an increase in 5T4-specific T cell responses. Although the baseline frequency of Tregs was elevated in all patients, those with stable disease showed a trend toward increased effector CD8+ T cells and a decrease in Tregs. CONCLUSION: Vaccination with MVA-5T4 did not improve objective response rates of IL-2 therapy but did result in stable disease associated with an increase in the ratio of 5T4-specific effector to regulatory T cells in selected patients. TRIAL REGISTRATION NUMBER: ISRCTN83977250.

Vaccination of renal cell cancer patients with modified vaccinia ankara delivering tumor antigen 5T4 (TroVax) administered with interleukin 2: a phase II trial.

PURPOSE: The attenuated vaccinia virus modified vaccinia ankara (MVA) has been engineered to deliver the tumor antigen 5T4 (TroVax). TroVax has been evaluated in an open-label phase II trial in metastatic renal cell cancer patients in which the vaccine was administered in combination with interleukin-2 (IL-2). The safety, immunologic, and clinical efficacy of TroVax in combination with IL-2 was determined. EXPERIMENTAL DESIGN: Twenty-five patients with metastatic renal cell cancer were treated with TroVax plus IL-2. 5T4-specific cellular and humoral responses were monitored throughout the study. Clinical responses were assessed by measuring changes in tumor burden by computed tomography or magnetic resonance imaging scan. RESULTS: TroVax was well tolerated with no serious adverse event attributed to vaccination. Of 25 intention-to-treat patients, 21 mounted 5T4-specific antibody responses. Two patients showed a complete response for > 24 months and one a partial response for > 12 months. Six patients had disease stabilization from 6 to > 21 months. Median progression-free survival (PFS) and overall survival (OS) were > 3.37 months (range, 1.50- > 24.76) and > 12.87 months (range, 1.90- > 24.76), respectively. A statistically significant relationship was detected between the magnitude of 5T4-specific antibody responses and PFS and OS. CONCLUSION: TroVax in combination with IL-2 was safe and well tolerated in all patients. The high frequency of 5T4-specific immune responses and good clinical response rate are encouraging and warrant further investigation.

Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM.

Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37 degrees C and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37 degrees C may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.