Pigmentary mosaicism as a recurrent clinical manifestation in three new patients with mosaic trisomy 12 diagnosed postnatally: cases report and literature review.

BACKGROUND: To date, only twenty-one cases diagnosed postnatally with mosaic trisomy 12 have been reported. The most frequent phenotypic manifestations are developmental delay, dysmorphic facial features, congenital heart defects, digital alterations, and pigmentary disorders. In the present report, detailed clinical and genetic profiles of three unrelated new patients with mosaic trisomy 12 are described and compared with previously reported cases. CASE PRESENTATION: In the present report, we include the clinical, cytogenetic, and molecular description of three Mexican patients diagnosed postnatally with mosaic trisomy 12. At phenotypic level, the three patients present with developmental delay, dysmorphic facial features, congenital heart defects and skin pigmentary anomalies. Particularly, patient 1 showed unique eye alterations as bilateral distichiasis, triple rows of upper lashes, and digital abnormalities. In patient 2 redundant skin, severe hearing loss, and hypotonia were observed, and patient 3 presented with hypertelorism and telecanthus. Hyperpigmentation with disseminated pigmentary anomalies is a common trait in all of them. The cytogenetic study was carried out under the strict criteria of analysis, screening 50-100 metaphases from three different tissues, showing trisomy 12 mosaicism in at least one of the three different tissues analyzed. With SNParray, the presence of low-level mosaic copy number variants not previously detected by cytogenetics, and uniparental disomy of chromosome 12, was excluded. STR markers allowed to confirm the absence of uniparental disomy as well as to know the parental origin of supernumerary chromosome 12. CONCLUSIONS: The detailed clinical, cytogenetic, and molecular description of these three new patients, contributes with relevant information to delineate more accurately a group of patients that show a heterogeneous phenotype, although sharing the same chromosomal alteration. The possibility of detecting mosaic trisomy 12 is directly associated with the sensitivity of the methodology applied to reveal the low-level chromosomal mosaicism, as well as with the possibility to perform the analysis in a suitable tissue.

SARS-CoV-2 IgG response in symptomatic and asymptomatic COVID-19-infected healthcare workers.

BACKGROUND: Healthcare workers (HCWs) accounted for a significant proportion of COVID-19 infections worldwide. Retrospective seroprevalence surveys are often used to screen for unidentified previous infection with SARS-CoV-2. However, the rate of humoral response in HCWs affected by COVID-19 is not well-defined. AIMS: To assess the specific IgG humoral response in symptomatic and asymptomatic SARS-CoV-2-infected HCWs and identify potential factors associated with humoral response. METHODS: We prospectively recruited 204 HCWs with RT-PCR-confirmed COVID-19 infection to evaluate SARS-CoV-2 humoral response. Serum-IgG antibodies against SARS-CoV-2 were analysed using two commercially available serological assays. A logistic regression was performed to identify independent factors associated with positive IgG serology test. RESULTS: Overall, the SARS-CoV-2 IgG seropositivity rate was 77%. This seropositivity rate was higher in symptomatic than in asymptomatic COVID-19 infection (83% versus 57%; P < 0.001) and in older HCWs.. The seropositivity rate did not diminish with time. In logistic regression, only a history of COVID-19 symptoms and age were identified as independent factors associated with the detection of anti-SARS-CoV-2 IgG antibodies. CONCLUSIONS: SARS-CoV-2 IgG antibodies are found significantly more frequently in symptomatic and in older HCWs. The fact that not all COVID-19 HCWs develop detectable IgG is vital for the interpretation of COVID-19 seroprevalence surveys.

Curcumin induces p53-independent inactivation of Nrf2 during oxidative stress-induced apoptosis.

The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a master regulator of a battery of antioxidant and detoxificant genes with cytoprotective function. Since Nrf2 inactivation is necessary for the complete execution of apoptosis in the presence of extensive cellular damage caused by oxidative stress, constant activation of Nrf2 may protect tumoral cells from apoptosis. The tumor suppressor gene p53 has been suggested to participate in apoptosis-related repression of Nrf2. Thus, we studied the inactivation of Nrf2 during oxidant-induced apoptosis in a p53 dysfunctional cellular model. Using curcumin dose-response assay and time-response assay in an immortalized lymphoblastoid cell line (control line 45), we observed a time-dependent increase in apoptotic markers such as deoxyribonucleic acid (DNA) fragmentation, phosphatidylserine exposure, and caspase-3, caspase-9 and poly (ADP-ribose) polymerases (PARP) cleavage. Interestingly, at early times of exposure to a proapoptotic dose of curcumin (15 µM), we observed nuclear accumulation of Nrf2 and the expression of Nrf2 target genes, whereas at late exposure times we found a reduction of total and nuclear protein levels of Nrf2 as well as downregulation of Nrf2 target genes in the absence of p53 activation. These data suggest that apoptosis-related inactivation of Nrf2 could occur in a p53 dysfunctional background, opening the possible occurrence of p53-independent mechanism to explain Nrf2 inactivation during apoptosis.

[Bacteremia caused by Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae. A retrospective study of 7 years]. / Bacteriemia por Klebsiella pneumoniae productora de carbapenemasa tipo KPC. Estudio comparativo y evolución en 7 años.

OBJECTIVE: Bacteremia caused by Klebsiella pneumoniae carbapenemase-producing strains (Kp-KPC) is associated with high mortality. The hypothesis of our work is that there was an increase in the levels of resistance to different antimicrobials in Kp-KPC isolated from bacteremia. METHODS: Retrospective and descriptive study in two periods: Period 1 (P1) 2010-2014 and period 2 (P2) 2015-2016. We included patients ≥18 years old with bacteremia caused by Kp-KPC in a General Hospital. We defined active drug (AD) if it was in vitro susceptible and in the case of meropenem if it had a MIC ≤ 8 mg/L in combination treatment. RESULTS: Fifty episodes of bacteremia caused by Kp-KPC were analyzed in 45 patients. (P1: 21 and P2: 29). The following variables were similar in both periods: median age (53 vs. 52 years); male sex (45 vs. 62%); site of infection: primary bacteremia (52 vs.45%), bacteremia associated with catheter (24 vs.17%), and other (24 vs. 38%). During P2 there was a significant increase in colistin resistance (28 vs. 69%) (p <0.01), an increase in MIC to meropenem ≥ 16 mg/L (74 and 97%) (p = 0.02), and decrease in tigecycline resistance (29 vs. 4%) (p = 0.02). The overall mortality was 40 in P1 and 32% in P2 (p=0.7). There was not difference in mortality when the definitive treatment was with an active antimicrobial vs. two active antimicrobials, as well as between the different antimicrobials used. CONCLUSIONS: There was a significant increase in bacteremia caused by Kp-KPC and the level of colistin resistance and MIC to meropenem. Overall mortality was high in both periods.

Hemorragia uterina anormal / Uterine abnormal hemorrhage

La hemorragia uterina anormal (HUA) es un signo y síntoma clínico frecuente en el consultorio de ginecología, manifestándose en cualquier etapa de la vida de la mujer. Se presenta en el 25% de adolescentes y en el 50% en mayores de 40 años; además represen-ta un 25% de las cirugías ginecológicas. Tiene como factores predisponente la raza, gemelar, agregación familiar, asociación a enfermedades geneticas. La cantidad y duración de las menstruaciones es muy subjetiva, por lo que hay problemas para valorar las pérdidas sanguíneas de las pacientes. El diagnóstico de hemorragia uterina disfuncional es por exclusión, primero se debe descartar las causas orgánicas. Por ello la HUA es un problema frecuente que se puede presentar a cualquier edad repercutiendo en varios aspectos como el sanitario y social, su tratamiento debe ser multidisciplinario, ya que no debemos olvidar que es una de las principales causas de anemia en la mujer y también de histerectomías. (AU)

The HUA is a common clinical signs and symptoms in gynecology clinic, being a disorder in the female genital tract, can occur at any age of life of women. It comes in a 25% in adolescents and 50% older than 40 years and 25% of gynecological surgeries. It has an ethnic predisposition, twins, familial aggregation, association with genetic diseases. The amount and duration of menses is very subjective, so there are problems in assessing blood loss of patients. The diagnosis of dysfunctional uterine bleeding is by exclusion, you must first rule out organic causes. Thus the HUA is a common problem that can occur at any age impacting several aspects such as health and social, treatment should be multidis-ciplinary, and we must not forget that it is a major cause of anemia in women and hysterectomy. (AU)

Hepatitis B vaccination in haemodialysis patients: an underestimated problem. Factors influencing immune responses in ten years of observation in an Italian haemodialysis centre and literature review.

OBJECTIVES: Despite universal infection control precautions, the risk of hepatitis B virus (HBV) infection in patients on chronic haemodialysis (HD) remains high. For this reason anti-HBV vaccination is recommended in these subjects. In hemodialyzed patients vaccinal response is often suboptimal and it's not clear what factors may influence it. STUDY DESIGN: The aim of our study is to assess the influence of some clinical and laboratory factors on seroconversion rate after anti HBV vaccination in a cohort of patients on maintenance HD. METHODS: We analysed 60 patients on regular HD, 40 men and 20 women (age 64±12 years, range 40-88 years), immunized with Engerix B ® vaccine, followed for an average time of 62 month (12-120 months). For each patient the following data were collected: age, serum albumin (sAlb), Blood urea nitrogen before HD session (BUN), age at vaccination, dialysis vintage, presence of systemic disease, type of vascular access, dialysis modality. Correlation between these factors and anti Hbs titer was estimated with multiple regression analysis. RESULTS: Anti-Hbs seroconversion rate ( Anti Hbs > 10 IU/l) was 77%. Better rate of seroconversion (86%) was observed in patients with arteriovenous fistula (AVF) and serum albumin > 3,5 g/dL (93%), while higher rate of not responders (50%) in patients with systemic diseases. The only parameter correlated to anti Hbs titer was sAlb (p =0,0012). sAlb was correlated to age in all patients (p=0,01) and age was correlated to higher anti Hbs titer in the responder group (p=0,018). DISCUSSION: In our experience an early vaccination, when patients on chronic HD are younger and in better nutritional conditions, improves anti-HBV response.

[Antimicrobial susceptibility of Gram-negative bacilli of community acquired intra-abdominal infections in a hospital at Buenos Aires, Argentina]. / Sensiblidad antimicrobiana de bacilos gramnegativos de infecciones intraabdominales de la comunidad en un hospital de la ciudad de Buenos Aires, Argentina.

OBJECTIVE: Community acquired complicated intra-abdominal infections (cIAI) are a common condition. Few data are available about the level of antimicrobial resistance of Gram-negative bacteria isolated from community acquired cIAIs in Argentina. METHODS: Retrospective-prospective observational study (March 2010 to February 2012). Gram-negative bacteria antimicrobial susceptibility of isolates from community acquired cIAIs were evaluated. RESULTS: During this period, a total of 85 patients were included and 138 pathogens were collected. Male sex: 58%. Median age: 33. Monomicrobial cultures were obtained in 49% of the cases. Ninety (65%) corresponded to Gram-negative organisms, and 48 (38%) to Gram-positive cocci. Gram-negative organisms most frequently observed were: Escherichia coli 76%, Klebsiella pneumoniae 8%, Pseudomonas aeruginosa 7% and Enterobacter spp. 6%. E. coli and K. pneumoniae showed a high percentage of strains resistance to ciprofloxacin of 37% and 29%, respectively. Similarly, resistance to ampicillin/sulbactam was observed in a 16% of the E. coli isolates. The prevalence of multiresistant Gram-negative organisms was 38%. CONCLUSIONS: A high level of resistance to antimicrobials was observed in community acquired cIAIs, mainly to ciprofloxacin and ampicillin/sulbactam two of the most used antimicrobial for empirically treatment of cIAIs in our country. In addition a significant proportion of multiresistant Gram-negative organisms were identified.

Nuclear factor erythroid 2-related factor gene variants and susceptibility of arsenic-related skin lesions.

Inorganic arsenic (iAs) is an important pollutant associated with various chronic-degenerative diseases. The cytoprotective protein nuclear factor erythroid 2-related factor (NRF2) has been proposed as an important responsive mechanism against iAs exposure. The aim of this study was to determine whether the risk of skin lesions in people exposed to iAs-contaminated water could be modified by the presence of single nucleotide polymorphisms in the NRF2 coding gene. We studied 117 individuals with long-term iAs exposure and 120 nonexposed individuals. Total As was determined in water, meanwhile iAs and its metabolites were measured in urine. The iAs-induced skin lesion status was evaluated by expert dermatologists. We sequenced the promoter region of NRF2 in a sample of 120 healthy donors. We found four polymorphisms previously reported and one novel polymorphism in the 5' regulatory region of the NRF2. In this study, we did not find allelic and genotype association of NRF2 polymorphisms with iAs-related skin lesion. However, the analysis of haplotypes composed by -653GA, and -617CA NRF2 single nucleotide polymorphisms showed a significant association with protection against skin lesions in the low-As exposure group. This is the first report studying the association between NRF2 polymorphisms and susceptibility of As-related skin lesions. Increasing the sample size will allow us to confirm this data.

Intraprostatic distribution and long-term follow-up after AdV-tk immunotherapy as neoadjuvant to surgery in patients with prostate cancer.

A phase I-II study to evaluate gene-mediated cytotoxic immunotherapy in newly diagnosed prostate cancer before radical prostatectomy was conducted in Monterrey, Mexico. First, to investigate delivery of adenovirus to the prostate, fluorescently labeled vector was injected into fresh prostatectomy specimens and distribution was visually analyzed. The optimal volume and site instillation was then used for transrectal ultrasound guided intraprostatic injection in 10 patients with adenocarcinoma scheduled for radical prostatectomy. Each received two apical and two basal 0.5 ml injections of AdV-tk for a total of 1 × 10(11) vp followed by 14 days of prodrug. Nine patients continued to tumor resection: six high risk, one intermediate and two low risk. In vivo vector distribution was analyzed from the resected tissue of four patients. Patients were monitored for tumor progression and acute and long-term safety. For vector delivery, two apical and two basal injections of 0.5 ml led to optimal organ-wide distribution ex vivo and in vivo. Cytotoxicity was evidenced by transient rise in PSA and tumor histology. There were no significant adverse events deemed related to the treatment and no late toxicities after median follow-up of 11.3 years. All six high-risk patients had positive surgical margins and one had seminal vesicle involvement. Despite slow PSA rise post surgery in three of these patients, none developed metastases. The intermediate- and low-risk patients had complete resections and none have progressed. In conclusion, in vivo transrectal ultrasound guided instillation of an adenoviral vector into four sites in the prostate was practical as an outpatient procedure, well tolerated and led to distribution throughout the intraprostatic tumor mass. AdV-tk demonstrated no significant acute or late toxicities. Trends in PSA and disease progression conveyed the possibility of a sustained immune response against residual disease.

Human Mycobacterium bovis infection in Buenos Aires: epidemiology, microbiology and clinical presentation.

We performed a retrospective study of clinical, epidemiological and microbiological characteristics of patients with confirmed Mycobacterium bovis infection treated at Francisco Muñiz Hospital, Buenos Aires, Argentina, between 1996 and 2008. A total of 39 patients were included, accounting for 0.4% of tuberculosis cases in our hospital. Of these, 93% had at least one risk factor for M. bovis; the most frequent was occupational exposure (65%), followed by history of living in a rural area (31%) and consumption of unpasteurised milk (4%). Pulmonary disease was the most frequent clinical presentation. Rifampicin resistance and multidrug resistance were seen in two patients, both of whom had human immunodeficiency virus infection.

Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models.

Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRAS(G12V) and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma.

The NRF2 gene variant, -653G/A, is associated with nephritis in childhood-onset systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with oxidative stress and characterized by chronic inflammation. Kidney malfunction, an aggressive characteristic of this disease, is not present in all affected individuals. The Nrf2-Keap1 pathway is important in protecting against oxidative stress and inflammation. Mouse models and genome-wide scans have suggested NRF2 (Nuclear factor (erythroid-derived 2)-like 2) as a candidate gene for susceptibility to SLE. We therefore investigated whether NRF2 polymorphisms are associated with childhood-onset SLE in a Mexican Mestizo population. Two single nucleotide polymorphisms (SNPs) were genotyped by TaqMan((R)) assays in 362 patients with childhood-onset SLE and 379 controls. We found no significant association between susceptibility to SLE and NRF2 polymorphisms. However, after population stratification by gender, the heterozygous genotype of the -653G/A SNP was significantly associated with nephritis in females only [OR = 1.81, CI (1.04-3.12), p = 0.032]. This association was stronger in females affected with severe nephritis [classes IV-VI; OR = 2.16, CI (1.12-4.15), p = 0.019]. Our results suggest that NRF2 is not associated with susceptibility to childhood-onset SLE, but it could confer a risk for developing kidney malfunction in SLE-affected individuals.

Virus specific immune responses after human neoadjuvant adenovirus-mediated suicide gene therapy for prostate cancer.

PURPOSE: Neoadjuvant gene therapy potentially improves the outcome of primary treatment of prostate cancer by radical prostatectomy in patients with high risk of recurrence. We conducted a Phase I escalating dose study with a replication-defective adenovirus expressing the herpes simplex virus-thymidine kinase gene (Adv-HSV-tk vector). The primary end point was toxicity, while the evaluation of the patients' cellular and humoral immune responses served as a secondary endpoint. MATERIAL AND METHODS: The Adv-HSV-tk vector was injected into the prostate in two doses (2x10(10) to 2x10(11) viral particles), followed by ganciclovir twice daily for 14 days and retropubic radical prostatectomy on day 21. Adenovirus-specific neutralizing, IgG and IgA antibodies were evaluated. Peripheral blood mononuclear cells (PBMC) were stimulated by Adv-HSV-tk and analysed for IFN-gamma production and 3H-thymidine incorporation. Prostate specimens were immunostained for B (CD20+) and for T (CD3+) lymphocytes. RESULTS: Toxicity was minor in all 8 patients treated. In the prostate, no virus related cytopathic effect could be observed. Dose-dependent infiltration of T and B lymphocytes in the whole prostate and in tumor areas was observed. Boosting of adenovirus-specific antibody responses was observed in 7 patients, and an increased adenovirus-specific PBMC proliferation and IFN-gamma production was seen after Adv-HSV-tk stimulation. CONCLUSION: Neo-adjuvant adenovirus-mediated cytotoxic gene therapy prior to prostatectomy for prostate cancer is feasible and safe in an outpatient setting for intraprostatic vector doses up to 2x10(11) viral particles. Activation of the immune system was observed. Application of higher vector doses may be considered.

Effects of arsenite on cell cycle progression in a human bladder cancer cell line.

Bladder cancer is one of the most important diseases associated with arsenic (As) exposure in view of its high prevalence and mortality rate. Experimental studies have shown that As exposure induces cell proliferation in the bladder of sodium arsenite (iAsIII) subchronically treated mice. However, there is little available information on its effects on the cell cycle of bladder cells. Thus, our purpose was to evaluate the effects of iAsIII on cell cycle progression and the response of p53 and p21 on the human-derived epithelial bladder cell line HT1197. iAsIII treatment (1-10 microM) for 24 h induced a dose-dependent increase in the proportion of cells in S-phase, which reached 65% at the highest dose. A progressive reduction in cell proliferation was also observed. BrdU was incorporated to cellular DNA in an interrupted form, suggesting an incomplete DNA synthesis. The time-course of iAsIII effects (10 microM) showed an increase in p53 protein content and a transient increase in p21 protein levels accompanying the changes in S-phase. These effects were correlated with iAs concentrations inside the cells, which were not able to metabolize inorganic arsenic. Our findings suggest that p21 was not able to block CDK2-cyclin E complex activity and was therefore unable to arrest cells in G1 allowing their progression into the S-phase. Further studies are needed to ascertain the mechanisms underlying the effects of iAsIII on the G1 to S phase transition in bladder cells.

Intravenous administration of replication-incompetent adenovirus to rhesus monkeys induces thrombocytopenia by increasing in vivo platelet clearance.

A replication-incompetent adenovirus vector was administered to rhesus macaques at 1, 3 and 6 x 1012 particles/kg doses to investigate its toxicity. Platelet count decrements of 28%, 82% and 90%, respectively, were observed, with corresponding platelet half-lives of 69.0, 25.2 and 22.2 h (compared with 111 h in untreated animals). The platelet decline was equivalent for all three doses for 8 h, and platelet count recovery began as early as 8 h after infusion for low-dose recipients, or as late as 24 h for the medium and high dose recipients. These observations suggest that thrombocytopenia is a saturable, reversible consumptive process.

Adenovirus-mediated thymidine kinase gene therapy for recurrent ovarian cancer: expression of coxsackie-adenovirus receptor and integrins alphavbeta3 and alphavbeta5.

OBJECTIVE: Ten patients with recurrent ovarian cancer received a combined treatment of optimal tumor debulking, adenovirus-mediated herpes simplex virus thymidine kinase gene therapy (GT), and systemic application of acyclovir or valacyclovir and topotecan. Biopsies were taken at the time of secondary debulking about 1 month after the application of GT and chemotherapy and were analyzed for expression of coxsackie-adenovirus receptor (CAR) and integrins alphavbeta3 and alphavbeta5 with respect to treatment response. METHODS: Treatment modalities and study design have been described recently. Immunohistochemistry was used to visualize expression of CAR and integrins alphavbeta3 and alphavbeta5 in tumor samples taken before and after application of GT. RESULTS: Before GT six of ten patients presented with CAR-positive and four with CAR-negative tumors. After GT all tumors showed CAR expression. Integrin alphavbeta3 was found in all tumors before and after GT. Expression of integrin alphavbeta5 was seen in eight of ten tumor samples before GT and in all samples after GT. CONCLUSION: Despite the importance of CAR and integrin expression for successful adenovirus internalization, other cell surface receptors might be involved in this process. It is too early to decide whether expressions of CAR and integrin alphavbeta3/alphavbeta5 on tumor cells are appropriate additional inclusion criteria for the enrollment of patients in GT trials. Further research is necessary to evaluate the effect of GT plus chemotherapy on CAR and integrin expression.

Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion.

Adenoviral vectors efficiently target normal liver cells; however, a clear-cut description of the safety boundaries for using adenovectors in hepatic cirrhosis has not been settled. With this in mind, we used a first-generation, replication-deficient adenoviral vector carrying the E. coli lacZ gene (Ad5betaGal) to monitor therapeutic range, biodistribution, toxicity and transduction efficiency in Wistar rats made cirrhotic by two different experimental approaches resembling alcoholic cirrhosis and biliary cirrhosis in humans. Further, we show proof of concept on fibrosis reversion by a 'therapeutic' Ad-vector (AdMMP8) carrying a gene coding for a collagen-degrading enzyme. Dose-response experiments with Ad5betaGal ranging from 1 x 10(8)-3 x 10(12) viral particles (vp) per rat (250 g), demonstrated that adenovirus-mediated gene transfer via iliac vein at 3 x 10(11 )vp/rat, resulted in an approximately 40% transduction in livers of rats made cirrhotic by chronic intoxication with carbon tetrachloride, compared with approximately 80% in control non-cirrhotic livers. In rats made cirrhotic by bile-duct obstruction only, 10% efficiency of transduction was observed. Biodistribution analyses showed that vector expression was detected primarily in liver and at a low level in spleen and kidney. Although there was an important increase in liver enzymes between the first 48 h after adenovirus injection in cirrhotic animals compared to non-transduced cirrhotic rats, this hepatic damage was resolved after 72-96 h. Then, the cDNA for neutrophil collagenase, also known as Matrix Metalloproteinase 8 (MMP8), was cloned in an Ad-vector and delivered to cirrhotic rat livers being able to reverse fibrosis in 44%. This study demonstrates the potential use of adenoviral vectors in safe transient gene therapy strategies for human liver cirrhosis.

Histologic and immunohistochemical analysis of tissue response to adenovirus-mediated herpes simplex thymidine kinase gene therapy of ovarian cancer.

Herpes simplex virus (HSV) thymidine kinase (tk) gene incorporated into adenovirus was delivered intraperitoneally (ip) followed by an antiherpetic prodrug and topotecan in patients with recurrent epithelial ovarian cancer. Tissue response was evaluated. Ten patients underwent secondary debulking with subsequent delivery of ADV-HSV-tk therapy. Two patients each were treated at dose level 1 (2 x 10(10) vector particles = VP), 2 (2 x 10(11) VP), and 3 (2 x 10(12) VP); four patients were treated at dose level 4 (2 x 10(13) VP). Five patients underwent second-look surgery about one month after gene therapy (GT). Treatment response, presence of vector DNA, protein expression of steroid hormone receptors, p53, c-erbB2 and Ki67 protein were analyzed. At second-look, two out of five patients were tumor-free and none of their peritoneal biopsies showed vector DNA. After GT, the vital tumor mass was smaller, desmoplastic reaction had increased, and tumors were less differentiated with an increase of Ki67 expression. There was no change in expression of hormone receptors, p53, or c-erbB2. ADV-HSV-tk GT appears to eliminate cells with higher differentiation first and might induce fibrosis. Dedifferentiation might render residual cells more sensitive to chemotherapy secondary to their subsequent higher mitotic activity.

Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters.

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Adenovirus-mediated thymidine kinase gene therapy in combination with topotecan for patients with recurrent ovarian cancer: 2.5-year follow-up.

OBJECTIVE: Patients with recurrent ovarian cancer were treated intraperitoneally (ip) with a replication-deficient recombinant adenovirus (ADV) containing the herpes simplex virus thymidine kinase gene. Vector delivery was followed by intravenous administration of an antiherpetic prodrug and a topoisomerase I inhibitor. METHODS: Ten patients with stage IIIc epithelial ovarian cancer underwent secondary debulking to < or =0.5 cm residual tumor. Patients with normal ip flow received delivery of ip ADV. Two patients each were treated on dose level 1 (2 x 10(10) vector particles), dose level 2 (2 x 10(11)), and dose level 3 (2 x 10(12)); four patients were on dose level 4 (2 x 10(13)). Acyclovir and topotecan were started 24 h after vector delivery. Five patients underwent second-look surgery about 4 weeks after application of gene therapy (GT). RESULTS: At the time of the second-look surgery, two out of five patients were free of tumor. Four weeks after GT none of the peritoneal biopsies showed residual vector DNA. Patients pretreated with an average of three different chemotherapeutic drugs and two different chemotherapy regimens had a median overall survival (OS) of 18.5 months. Three patients are still alive 30, 30, and 31 months after GT. CONCLUSION: With the combination of secondary optimal debulking, GT, and topotecan, median OS was about one-third longer than in previously reported second-and third-line trials. Survival was comparable to that of patients of other studies with secondary cytoreductive surgery in combination with chemotherapy.