Seroepidemiologic survey for antibodies to human retroviruses in human and non-human primates in Brazil

The prevalence of antibodies to HTLV-I and HIV-I in Brazil was determined by testing sera from: (a) 119 members of an isolated Amazonian community of African origin; (b) 100 voluntary blood donors in Rio de Janeiro; (c) 215 patients treated at the Hematology Service, National Cancer Institute, Rio de Janeiro, and (d) 44 Cebus apella New World monkeys, wild-caught in Amazonia. Anti-HTLV-I was detected in I (0.84%) of 119 Amazonians, in 8 (3.72%) of the 215 patients and in none of the blood donors or monkeys. The high prevalence found in patients included 4 (5.79%) of 69 with non-Hodgkin lymphoma, 2 (5.88%) of 34 with Hodgkin lymphoma, I (16.66%) of 6 patients with diagnosis of anemia and I (20%) of 5 with HIV-I infection. Anti-HIV-l was found in 7 (14.89%) of 47 patients and in none of the other groups. The high incidence of HTLV-I infection in the patient group suggests that this retrovirus is endemic in parts of Brazil.     

SYNERGISTIC EFFICACY OF ADENOVIRUS-MEDIATED BCL-XS GENE TRANSFER AND TOPOTECAN IN OVARIAN CANCER CELL

Ovarian cancer is one of the three most frequent malignancy tumors in the female reproductive organ. The mortality of ovarian cancer is highest in the three malignancies. Recently, its diagnosis and therapy are improved, but the survival rate of 5 years is still poor. So it is important to improve the curative effect by molecule genetic methods. The genes of bcl-2 family can regulate apoptosis and influence the sensitivity of tumor to chemotherapy- induced apoptosis[1-6] In the bcl-2 famil…     

Isolation of simian retroviruses closely related to human T-cell leukemia virus by establishment of lymphoid cell lines from various non-human primates

Simian retroviruses closely related to human T-cell leukemia virus (HTLV) were isolated by establishing virus-producing lymphoid cell lines from 7 species of non-human primates. By co-cultivation with human umbilical cord-blood cells and/or in the presence of interleukin-2, lymphoid cell lines were successfully established from the chimpanzee. African green monkey, pig-tailed macaque, red-faced macaque, Formosan monkey, Japanese monkey and bonnet monkey that had antibodies against HTLV antigens. These cell lines reacted with human sera of ATL patients and monoclonal antibodies against p19 and p24 of HTLV antigens. Cellular DNAs contained the provirus sequences homologous to HTLV-I by Southern blot hybridization. Moreover, they produced extracellular type-C virus particles and RNA-dependent DNA polymerase. All of these lymphoid line cells had Tac antigen, interleukin-2 receptor, and those of chimpanzee and red-faced macaque had helper/induced T-cell markers, while those derived from African green monkey had suppressor/cytotoxic T-cell markers. Furthermore, simian HTLV-related viruses of pig-tailed macaque, red-faced macaque and Japanese monkey were transmitted to human lymphocytes on co-cultivation.     

Structural and functional characteristics of irradiation damage to parotid glands in the miniature pig

PURPOSE: To evaluate the effects of a solitary megadose protocol of ionizing radiation (IR) on the structure and function of the miniature pig (minipig) parotid gland. METHODS AND MATERIALS: Fourteen minipigs were subjected to either 15 or 20 Gy to one parotid gland with a linear accelerator, whereas another four minipigs served as non-IR controls. Salivary flow rates and salivary chemistries were measured pre-IR and 4 and 16 weeks post-IR. A quantitative assessment of gland weight and acinar area and detailed serum chemistry and hematologic analyses were also performed. RESULTS: Parotid flow rates decreased by approximately 50% either with 20 Gy at 4 weeks, or 15 Gy at 16 weeks post-IR. In the 20 Gy group, salivary flow rates were reduced by approximately 80% at 16 weeks post-IR. A significant decrease in salivary calcium and amylase and an increase of salivary potassium levels were found in both IR groups. There were also transient alterations in serum chemistry and hematology parameters post-IR. Parotid gland weights were significantly decreased (-50%) in the 15 and 20 Gy groups at 4 and 16 weeks post-IR. Additionally, the acinar cell area in glands of both IR groups was significantly reduced from that in control glands at both the 4 and 16 weeks time points. CONCLUSION: Structural changes in salivary gland parenchyma occurred relatively early after IR, whereas the alterations in salivary output were relatively delayed. Further, reductions in salivary flow were not proportional to acinar cell area loss. Together, these findings suggest that nonparenchymal IR damage likely contributes to IR-induced salivary hypofunction.     

Additive effect of adenovirus-mediated E2F-1 gene transfer and topoisomerase II inhibitors on apoptosis in human osteosarcoma cells

Recently, it has been demonstrated that Etoposide, a topoisomerase II inhibitor, can induce apoptosis in MDM2-overexpressing tumor cells by inhibition of MDM2 synthesis. We have previously shown that E2F-1 overexpression induces apoptosis of MDM2-overexpressing sarcoma cells, which is related to the inhibition of MDM2 expression. Therefore, the present study was designed to investigate the in vitro and in vivo effect of combined treatment of adenovirus-mediated E2F-1 and topoisomerase II inhibitors on the growth inhibition and apoptosis in human sarcoma cells. Two human sarcoma cell lines, OsACL and U2OS, were treated with topoisomerase II inhibitors (Etoposide and Adriamycin), alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ) or E2F-1 (Ad-E2F-1). E2F-1 expression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at a low dose (multiplicity of infection, 2) markedly increased the sensitivity of human sarcoma cells to topoisomerase II inhibitor treatment. This cooperative effect of E2F-1 and topoisomerase II inhibitors was less marked in SAOS-2 cells (p53 and pRb null). Topoisomerase II inhibitors also cooperated with E2F-1 overexpression to enhance tumor cell killing in an in vivo model using xenografts in nude mice. When combined with Adriamycin or Etoposide, E2F-1 adenovirus therapy resulted in approximately 95% and 85% decrease in tumor size, respectively, compared to controls (P<.05). These results suggest a new chemosensitization strategy that is effective in MDM2-overexpressing tumors and may have clinical utility.     

Detection of serum antibodies to adult T-cell leukemia virus in non-human primates and in people from Africa

The distribution of serum antibodies to adult T-cell leukemia virus (ATLV) was examined as a marker for virus infection among non-human primates as well as people from Africa and Germany. The virus is present in Africa in certain primate species including man. Altogether, 468 sera from 27 monkey species were examined. Only African green monkeys, less frequently also chimpanzees and crab-eating monkeys, were found to be infected. About 1-2% of people from Kenya have antibodies, while ATLV-antibodies may be present in well below 0.1% of the German population.     

EFFECT OF ADENOVIRUS—MEDIATED p53 GENE TRANSFER ON APOPTOSIS AND RADIOSENSITIVITY OF HUMAN

To evaluate the effect of adenovirus-mediated p53 gene(Adp53) on apoptosis and radiosensitivity of human gastric carcinoma cell lines.Methods:Recombinant adenovirus expressing wild-type p53 lines with different p53 genetic status.p53 protein expression was detected by immunohistochemistry assay and western blot assay.Cell survival was assessed using a clonogenic assay.TUNEL assay was used in determination of apoptosis.Four human gastric carcinoma cells infected with Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flow cytometry.Results:G2/M arrest,apoptosis and inhibition of tumor cell proliferation were induced by infection at Adp53 at 100 MOI which caused high transfer rate of wild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells.The radio-enhancement ratio of Adp53 at 4Gy were3.0 for W cell,3.6 for M cell,2.2 for neo cell and 2.5 for 823 cell in vitro.Conclusion :This study demonstrated that Adp53 transfer increased cellular apoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53 status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.     

Safety and efficacy of intracranial vascularized submental lymph node transfer for treating hydrocephalus

Hydrocephalus is routinely treated with ventriculoperitoneal shunt drainage of cerebrospinal fluid (CSF), a procedure plagued by high morbidity and frequent revisions. Vascularized submental lymph node (VSLN) transplants act as lymphatic pumps to drain interstitial fluid (ISF) from lymphedematous extremities. As the field of neuro-lymphatics comes to fruition, we hypothesize the efficacy of VSLN in the drainage of intracranial CSF-ISF. We report novel placement of VSLN in the temporal subdural space in two patients diagnosed with symptomatic communicating hydrocephalus. At a minimum follow-up of 1 month postoperatively, both experienced radiological and clinical improvements.     

1982 resident's essay award: the immunologic effects of lymphoid irradiation in human and non-human primates: cellular changes and the potential for renal transplantation

Previous reports have suggested that the immunosuppressive effects of lymphoid irradiation (LI) are related to changes in T cell peripheral blood levels. We prospectively studied 13 patients with Stages I and II Hodgkin's disease before, during, and after a course of Lt. Forty to 45Gy delivered to the mantle field in $\text{4}\text{1}\text{2}\text{–5}$ weeks was followed by a 3–4 week break and 35–40Gy to the paraaortic field. Daily fraction size was 1.1–1.8Gy. Peripheral venous samples were drawn weekly. T cell subsets were identified by fluorescent staining of lymphocyte surface antigens using murine monoclonal antibodies OKT3, OKT4, and OKT8 (reactive with all T cells, helper/inducer cells (H/I), and cytotoxic/suppressor cells (C/S), respectively), and quantitated by flow cytometry. The absolute lymphocyte count prior to LI was 1400 ± 270/mm3. This fell to 597 ± 280/mm3 at the conclusion of LI and returned to 1490 ± 800/mm3 at 7–8 months post-LI. The percent total T cells and H/I were reduced from pre-LI values of 66 ± 8 % and 48 ± 10%, respectively, to 7–8 month post-LI values of 36 ± 16% and 19 ± 8%. The percent C/S cells was relatively unchanged by Lt: 20 ± 4% before treatment and 26 ±10 % at 7–8 months after Lt.Seven previously spleenectomized cynomolgus monkeys received 20Gy of LI in 20 equal daily fractions over a 4 week period through a single anterior field including the cervical, axillary, mediastinal, paraaortic iliac, and inguinal lymph node chains at a dose rate of 4.6–4.8cGy/minute. Renal allografts from unrelated monkey donors were placed following Lt. Graft function was monitored by urine output, serum BUN and creatinine, and renal biopsy. Low dose rate LI was well tolerated. The absolute lymphocyte count fell from a pre-LI level of 3080 ± 1227/mm3 to 534 ± 478/mm3 at 1 month post-LI. The percent H/I fell slightly during LI from a pre-treatment value of 28 ± 5% to 20 ± 10% at 1 month post-LI. There was a slight increase in percent C/S cells from a pre-LI value of 29 ± 13% to a 1 month post-LI value of 36 ± 19%.In 4 animals that received no LI (controls), survival post-transplant was 8–11 days prior to death from acute rejection. Five animals received transplants immediately following Lt. Four survived 11–56 days prior to death of presumed infection. One died at day 45 with rejection. Another group of two animals received transplants $\text{3}\text{1}\text{2}$ weeks after Lt. One died of rejection at day 42. One remains well at day 394 post-transplant.     

Prevalence of antibody to adult T-cell leukemia virus-associated antigens (ATLA) in Japanese monkeys and other non-human primates

The prevalence of adult T-cell-leukemia virus (ATLV) infection was examined in Japanese monkeys living naturally in various parts of Japan and in other species of non-human primates imported into and kept in Japan. Sera of 2,650 Japanese monkeys from 41 troops throughout Japan were tested. High incidences of anti-ATLV-associated antigen (ATLA)-positive monkeys were found in most troops, not only in the endemic area of human ATL(Southwestern Japan), but also in non-endemic areas. The incidence of sero-positive individuals increased gradually with age, reaching a maximum when the animals became adult, indicating age dependency, like that found by epidemiological studies on humans. Anti-ATLA antibodies were also detected in 90 of 815 sera of imported non-human primates of 33 species other than Japanese monkeys. All the anti-ATLA sero-positive monkeys were Catarrhines (Old World monkeys), mainly macaques of Asian origin. Some sero-positive monkeys were also found among animals of African origin, but no antibody was detected in Prosimians and Platyrrhines (New World monkeys). The clear-cut difference between the geographical distribution of sero-positive simians and that of humans indicates the improbability of direct transmission of ATLV from simians to humans.     

The plasma and cerebrospinal fluid pharmacokinetics of the platinum analog satraplatin after intravenous administration in non-human primates

Background  

Satraplatin is an orally bioavailable platinum analog with preclinical activity in cisplatin resistant models and clinical activity in adults with refractory cancers. The cerebrospinal fluid (CSF) penetration of cisplatin and carboplatin in non-human primates (NHP) is limited (3.7 and 2.6%, respectively). We evaluated the plasma and CSF pharmacokinetics (PK) of satraplatin after an intravenous (IV) dose in NHP.     

Gene transfer of endostatin enhances the efficacy of doxorubicin to suppress human hepatocellular carcinomas in mice

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death, and is chemoresistant to anticancer drugs. Anti-angiogenic therapy has been shown to enhance the efficacy of chemotherapy to treat solid tumors. The aim of the present study was to determine whether endostatin, a potent antiangiogenic agent, could enhance the efficacy of doxorubicin to combat HCC. An endostatin expression plasmid was constructed and its expression in vitro and in vivo was detected after gene transfer. Recombinant endostatin inhibited angiogenesis in the chorioallantoic membrane assay, and showed synergistic effects with doxorubicin in inhibiting the in vitro proliferation of endothelial cells, but not that of tumor cells. Both endostatin gene therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, and tumor angiogenesis. Combination therapy with endostatin gene therapy and doxorubicin showed a stronger effect in suppressing tumor growth, and tumor angiogenesis, than the respective monotherapies. Gene transfer of endostatin down-regulated the expression of both hypoxia-inducible factor-1alpha and vascular endothelial growth factor (VEGF), whereas doxorubicin only down-regulated VEGF expression. Endostatin and doxorubicin synergized to down-regulate VEGF expression. Endostatin and doxorubicin combination therapy warrants investigation as a therapeutic strategy to combat HCC.     

Whole brain radiotherapy for brain metastases: the technique of irradiation influences the dose to parotid glands

In the treatment of brain metastases, whole brain radiotherapy can be carried out according two distinct methods: one using multileaf collimator for field shaping and protection of organs at risk, and a second one is to make a rotation of the field to avoid the eyes. The aim of the study was to compare for 10 patients the dose distributions at organs at risk for each method. Patients received 30 Gy in 10 fractions. Except for parotid glands, the dose received by organs at risk and the planning target volume was the same with each method. For whole brain radiotherapy, excluding the cisterna cerebellomedullaris, the mean parotid dose was 9.63 Gy using the multileaf collimator versus 12.32 Gy using the field rotation (P=0.04). For whole brain radiotherapy including the cisterna cerebellomedullaris, the mean parotid dose was 11.12 Gy using the multileaf collimator versus 20.06 Gy using field rotation (P<0.001). Using the multileaf collimator seems recommended for whole brain radiotherapy, to reduce the dose to the parotids.     

Alteration of drug chemosensitivity caused by the adenovirus-mediated transfer of the wild-type p53 gene in human lung cancer cells

The aim of the present study is to identify the optimal anticancer agents for use in combination with gene therapy using wild-type (wt) p53 gene transfer. We used adenoviral vectors expressing human wt p53 (AdCAp53) and investigated the effects of wt p53 gene transfer in combination with 12 anticancer agents on a human pulmonary squamous cell carcinoma cell line, NCI-H157, and a human pulmonary large cell carcinoma cell line, NCI-H1299. Solutions containing anticancer agents at various concentrations were added followed by the addition of recombinant adenovirus solutions; after a 5-day incubation period, the anticancer activity was then evaluated by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbo xanilide assay. Each 50% inhibitory concentration was calculated based on the dose-response curves. The agents showing a high degree of effectiveness on NCI-H157 cells were cisplatin (CDDP), 5-fluorouracil (5-FU), bleomycin, and 7-ethyl-10-hydroxy-camptothecin (SN-38), an active metabolite of irinotecan (CPT-11); conversely, cyclophosphamide and paclitaxel showed a low degree of effectiveness. Based on these data, an isobologram was performed to investigate the interaction between AdCAp53 and some anticancer agents. A supra-additive effect was thus observed for 5-FU and SN-38 on NCI-H157 cells. An additive effect was also observed for CDDP, paclitaxel, bleomycin, and cyclophosphamide on NCI-H157 cells. CDDP, paclitaxel, 5-FU, and SN-38 had an additive effect on NCI-H1299 cells. No drug showed any subadditive or protective effects. These findings suggest that CPT-11 and 5-FU may thus be useful as possible anticancer agents for use in a combination therapy regimen using wt p53 gene transfer. CDDP and CPT-11 had a significant antitumoral effect on H157 cell xenografts of nude mice in vivo. These results indicate that CPT-11 as well as CDDP would be a candidate for the combination of chemotherapy and gene therapy for non-small cell lung cancer.     

Gene therapy for human nasopharyngeal carcinoma by adenovirus-mediated transfer of human p53, GM-CSF, and B7-1 genes in a mouse xenograft tumor model

Incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions. Prevention of tumor recurrences and metastases is a crucial approach to improve therapeutic outcome in NPC patients. In this study, we investigated the effects of the cotransfer of the tumor suppressor gene, p53, in combination with the immunostimulatory genes, GM-CSF and B7-1, on tumor regression and subsequent tumor recurrence. We constructed a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes (Ad-p53/GM-CSF/B7-1), which mediated high-level expression of these three genes in NPC CNE-1 cells. Ad-p53/GM-CSF/B7-1 infection inhibited the growth of CNE-1 cells and induced tumor-specific cytotoxic T-lymphocytes (CTLs) in vitro. In CNE-1 xenograft tumor models in huPBL-nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, an intratumoral injection of Ad-p53/GM-CSF/B7-1 resulted in a reduced tumor burden, compared to normal saline (NS) and Ad-p53 controls. Tumors in the Ad-p53/GM-CSF/B7-1 group displayed diffuse necrosis and infiltration of human T-cells. Further, the tumor occurrence of CNE-1 cell rechallenge largely decreased after the primary tumor was intratumorally injected with Ad-p53/GM-CSF/B7-1 in the HuPBL-NOD/SCID mice model. Only 2 of 8 (25%) animals in the Ad-p53/GM-CSF/B7-1 group had developed measurable tumors, which demonstrated extensive necrosis and much more human T-cell infiltration, compared to 5 of 7 (71%) in the NS and Ad-p53 groups. Therefore, the adenovirus-mediated introduction of p53, GM-CSF, and B7-1 genes could improve local control and prevent the recurrence or metastases of NPC tumors, which suggests a potential therapeutic value in NPC treatment.     

CpG oligodeoxynucleotides enhance the capacity of adenovirus-mediated CD154 gene transfer to generate effective B-cell lymphoma vaccines

Activation of CD40 by CD154 induces antigen-presenting cells (APC) to express immune costimulatory molecules, thereby enhancing their APC activity. Oligonucleotides (ODN), containing immunostimulatory DNA sequences (ISS) with nonmethylated CpG dinucleotides in a defined motif, also can induce similar changes in APC. In this study, we examined whether infection with recombinant adenovirus (Ad) encoding CD154 and/or treatment with ISS-ODN could enhance the capacity of A20 murine B lymphoma cells to function as APCs capable of inducing a syngeneic antilymphoma immune response. High-level expression of CD154 after infection with Ad-CD154 induced up-regulation of immune costimulatory molecules on A20 cells, as did incubation with ISS-ODN. Treatment of A20 cells with ISS-ODN also enhanced surface expression of alphav integrins, making them significantly more susceptible to Ad infection than nontreated A20 cells. In syngeneic mixed-lymphocyte reactions with BALB/c splenocytes, A20 cells activated with ISS-ODN and then transduced with Ad-CD154 were significantly more effective APCs than Ad-CD154 transduced cells, which, in turn, were significantly more effective than A20 cells treated with ISS-ODN alone. Also, injection of mice with ISS-activated, Ad-CD154-infected cells induced significantly better A20-specific immune responses against A20 cells, as assessed via enzyme-linked immunospot analysis in vitro and immune prophylaxis against subsequent challenge with A20 lymphoma cells in vivo. These data demonstrate that CpG-containing oligonucleotides can serve as an adjuvant for Ad-mediated gene therapy of B-cell malignancies.