Lymphokine-activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites.

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus-induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine-activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus-loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand-off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFNÉ£, IL-12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune-mediated tumor cell killing.

Viral warfare! Front-line defence and arming the immune system against cancer using oncolytic vaccinia and other viruses.

BACKGROUND: Despite mankind's many achievements, we are yet to find a cure for cancer. We are now approaching a new era which recognises the promise of harnessing the immune system for anti-cancer therapy. Pathogens have been implicated for decades as potential anti-cancer agents, but implementation into clinical therapy has been plagued with significant drawbacks. Newer 'designer' agents have addressed some of these concerns, in particular, a new breed of oncolytic virus: JX-594, a genetically engineered pox virus, is showing promise. OBJECTIVE: To review the current literature on the use of oncolytic viruses in the treatment of cancer; both by direct oncolysis and stimulation of the immune system. The review will provide a background and historical progression for the surgeon on tumour immunology, and the interplay between oncolytic viruses, immune cells, inflammation on tumourigenesis. METHODS: A literature review was performed using the Medline database. CONCLUSIONS: Viral therapeutics hold promise as a novel treatment modality for the treatment of disseminated malignancy. It provides a multi-pronged attack against tumour burden; direct tumour cell lysis, exposure of tumour-associated antigens (TAA), induction of immune danger signals, and recognition by immune effector cells.

Measles virus causes immunogenic cell death in human melanoma.

Oncolytic viruses (OV) are promising treatments for cancer, with several currently undergoing testing in randomised clinical trials. Measles virus (MV) has not yet been tested in models of human melanoma. This study demonstrates the efficacy of MV against human melanoma. It is increasingly recognised that an essential component of therapy with OV is the recruitment of host antitumour immune responses, both innate and adaptive. MV-mediated melanoma cell death is an inflammatory process, causing the release of inflammatory cytokines including type-1 interferons and the potent danger signal HMGB1. Here, using human in vitro models, we demonstrate that MV enhances innate antitumour activity, and that MV-mediated melanoma cell death is capable of stimulating a melanoma-specific adaptive immune response.

Immune activation by combination human lymphokine-activated killer and dendritic cell therapy.

BACKGROUND: Optimal cellular immunotherapy for cancer should ideally harness both the innate and adaptive arms of the immune response. Lymphokine-activated killer cells (LAKs) can trigger early innate killing of tumour targets, whereas long-term adaptive-specific tumour control requires priming of CD8+ cytotoxic lymphocytes (CTLs) following acquisition of tumour-associated antigens (TAAs) by antigen-presenting cells such as dendritic cells (DCs). As DCs stimulate both innate and adaptive effectors, combination cell therapy using LAKs and DCs has the potential to maximise anti-tumour immune priming. METHODS: Reciprocal activation between human clinical grade LAKs and DCs on co-culture, and its immune consequences, was monitored by cell phenotype, cytokine release and priming of both innate and adaptive cytotoxicity against melanoma targets. RESULTS: Co-culture of DCs and LAKs led to phenotypic activation of natural killer (NK) cells within the LAK population, which was associated with increased production of inflammatory cytokines and enhanced innate cytotoxicity against tumour cell targets. The LAKs reciprocally matured DCs, and the combination of LAKs and DCs, on addition of melanoma cells, supported priming of specific anti-tumour CTLs better than DCs alone. CONCLUSION: Clinical-grade LAKs/DCs represents a practical, effective combination cell immunotherapy for stimulation of both innate and adaptive anti-tumour immunity in cancer patients.

Immunophenotypic differences between normal glia, astrocytomas and malignant gliomas: correlations with karyotype, natural history and survival.

The karyotypic and antigenic phenotypes of early passage normal and malignant glial cultures were correlated in vitro. Astrocytomas (4) were distinguished from the normal glia (8) by a mixed near-diploid karyotype and anchorage-independent growth. Malignant gliomas (41) demonstrated cytogenetic abnormalities ranging from mixed normal G- and Q-banded and near-diploid cultures, through mixed near-diploid/hyperdiploid to predominantly hyperdiploid stem-lines. This correlated with the differential expression of certain antigens and established qualitative antigenic differences from normal glia. Associations were found between histopathologic grade of glial neoplasm and the expression of antigens 5.1H11 (p = 0.0002), CNT/11 (p = 0.001), CNT/10 (p = 0.004), CAT301 (p = 0.014), M111 (p = 0.024), and L101 (p = 0.044). An ominous association was demonstrated between the duration of clinical survival and the expression of antigens 5.1H11 (p = 0.0007), CNT/10 (p = 0.027) and B2.6 (p = 0.038). Correcting for diagnosis and age, multivariate analysis demonstrated that HLA-DR (p = 0.050) and 5.1H11 (p = 0.069) were unfavorably correlated with patient survival. This suggests the application of the in vitro immunophenotype for its predictive utility, as well as a novel method of selection of tumor-associated antigens for monoclonal antibody-mediated immunotherapy.

Adaptation of a chloroquine-resistant strain of Plasmodium vivax from Indonesia to New World monkeys.

The spread of chloroquine-resistant Plasmodium vivax from Papua New Guinea and Indonesia poses a serious health threat to areas of Southeast Asia where this species of malaria parasite is endemic. A strain of P. vivax from Indonesia was adapted to develop in splenectomized Aotus lemurinus griseimembra, Aotus vociferans, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission to splenectomized Saimiri monkeys was obtained via sporozoites. Chemotherapeutic studies indicated that the strain was resistant to chloroquine and amodiaquine while sensitive to mefloquine. Infections of chloroquine-resistant P.vivax in New World monkeys should be useful for the development of alternative treatments.

Efficacy of vaccines containing rhoptry-associated proteins RAP1 and RAP2 of Plasmodium falciparum in Saimiri boliviensis monkeys.

A vaccine trial was conducted with rhoptry-associated proteins 1 and 2 (RAP1 and RAP2) of Plasmodium falciparum in Saimiri boliviensis monkeys to compare the ability of parasite-derived (PfRAP1 and 2) and recombinant proteins (rRAP1 and 2) to induce protective immune responses and to find adjuvants suitable for use in humans. Eight groups of 6 monkeys each were immunized with parasite-derived or recombinant RAP1 and 2 with Freund's complete adjuvant (FCA) followed by Freund's incomplete adjuvant (FIA), Montanide ISA720 adjuvant, or CRL1005 adjuvant. Recombinant RAP1 and RAP2 were also administered separately, with Montanide ISA720. After 3 immunizations, monkeys were challenged by iv inoculation of 50,000 parasites of the Uganda Palo Alto strain of P. falciparum. Of the animals vaccinated using FCA/FIA, 1 of 6 control monkeys, 3 of 6 immunized with PfRAP1 and 2, and 2 of 6 with rRAP1 and 2 did not require drug treatment. Of the monkeys vaccinated with Montanide ISA720 adjuvant, 0 of the 6 control monkeys, 2 of 6 immunized with RAP1 and 2, 1 of 6 immunized with rRAP1, and 4 of 6 immunized with RAP2 did not require drug treatment. Two of 6 monkeys immunized with PfRAP1 and 2 with CRL1005 did not require treatment. All groups receiving RAP1, RAP2, or both had a significant decrease in initial parasite multiplication rates and there was a significant negative correlation between anti-RAP2 antibody and multiplication rates. Animals were rechallenged with the homologous parasite 126 days after the first challenge. Of the monkeys that did not require drug treatment after the first challenge, none developed detectable parasitemia following rechallenge.

Five novel cell surface antigens of CNS neoplasms.

Optimal monoclonal antibody-mediated immunotherapy requires the identification of tumor-restricted cell surface antigens. We have identified and partially characterized 5 new monoclonal antibodies generated against malignant astrocytoma, medulloblastoma, neuroblastoma and melanoma which were used to define 5 neuroectodermal tumor antigenic systems. CNT/1 identifies a 57-kDa, heat-stable, trypsin-sensitive neuroblastoma surface antigen, which is expressed intracellularly in many malignant gliomas, medulloblastomas, ependymomas, breast and ovarian carcinomas. CNT/2 reacts with a 130-kDa, heat-labile, trypsin- and neuraminidase-resistant antigen restricted to low-grade astrocytomas and malignant gliomas. CNT/11 reacts with a 70-kDa, heat-labile, trypsin-sensitive antigen coded for by a gene on chromosome 12, and is restricted to astrocytomas, neuroblastomas and sarcomas. CNT/8 identifies a heat-labile, trypsin-sensitive antigen whose gene has been localized to chromosome 15 and is expressed by neuroectodermal and mesodermally derived tumors and few epithelial cancers. The B2.6 antigen is identified only in terms of serologic reactivity with a subset of cultured astrocytomas and melanomas. Neuroectodermal tumor-associated antigens may be categorized as lineage-consistent, lineage-independent and putatively tumor-restricted in their expression. These restricted antibodies may be potentially useful reagents to consider for monoclonal antibody-mediated immunotherapy of CNS neoplasms.

Antigenic phenotypes of cultured malignant astrocytomas: identification of lineage-consistent, lineage-independent and putative tumor-restricted antigenic expression.

The treatment of CNS neoplasms with monoclonal antibody-mediated immunotherapy optimally requires the identification of tumor restricted cell surface antigens. However, little is known regarding the antigenic phenotype(s) of malignant astrocytomas. The interrelated expression of four neuroectodermal tumor antigens, CNT/11, AJ8, A010 and CNT/2, has been studied in cultured malignant gliomas and correlated with anchorage independent growth, morphology, glial fibrillary acidic protein, and the surface expression of other antigens. Many of these latter antigens have been reported to be expressed by specific fetal and differentiated adult cell lineages or tissues, as well as certain classes of malignant tumors. The tumor-associated expression of these antigens may be broadly classified as lineage-consistent, lineage-independent or putatively tumor-restricted. Malignant glioma tumor antigenic heterogeneity represents the expression of neuroectodermal and non-neuroectodermal cell surface markers. The importance of this observation is 2-fold. Lineage-independent antigen expression may be an indication of altered genome regulatory processes within tumor cells, and thus reflect the degree of anaplasia. The identification of lineage-consistent and lineage-independent tumor associated antigens may contribute to the selection of "target" antigens and the prediction of toxicity for monoclonal antibody mediated immunotherapy.

Further analysis of the theoretical effectiveness of the TwoDay method of family planning.

This article validates the theoretical effectiveness of a simple approach to identify the fertile window of the menstrual cycle. The TwoDay method identifies all days in the cycle in which the woman notices cervical secretions, and the days immediately following these days, as the period in which the woman should consider herself fertile. Women who use this method are counseled to avoid unprotected intercourse on these days. The theoretical effectiveness of the TwoDay method was tested previously by applying the method rules to the menstrual cycles of women from a large data set from the World Health Organization (WHO). For the current study, we administered the same analysis to a data set from an Italian Ovulation Method center. These data are better suited for the analysis than were the WHO data because they identify all days with secretions. Results suggest that the method can be highly effective in helping women to identify correctly the days on which they should avoid unprotected intercourse if they do not wish to become pregnant, although some users may identify a few days as fertile that actually are not.

The TwoDay Algorithm: a new algorithm to identify the fertile time of the menstrual cycle.

Women who monitor their fertility signs and recognize when they are fertile can use this knowledge to conceive or to avoid pregnancy. Studies have shown that there is a rather small fertile window of several days during each menstrual cycle. Established methods of identifying the fertile window, such as the Ovulation and the Symptothermal methods of Natural Family Planning, can be very effective in helping couples avoid pregnancy. A new algorithm for identifying the fertile window has been developed, based on monitoring and recording of cervical secretions. The TwoDay Algorithm appears to be simpler to teach, learn, and use than current natural methods. A large existing data set from a World Health Organization study of the Ovulation Method, along with Natural Family Planning charts from women using the Ovulation Method and the Symptothermal Method, were used to determine the potential effectiveness of the TwoDay Algorithm in identifying the fertile window. Results suggest that the algorithm can be an effective alternative for low literacy populations or for programs that find current Natural Family Planning methods too time consuming or otherwise not feasible to incorporate into their services. Further studies are needed to determine the efficacy of the TwoDay Algorithm in avoiding pregnancy and to assess its acceptability to users and providers.

A fixed formula to define the fertile window of the menstrual cycle as the basis of a simple method of natural family planning.

A significant number of women worldwide use periodic abstinence as their method of family planning. Many of them use some type of calendar-based approach to determine when they should abstain from unprotected intercourse to avoid pregnancy; yet they often lack correct knowledge of when during their menstrual cycle they are most likely to become pregnant. A simple method of natural family planning (NFP) based on a fixed formula to define the fertile window could be useful to these women. This article reports the results of an analysis of the application of a fixed formula to define the fertile window. A large existing data set from a World Health Organization study of the Ovulation Method was used to estimate the theoretical probability of pregnancy using this formula. Information about the variable probability of pregnancy on different cycle days relative to ovulation also was considered in the analysis. Results suggest that a fixed formula in which days 8-19 of the menstrual cycle are considered to be the fertile window would provide the appropriate basis of a simple, effective, family planning method.

Titrimetric study of the reaction of chloramine-T with ammonia.

A titrimetric study of the reaction between chloramine-T (CAT) and ammonia is described. The effects of the presence of bromide, the ratio of CAT to ammonia concentrations, the time for reaction and the pH of the reaction media are all significant in the quantitativeness of the reaction that occurs.

Semi-automated determination of aluminium with Xylenol Orange.

The photometric determination of aluminium by automated colour development with Xylenol Orange is described. The reagent has been found to act faster if used in alcohol solution.

Coulometric titration of potassium hydrogen phthalate in a non-aqueous solution, with a vitreous carbon anode.

A vitreous carbon anode has been used as working electrode in the coulometric titration of potassium hydrogen phthalate in glacial acetic acid-acetic anhydride medium with protous generated electrochemical oxidation of quinol.

Use of vitreous carbon as a working electrode in coulometric titration of potassium hydrogen phthalate.

The use of a vitreous carbon electrode as a cathode in the amperostatic coulometric titration of aqueous potassium hydrogen phthalate solution is described. It is shown that 10 mg of the phthalate can be titrated with a precision better than 0.5%. Current-voltage curves for platinum and vitreous carbon cathodes show that there is an overpotential on the latter relative to the former.

Assessing chemical toxicity with the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial systems.

The inhibition of light emitted by the bioluminescent bacterium, Vibrio fischeri, is the basis for several toxicity bioassays. The inhibitory effects of 81 chemicals, after 5 min contact time, were studied at eight concentrations using reagents from three commercial assay systems (ToxAlert 10, Microtox and LUMIStox). Solubility in water was the limiting factor in determining the selection of chemicals for study. The effective nominal concentrations (EC) resulting in 20, 50 and 80% inhibition were determined using Ln dose/Ln gamma plots and the results obtained for each system were compared by linear regression. The chemical concentrations producing 10-90% inhibition extended over 9 orders of magnitude and ranged from a minimum of 0.001 ppm to a maximum of 1,000,000 ppm. The toxicity of many chemicals was apparently related to their pH in solution and at high chemical concentrations, to osmotic imbalance. The fact that the same operator tested the same solutions simultaneously on three different systems reduced sources of error and variability and improved the consistency and reliability of the results. Only five compounds gave EC 50s that varied more than three-fold between assays. These data provide comparisons of toxicity that have not been previously available and demonstrate that, when used under standardised conditions, these bioluminescence-based toxicity assays produce very similar results.

Adaptation of the AMRU-1 strain of Plasmodium vivax to Aotus and Saimiri monkeys and to four species of anopheline mosquitoes.

A chloroquine-resistant strain of Plasmodium vivax (AMRU-1) from Papua New Guinea has been adapted to grow in 4 species of Aotus monkeys (Aotus lemurinus griseimembra, Aotus vaciferans, Aotus nancymai, and Aotus azarae boliviensis), hybrid Aotus monkeys, and Saimiri boliviensis monkeys. Whereas it was possible to infect Saimiri monkeys with this parasite by inoculation of parasitized erythrocytes, only 42% of Saimiri monkeys became infected, compared to 92% of Aotus monkeys attempted. Comparative mosquito feedings showed that only A. vociferans, A. l. griseimembra, and Saimiri boliviensis monkeys produced infections in mosquitoes. Oocysts were observed on the guts of the 4 species of mosquitoes used (Anopheles gambiae, Anopheles stephensi, Anopheles freeborni, and Anopheles dirus), but sporozoite transmission was effected only with the intravenous inoculation of sporozoites from An. dirus into an A. l. griseimembra monkey.

Breastfeeding promotion in family planning programs.

Family planning programs serve large numbers of women of childbearing, and thus breastfeeding, age. Although these programs appear to have a number of characteristics which would be useful for promoting and supporting breastfeeding, most family planning programs have done very little in this area. The Agency for International Development (A.I.D.) and the Institute for International Studies in Natural Family Planning are working to find ways to remove barriers to family planning breastfeeding promotion efforts. Such barriers include lack of or conflicting measures of program success and lack of information on the breastfeeding/fertility relationship. The Institute is developing guidelines to help family planning programs promote breastfeeding.