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.

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.

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.

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.

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.

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.

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.

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.

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.

Knowledge of likely time of ovulation and contraceptive use in unintended pregnancies.

This investigation analyzed social and demographic characteristics of women having an unwanted or mistimed pregnancy (unintended pregnancies at the current time) in South America. A sample of 5135 women having had a normal non-malformed live-born infant were interviewed immediately postpartum at 18 hospitals participating in the Latin American Collaborative Study of Congenital Malformations (Spanish acronym: ECLAMC). Half (2568/5135 = 50%) reported that their pregnancies had been unintended, and, of those, 59.3% (1522/2568 = 59.3%) declared that they were trying to avoid conception. The latter group (n = 1522) was the main sample for this study. Patients were asked about their knowledge of when during the menstrual cycle conception is most likely to occur, their biomedical and social characteristics, the type of contraceptive methods used, their opinion of reasons for contraceptive failure, and their reasons for not using contraceptive methods. Among women with unintended pregnancies who attempted to avoid conception, only 61.6% were using contraceptive methods. Reasons given for not using contraceptives included health problems, lack of knowledge and lack of access to contraception. Women with unintended pregnancies who had not attempted to avoid conception were younger, often primigravid, less educated, and less knowledgeable concerning when during the cycle pregnancy is most likely to occur. Thus, reproductive health policies should be aimed at this target group.

PIP: This study analyzes the social and demographic characteristics of women having an unwanted or unintended pregnancy in South America between 1992 and 1994. The study was conducted among 5135 women who had a normal non-malformed live-born infant and were interviewed at 18 hospitals participating in the Latin American Collaborative Study of Congenital Malformations. Patients were asked about their knowledge of when conception is most likely to occur during the menstrual cycle, their biomedical and social characteristics, the type of contraceptive methods used, their opinion and reasons for contraceptive failure, and their reasons for failing to use contraceptive methods. Results indicate that 50% of the respondents had unintended pregnancies, and about 59% declared that they were trying to avoid conception. However, among women with unintended pregnancies who attempted to avoid conception, only 61.6% were using contraceptive methods. Reasons for failure to use contraceptives include health problems, lack of knowledge, and lack of access to contraception. Meanwhile, women with unintended pregnancies who had not attempted to avoid conception were younger, often primigravid, less educated, and less knowledgeable concerning the likelihood that pregnancy will occur during menstrual cycle. This study implies that reproductive health policies should be aimed at this target group.

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.

Evidence for multiple pathologic and protective mechanisms of murine cerebral malaria.

Murine cerebral malaria (CM) induced by Plasmodium berghei ANKA kills susceptible mice within 24 to 48 h of onset of symptoms and is characterized by the production of inflammatory cytokines in the brain. C57BL/6J mice are sensitive to lethal CM, while A/J mice are resistant. These strains of mice were immunized with an adjuvant vaccine of killed whole-blood-stage parasites. The immunization protected C57BL/6 mice from lethal CM following virulent challenge. The same immunization increased the incidence of lethal CM in A/J mice challenged similarly. Histopathologic examination of the brains of mice from these studies revealed two distinct types of lesions. Type I CM is acute in onset; usually lethal; and characterized by widespread microglial activation, endothelial cell damage, and microvascular disruption in the brain. Type II CM is characterized by intense, but focal, mononuclear cell inflammation without endothelial cell damage or microvascular destruction. Animals with type II lesions were clinically normal and protected from type I lesions. Available clinical, epidemiological, and biochemical evidence suggests that type I and type II lesions might exist in human CM as well.

Unintended pregnancies in women delivering at 18 South American hospitals. NFP-ECLAMC Group. Latin American Collaborative Study of Congenital Malformations.

Unintended pregnancies are accepted as associated with social, maternal and perinatal risks, but few data exist in South America. In a selected network of hospitals participating in the ECLAMC (Spanish acronym for Latin American Collaborative Study of Congenital Malformations), the frequency of unintended pregnancies was 49.8% in 5155 mothers of normal liveborns, as interviewed in the post-partum period (1992-1994). Compared with the intended pregnancy group, these mothers were more frequently multiparous, conceived easily, had a surprisingly higher mean maternal age, lower educational level, and Black ancestors. The frequency of mistimed pregnancies was the highest among primiparae. No adverse perinatal outcome could be found with regard to low birthweight (< 2500 g), prematurity (< 37 weeks), and early neonatal death. The rates of Caesarean delivery, twinning and sex ratio were similar in intended and unintended groups. Logistic regression analysis showed that maternal education could be a confounding factor associated with other maternal variables. The rate of unintended pregnancies in the present study is significantly higher than that described for other regions. Knowledge of the characteristics of women experiencing unintended pregnancies would allow proper public health strategies.

Sex ratio associated with timing of insemination and length of the follicular phase in planned and unplanned pregnancies during use of natural family planning.

This was a multicentred, prospective study of pregnancies among women using natural family planning. The women maintained natural family planning charts of the conception cycle, recording acts of intercourse and signs of ovulation (cervical mucus changes, including peak day and basal body temperature). Charts were used to assess the most probable day of insemination relative to the day of ovulation and length of the follicular phase of the cycle. The sex ratio (males per 100 females) for 947 singleton births was 101.5, not significantly different from the expected value of 105. The sex ratio did not vary consistently or significantly with the estimated timing of insemination relative to the day of ovulation, with the estimated length of the follicular phase or with the planned or unplanned status of the pregnancy. Although these findings may be affected by imprecision of the data, the study suggests that manipulation of the timing of insemination during the cycle cannot be used to affect the sex of offspring.

PIP: In the context of ongoing debate over the determinants of sex ratio, the authors used data from a multinational study of pregnancies among natural family planning (NFP) users to investigate the association between timing of conception or follicular phase and length and the sex ratio at birth. They also explored whether a pregnancy's planned or unplanned status affects those associations. A multicenter, prospective study of pregnancies among women using NFP was conducted. The women maintained NFP charts of their conception cycles, recording acts of intercourse and signs of ovulation such as cervical mucus changes and basal body temperature. Charts were used to identify the most probable day of insemination relative to the day of ovulation and length of the follicular phase of the cycle. The sex ratio (number of males per 100 females) for 947 singleton births was 101.5, not significantly different from the expected value of 105. The sex ratio did not vary consistently or significantly with the estimated timing of insemination relative to the day of ovulation, with the estimated length of the follicular phase, or with the planned or unplanned status of the pregnancy. Study findings suggest that manipulating the timing of insemination during the cycle cannot be used to affect the sex of offspring.

Cytokine profile suggesting that murine cerebral malaria is an encephalitis.

Cerebral malaria (CM) remains a poorly understood and life-threatening complication of malaria caused by the parasite Plasmodium falciparum. The discovery that murine CM caused by Plasmodium berghei ANKA and human CM are both characterized by production of inflammatory cytokines, especially tumor necrosis factor alpha (TNF-alpha), led to a revival of the suggestion that P. berghei CM may have value as a model of the human disease. In this study, quantitative reverse transcription-PCR was used to measure levels of message for 18S rRNA of P. berghei and 10 cytokines in the brains, livers, and spleens of mice during the induction and course of CM. A coordinated increase in RNA of parasite and proinflammatory cytokines was observed in the brains of mice in parallel with onset of CM. Levels of message for parasite, TNF-alpha, and gamma interferon increased in the brains of mice from day 5 to death on day 7. These changes were observed only in the brain, and message for other cytokines remained near baseline levels. This demonstrated that parasite sequestration does take place in the brains of mice with CM. Histologically, CM was characterized by widespread damage to the microvasculature in the brain with focal infiltration of inflammatory cells. The pattern of cytokine production in the brain is characteristic of other murine encephalitides.

Effects of timing of conception on birth weight and preterm delivery of natural family planning users.

OBJECTIVE: Various birth defects and untoward perinatal outcomes have been claimed to be associated with pregnancies conceived by gametes aged in vivo before fertilization. Thus, these outcomes were systematically assessed in pregnancies occurring in natural family planning (NFP) users. Our international multicenter cohort study of NFP pregnancies (n = 877) is by far the largest systematic study designed to assess pregnancy outcome and is of sufficient power to allow us to address the concern of low birth weight (< 2500 g) and preterm delivery (< 37 weeks gestation). STUDY DESIGN: In addition to gathering baseline medical data, evaluation was performed at 16 weeks, 32 weeks and at term. Data were collected in a systematic cohort fashion, verified by the five collaborating international recruiting centers, and analyzed by investigators in the US. Most recruiting center principal investigators are obstetrician-gynecologists and, if not, have integral relationships with such specialists. Standard criteria could thus be applied within and among centers. In our cohort, birth weight was recorded accurately at delivery. Almost all of the deliveries occurred in hospitals; thus, data should be quite reliable. Neonatal examination for anomalies was usually conducted immediately after delivery, when birth weight was recorded. RESULTS: Analysis of risk factors for low birth weight and preterm delivery showed that this population had a low risk profile. Low birth weight infants (< 2500 g) and preterm deliveries were increased among women with a history of either prior low birth weight or preeclampsia in the index pregnancy. However, mean birth weight was unaffected by the timing of conception vis à vis ovulation or pregnancy history. Mean birth weight for the 877 singleton NFP pregnancies was 3349.6 g. The risk of preterm delivery was increased among older women who drank alcohol, but there were no significant effects of timing of conception vis à vis ovulation on preterm delivery. Results held when analysis was stratified according to whether NFP was being used for contraception or to achieve pregnancy. CONCLUSIONS: Our data do not appear to show striking differences between 877 NFP pregnancies and the general obstetric population. The timing of conception vis à vis ovulation does not exert significant effects on the birth weight or preterm delivery of resulting pregnancies, a reassuring finding for NFP users.

Pregnancy complications in natural family planning users.

A multicenter cohort study was designed to assess pregnancy outcome among natural family planning (NFP) users, and provide the opportunity to address complications in NFP users by planning status and by timing of conception with respect to day of ovulation. There were 877 singleton births in this sample. Complications evaluated were abnormal vaginal bleeding, urinary tract infection, vaginal infection, hypertension of pregnancy, proteinuria, glycosuria, and anemia. There was no significant difference in the mean age, number of prenatal visits or birth weight among optimally and non-optimally timed pregnancies or for planned and unplanned pregnancies. There were higher incidences of "parity 2 or more" and current smokers in the non-optimally timed pregnancies and lower incidences of prior pregnancy loss and "currently employed" in the non-optimally timed pregnancies. There was little difference in pregnancy complications with respect to pregnancy timing, with the exception of a significant increased risk of vaginal bleeding late in pregnancy among non-optimally timed conceptions (11.5%) compared to optimally timed pregnancies (5.2%, RR = 2.2, 95% CI 1.3-3.7). More differences were observed in pregnancy complication rates by planning status. Unplanned pregnancies were associated with significantly more late pregnancy bleeding, vaginal infections, proteinuria, glycosuria and medication use than planned pregnancies. Unplanned pregnancies had lower incidences of maternal anemia. Complications of pregnancy were low in this NFP population, irrespective of planned versus unplanned status. Women with planned pregnancies had even fewer complications during pregnancy than women with unplanned conceptions, suggesting that women using NFP to plan their reproduction may be at particularly low risk.

Pregnancy outcome in natural family planning users: cohort and case-control studies evaluating safety.

Conceptions involving aging gametes are of relevance to natural family planning (NFP) because women using NFP to avoid pregnancy abstain from intercourse during the fertile time of the cycle. To help verify the safety of pregnancies occurring among NFP practitioners, our group has, since 1986, conducted a large cohort study involving six experienced NFP centers. Timing of conception was determined from NFP charts, in which women recorded days on which intercourse occurred. The number of days from the most probable conception intercourse to probable day of ovulation was first determined, and used as an estimate of the time gametes remained in the genital tract before fertilization. Several studies have already been completed, cohort as well as case-control in nature.