Background Others and we have previously described the potent  in vivo  and  in vitro  activity of the broad-spectrum antiviral agent PAP (Pokeweed antiviral protein) against a wide range of viruses. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV).
Background
Others and we have previously described the potent  in vivo  and  in vitro  activity of the broad-spectrum antiviral agent PAP (Pokeweed antiviral protein) against a wide range of viruses. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV).in vivoin vitroMethods We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation.
Methods
We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation.Results PAP exhibits significant  in vivo  anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069).
Results
PAP exhibits significant  in vivo  anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069).in vivoConclusion Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.
Conclusion
Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.

Background The broad-spectrum anti-viral agent PAP (Pokeweed antiviral protein) [ 1 ] is a well-characterized 29-kDa plant-derived ribosome-inactivating protein (RIP) isolated from Phytolacca americana [ 2 ]. The anti-viral activity of PAP has been described against a wide range of viruses, including HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and polio virus [ 2 ] (For the most recent review, please see: [ 3 ]). The activity of PAP is attributed to its ability to inhibit protein synthesis by catalytically cleaving a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA [ 4 , 5 ] as well as from viral RNA[ 3 ]. The potent anti-HIV activity of PAP at nanomolar ranges taken together with the relative ease of large scale purification has led to the clinical use of PAP [ 2 ]. Since 1985, our group has studied the multifunctional efficacy of this potent agent [ 1 , 2 , 4 - 42 ]. The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on survival of mice challenged with intracerebral inoculations of lymphocytic choriomeningitis virus (LCMV). LCMV is a rodent-borne arena virus that can result in persistent neuronal infection on mice [ 43 , 44 ]. Alpha-Dystroglycan (alpha-DG) was recently identified as a receptor for LCMV as well as for several other arena viruses including Lassa fever virus [ 45 ]. The binding affinity of LCMV to alpha-DG determines viral tropism and the outcome of infection in mice [ 46 ]. LCMV has also been associated with both postnatal and intrauterine human disease. Infection in man is acquired after inhalation, ingestion or direct contact with virus found in the urine, feces and saliva of infected mice, hamsters and guinea pigs. Congenital LCMV infection is a significant, often unrecognized cause of chorioretinitis, hydrocephalus, microcephaly or macrocelphaly as well as mental retardation. Acquired LCMV infection, asymptomatic in approximately one third of individuals, is productive of central nervous system manifestations in one half of the remaining cases. Aseptic meningitis or meningoenceophalitis are the predominant syndromes, although transverse myelitis, a Guillain-Barre-type syndrome, as well as transient and permanent acquired hydrocephalus have also been reported [ 47 ]. In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.
Background
The broad-spectrum anti-viral agent PAP (Pokeweed antiviral protein) [ 1 ] is a well-characterized 29-kDa plant-derived ribosome-inactivating protein (RIP) isolated from Phytolacca americana [ 2 ]. The anti-viral activity of PAP has been described against a wide range of viruses, including HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and polio virus [ 2 ] (For the most recent review, please see: [ 3 ]). The activity of PAP is attributed to its ability to inhibit protein synthesis by catalytically cleaving a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA [ 4 , 5 ] as well as from viral RNA[ 3 ]. The potent anti-HIV activity of PAP at nanomolar ranges taken together with the relative ease of large scale purification has led to the clinical use of PAP [ 2 ]. Since 1985, our group has studied the multifunctional efficacy of this potent agent [ 1 , 2 , 4 - 42 ].The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on survival of mice challenged with intracerebral inoculations of lymphocytic choriomeningitis virus (LCMV). LCMV is a rodent-borne arena virus that can result in persistent neuronal infection on mice [ 43 , 44 ]. Alpha-Dystroglycan (alpha-DG) was recently identified as a receptor for LCMV as well as for several other arena viruses including Lassa fever virus [ 45 ]. The binding affinity of LCMV to alpha-DG determines viral tropism and the outcome of infection in mice [ 46 ]. LCMV has also been associated with both postnatal and intrauterine human disease. Infection in man is acquired after inhalation, ingestion or direct contact with virus found in the urine, feces and saliva of infected mice, hamsters and guinea pigs. Congenital LCMV infection is a significant, often unrecognized cause of chorioretinitis, hydrocephalus, microcephaly or macrocelphaly as well as mental retardation. Acquired LCMV infection, asymptomatic in approximately one third of individuals, is productive of central nervous system manifestations in one half of the remaining cases. Aseptic meningitis or meningoenceophalitis are the predominant syndromes, although transverse myelitis, a Guillain-Barre-type syndrome, as well as transient and permanent acquired hydrocephalus have also been reported [ 47 ].In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.Methods Purification of PAP PAP was purified from spring leaves of the pokeweed plant,  P. americanca , in four steps [ 22 , 38 ]. Briefly, spring leaves were homogenized in a neutral pH buffer and centrifuged to sediment the remaining cellular fragments. The supernatant was fractionated between 60–90% saturation of ammonium sulfate and the precipitate dialyzed against a low-ionic strength pH 7.5 buffer. The solution was passed through a DEAE cellulose column and the PAP-containing flow-through fraction was then applied to a cation exchange resin S-Sepharose column. The adsorbed PAP was eluted in a linear KCl gradient. The protein peak which eluted at 0.12 KCl was taken as PAP. This fraction was dialyzed extensively against water and lyophilized for storage at -20°C. The procedure resulted in homogeneous PAP, with a purity of >99% as measured by both SDS -12% PAGE and analytical cation- exchange high-performance liquid chromatography. Purified PAP induced concentration-dependent inhibition of HIV-1 replication in normal human peripheral blood mononuclear cells infected with the HIV-1 strain HTLV IIIB  with an IC 50  p24 of 14 ± 2 nM. Animal infection Animal infection was performed in an appropriate Animal BioSafety Level-3 Laboratory (ABL-3) at BRIEM (Research Institute for Epidemiology and Microbiology, MINSK, Belarus) with the technician wearing appropriate facility clothing. The culture was thawed in a water bath at 37°C and then diluted in normal saline to achieve the required concentration. In this study, all mice were challenged with 1000 PFU which is 100-times higher than the LD 50  dose. Each group of animals was placed in a separate cage. LCMV model Three week old CBA mice were intracerebrally infected with 1000 PFU of WE54 strain of LCMV that resulted in lethality of 100% of control (non-treated) animals in 6–8 days after infection. Control animals were given physiologic salt solution as a placebo instead of the compound. In general, for non-treated animals, clinical signs of the disease manifested on the 5 th  and 7 th  days by presenting: weight loss, immobility, disheveled hair, convulsions, severe decubitus paralysis and death. All subjective measurement of decreased mobility and scruffy fur were done in a blinded fashion as not to influence the results. The protective properties of the experimental anti-viral drugs were assessed by using the following treatment-preventative regimen: Mice were treated either with vehicle (n = 20) or PAP (n = 10) (0.25 mg/kg) administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Mice were then observed for 21-days post infection. The protective effect of the experimental anti-viral drugs was evaluated according to the rise of the survival rate and prolongation of mean life of the experimental animals as compared with the control animals. Statistical analysis Statistical significance was determined using the Kaplan Meier Log-Rank test.
Methods
Purification of PAP PAP was purified from spring leaves of the pokeweed plant,  P. americanca , in four steps [ 22 , 38 ]. Briefly, spring leaves were homogenized in a neutral pH buffer and centrifuged to sediment the remaining cellular fragments. The supernatant was fractionated between 60–90% saturation of ammonium sulfate and the precipitate dialyzed against a low-ionic strength pH 7.5 buffer. The solution was passed through a DEAE cellulose column and the PAP-containing flow-through fraction was then applied to a cation exchange resin S-Sepharose column. The adsorbed PAP was eluted in a linear KCl gradient. The protein peak which eluted at 0.12 KCl was taken as PAP. This fraction was dialyzed extensively against water and lyophilized for storage at -20°C. The procedure resulted in homogeneous PAP, with a purity of >99% as measured by both SDS -12% PAGE and analytical cation- exchange high-performance liquid chromatography. Purified PAP induced concentration-dependent inhibition of HIV-1 replication in normal human peripheral blood mononuclear cells infected with the HIV-1 strain HTLV IIIB  with an IC 50  p24 of 14 ± 2 nM.
Purification of PAP
PAP was purified from spring leaves of the pokeweed plant,  P. americanca , in four steps [ 22 , 38 ]. Briefly, spring leaves were homogenized in a neutral pH buffer and centrifuged to sediment the remaining cellular fragments. The supernatant was fractionated between 60–90% saturation of ammonium sulfate and the precipitate dialyzed against a low-ionic strength pH 7.5 buffer. The solution was passed through a DEAE cellulose column and the PAP-containing flow-through fraction was then applied to a cation exchange resin S-Sepharose column. The adsorbed PAP was eluted in a linear KCl gradient. The protein peak which eluted at 0.12 KCl was taken as PAP. This fraction was dialyzed extensively against water and lyophilized for storage at -20°C. The procedure resulted in homogeneous PAP, with a purity of >99% as measured by both SDS -12% PAGE and analytical cation- exchange high-performance liquid chromatography. Purified PAP induced concentration-dependent inhibition of HIV-1 replication in normal human peripheral blood mononuclear cells infected with the HIV-1 strain HTLV IIIB  with an IC 50  p24 of 14 ± 2 nM.P. americancaAnimal infection Animal infection was performed in an appropriate Animal BioSafety Level-3 Laboratory (ABL-3) at BRIEM (Research Institute for Epidemiology and Microbiology, MINSK, Belarus) with the technician wearing appropriate facility clothing. The culture was thawed in a water bath at 37°C and then diluted in normal saline to achieve the required concentration. In this study, all mice were challenged with 1000 PFU which is 100-times higher than the LD 50  dose. Each group of animals was placed in a separate cage.
Animal infection
Animal infection was performed in an appropriate Animal BioSafety Level-3 Laboratory (ABL-3) at BRIEM (Research Institute for Epidemiology and Microbiology, MINSK, Belarus) with the technician wearing appropriate facility clothing. The culture was thawed in a water bath at 37°C and then diluted in normal saline to achieve the required concentration. In this study, all mice were challenged with 1000 PFU which is 100-times higher than the LD 50  dose. Each group of animals was placed in a separate cage.LCMV model Three week old CBA mice were intracerebrally infected with 1000 PFU of WE54 strain of LCMV that resulted in lethality of 100% of control (non-treated) animals in 6–8 days after infection. Control animals were given physiologic salt solution as a placebo instead of the compound. In general, for non-treated animals, clinical signs of the disease manifested on the 5 th  and 7 th  days by presenting: weight loss, immobility, disheveled hair, convulsions, severe decubitus paralysis and death. All subjective measurement of decreased mobility and scruffy fur were done in a blinded fashion as not to influence the results. The protective properties of the experimental anti-viral drugs were assessed by using the following treatment-preventative regimen: Mice were treated either with vehicle (n = 20) or PAP (n = 10) (0.25 mg/kg) administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Mice were then observed for 21-days post infection. The protective effect of the experimental anti-viral drugs was evaluated according to the rise of the survival rate and prolongation of mean life of the experimental animals as compared with the control animals.
LCMV model
Three week old CBA mice were intracerebrally infected with 1000 PFU of WE54 strain of LCMV that resulted in lethality of 100% of control (non-treated) animals in 6–8 days after infection. Control animals were given physiologic salt solution as a placebo instead of the compound. In general, for non-treated animals, clinical signs of the disease manifested on the 5 th  and 7 th  days by presenting: weight loss, immobility, disheveled hair, convulsions, severe decubitus paralysis and death. All subjective measurement of decreased mobility and scruffy fur were done in a blinded fashion as not to influence the results. The protective properties of the experimental anti-viral drugs were assessed by using the following treatment-preventative regimen: Mice were treated either with vehicle (n = 20) or PAP (n = 10) (0.25 mg/kg) administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Mice were then observed for 21-days post infection. The protective effect of the experimental anti-viral drugs was evaluated according to the rise of the survival rate and prolongation of mean life of the experimental animals as compared with the control animals.Statistical analysis Statistical significance was determined using the Kaplan Meier Log-Rank test.
Statistical analysis
Statistical significance was determined using the Kaplan Meier Log-Rank test.Results In order to evaluate the anti-LCMV activity of PAP, mice were inoculated with intracerebral injections of LCMV at a dose level that is lethal to 100% of mice within 6–8 days. Mice were treated either with vehicle or PAP as described in the methods section. Of the 20 control mice, 3 died on day 1 immediately after intracerebral injection due to accidental brain injury and are excluded from the data analysis. All 17 of the remaining control mice developed clinical signs of LCMV infection between day 4 and 6, including weight loss, disheveled hair, decreased mobility and paralysis (Table  1 ). All control mice developed seizures between day 5 and day7 and died between day 6 and day 8 with a median survival of 7 days (Table  1 , Figure  1 ). Table 1 Anti-LCMV activity of PAP in CBA mice Disease Unset Days after inoculation with LCM Virus Survival (days) Weight loss (grams) Decreased mobility Convulsions Hair disheveled Vehicle   Mouse #1* <<1 NA 0 NA NA   Mouse #2* <<1 NA 0 NA NA   Mouse #3* <<1 NA 0 NA NA   Mouse #4 6 1.5 4.5 5.5 4.5   Mouse #5 6 2 4.5 5.5 4.5   Mouse #6 7 2 5 6 5   Mouse #7 7 1.5 5 6 5   Mouse #8 7 2.5 5 6 5   Mouse #9 7 4.5 5 6 5   Mouse #10 7 4.5 5 6 5   Mouse #11 7 4.5 5 6 5   Mouse #12 8 2 6 7 6   Mouse #13 8 2.5 6 7 6   Mouse #14 8 1 6 7 6   Mouse #15 8 1.5 6 7 6   Mouse #16 8 2 6 7 6   Mouse #17 8 2 6 7 6   Mouse #18 7 1.5 5 6 5   Mouse #19 8 2 6 7 6   Mouse #20 8 1.5 6 7 6 PAP 0.25 mg/kg   Mouse #1 <<1 NA NA NA NA   Mouse #2 <<1 NA NA NA NA   Mouse #3 6 1 4.5 5.5 5   Mouse #4 7 1.5 5 6 5   Mouse #5 8 1.5 6 7 5.5   Mouse #6 21 0 10 NO NO   Mouse #7 21 0 10 NO NO   Mouse #8 21 0 10 NO NO   Mouse #9 21 0 10 NO NO   Mouse #10 21 0 10 NO NO *Mouse died after intracerebral injection; NA = not applicable, NO = not observed Figure 1 Protective Activity of PAP in CBA Mice Challenged with LCM Virus . CBA mice were inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation. Mice were then observed twice daily for 21 days for morbidity and mortality. ( Top ) Shown are representative survival curves detailing the cumulative proportions (%) of mice surviving after virus inoculation. See Table 1 for more detailed information of the treatment outcome. ( Bottom ) Shown is Kaplan-Meir life-table analysis and statistical comparison using the log-rank test. Of the 10 mice treated with the broad-spectrum antiviral agent pokeweed antiviral protein (PAP), 2 died on day 1 immediately after intracerebral injection due to accidental brain injury and are invaluable. Five of the remaining 8 mice (62.5%) treated with PAP remained alive > 21 days post-LCMV inoculation  (median survival >21 days, p = 0.0069)  (Table  1 , Figure  1 ). These mice exhibited no LCMV infection-related weight loss or convulsions and showed no signs of scruffy fur. A significant improvement in mobility was also noted. Thus, PAP exhibited significant  in vivo  anti-LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV.
Results
In order to evaluate the anti-LCMV activity of PAP, mice were inoculated with intracerebral injections of LCMV at a dose level that is lethal to 100% of mice within 6–8 days. Mice were treated either with vehicle or PAP as described in the methods section. Of the 20 control mice, 3 died on day 1 immediately after intracerebral injection due to accidental brain injury and are excluded from the data analysis. All 17 of the remaining control mice developed clinical signs of LCMV infection between day 4 and 6, including weight loss, disheveled hair, decreased mobility and paralysis (Table  1 ). All control mice developed seizures between day 5 and day7 and died between day 6 and day 8 with a median survival of 7 days (Table  1 , Figure  1 ).Anti-LCMV activity of PAP in CBA miceDisease UnsetDays after inoculation with LCM VirusSurvival (days)Weight loss (grams)Decreased mobilityConvulsionsHair disheveledVehiclePAP 0.25 mg/kg*Mouse died after intracerebral injection; NA = not applicable, NO = not observedOf the 10 mice treated with the broad-spectrum antiviral agent pokeweed antiviral protein (PAP), 2 died on day 1 immediately after intracerebral injection due to accidental brain injury and are invaluable. Five of the remaining 8 mice (62.5%) treated with PAP remained alive > 21 days post-LCMV inoculation  (median survival >21 days, p = 0.0069)  (Table  1 , Figure  1 ). These mice exhibited no LCMV infection-related weight loss or convulsions and showed no signs of scruffy fur. A significant improvement in mobility was also noted. Thus, PAP exhibited significant  in vivo  anti-LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV.(median survival >21 days, p = 0.0069)in vivoDiscussion In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice. Future studies will examine the therapeutic activity of PAP against LCMV in post-challenge settings. As PAP is well described as an HIV agent, this observation is also relevant as HIV-1 infects the central nervous system (CNS) and it has been feared that the CNS may be a sanctuary site where HIV-1 could hide and continue to replicate despite otherwise effective antiretroviral treatment [ 48 , 49 ]. Antiretroviral therapy of HIV-infected children is associated with a decline in CSF HIV RNA and an improvement in neurological status. The development of genotypic mutations was different in CSF and plasma, suggesting discordant viral evolution. These results suggest that antiretroviral treatment in children should include agents with activity in the CNS [ 50 ]. While these results suggest that PAP crosses the blood brain barrier and may therefore be beneficial in other viral infections affecting CNS, they need to be interpreted with due caution for HIV infections involving the CNS since LCMV affects leptomeninges while HIV affects neurons. In future studies, we must also consider the possibility that PAP penetrates the blood brain barrier only when the latter is impaired by the intracerebral administration of the virus. If this is the case, PAP may not be useful in pre-challenge prophylaxis against LCMV-mediated CNS infection. While these results support the notion that the antiviral activity spectrum of PAP covers LCMV as well, an immunomodulatory effect of PAP may also contribute to the observed prophylactic efficacy of PAP against LCMV. Cases of LCMV infections have been reported in Europe, the Americas, Australia, and Japan. According to the Center for Disease Control, currently there is no specific drug therapy for LCMV. Although the anti-viral agent ribavirin is effective against LCMV in vitro, there is no established evidence to support its use for treatment of LCMV in humans [ 51 ]. The anti-viral activity of PAP has been described against numerous pathogenic viruses, which included poliovirus, HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and now, the negative-strand RNA virus Lymphocytic choriomeningitis virus. The ability of PAP to inhibit viral protein synthesis and depurinate viral RNA and DNA [ 15 , 16 ] as well as capped rRNA and mRNAs [ 52 ] and its ability to inhibit ribosomal frame shifting and retransposition, make it and ideal candidate for anti-viral strategies.
Discussion
In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice. Future studies will examine the therapeutic activity of PAP against LCMV in post-challenge settings. As PAP is well described as an HIV agent, this observation is also relevant as HIV-1 infects the central nervous system (CNS) and it has been feared that the CNS may be a sanctuary site where HIV-1 could hide and continue to replicate despite otherwise effective antiretroviral treatment [ 48 , 49 ]. Antiretroviral therapy of HIV-infected children is associated with a decline in CSF HIV RNA and an improvement in neurological status. The development of genotypic mutations was different in CSF and plasma, suggesting discordant viral evolution. These results suggest that antiretroviral treatment in children should include agents with activity in the CNS [ 50 ]. While these results suggest that PAP crosses the blood brain barrier and may therefore be beneficial in other viral infections affecting CNS, they need to be interpreted with due caution for HIV infections involving the CNS since LCMV affects leptomeninges while HIV affects neurons. In future studies, we must also consider the possibility that PAP penetrates the blood brain barrier only when the latter is impaired by the intracerebral administration of the virus. If this is the case, PAP may not be useful in pre-challenge prophylaxis against LCMV-mediated CNS infection. While these results support the notion that the antiviral activity spectrum of PAP covers LCMV as well, an immunomodulatory effect of PAP may also contribute to the observed prophylactic efficacy of PAP against LCMV.Cases of LCMV infections have been reported in Europe, the Americas, Australia, and Japan. According to the Center for Disease Control, currently there is no specific drug therapy for LCMV. Although the anti-viral agent ribavirin is effective against LCMV in vitro, there is no established evidence to support its use for treatment of LCMV in humans [ 51 ].The anti-viral activity of PAP has been described against numerous pathogenic viruses, which included poliovirus, HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and now, the negative-strand RNA virus Lymphocytic choriomeningitis virus. The ability of PAP to inhibit viral protein synthesis and depurinate viral RNA and DNA [ 15 , 16 ] as well as capped rRNA and mRNAs [ 52 ] and its ability to inhibit ribosomal frame shifting and retransposition, make it and ideal candidate for anti-viral strategies.Conclusion Treatment of CBA mice with the broad-spectrum anti-viral agent, PAP significantly improved the probability of survival following the LCMV challenge and decreased overall LCMV-related symptoms. The ability of PAP to exhibit anti-viral activity within the central nervous system is also encouraging within the framework of potential HIV-treatment. We have recently described the rational design and engineering of several recombinant PAP mutants with superior anti-HIV activity [ 11 , 17 ]. In our future studies, we plan to describe the potential anti-LCMV activity of these PAP mutants as well as optimizing the prophylactic and post-exposure treatment regimens. In addition, it will be important to determine if PAP or PAP mutants have activity against other viruses associated with lethal viral hemorrhagic fevers and/or encephalomyelitis, such as the Ebola viruses of the Filoviridae family [ 53 - 56 ].
Conclusion
Treatment of CBA mice with the broad-spectrum anti-viral agent, PAP significantly improved the probability of survival following the LCMV challenge and decreased overall LCMV-related symptoms. The ability of PAP to exhibit anti-viral activity within the central nervous system is also encouraging within the framework of potential HIV-treatment. We have recently described the rational design and engineering of several recombinant PAP mutants with superior anti-HIV activity [ 11 , 17 ]. In our future studies, we plan to describe the potential anti-LCMV activity of these PAP mutants as well as optimizing the prophylactic and post-exposure treatment regimens. In addition, it will be important to determine if PAP or PAP mutants have activity against other viruses associated with lethal viral hemorrhagic fevers and/or encephalomyelitis, such as the Ebola viruses of the Filoviridae family [ 53 - 56 ].List of abbreviations LCMV: Lymphocytic choriomeningitis virus PAP: Pokeweed anti-viral protein HIV and FIV: Human and Feline immunodeficiency virus, respectively
List of abbreviations
LCMV: Lymphocytic choriomeningitis virusPAP: Pokeweed anti-viral proteinHIV and FIV: Human and Feline immunodeficiency virus, respectivelyCompeting interests FMU is listed as an inventor on a number of PAP patents which are owned by PHI. FMU, AV and HET are salaried employees at PHI. PHI is a non-profit public charity.
Competing interests
FMU is listed as an inventor on a number of PAP patents which are owned by PHI. FMU, AV and HET are salaried employees at PHI. PHI is a non-profit public charity.Authors' contributions FMU designed the research project. LR, AP & LT conducted the Lassa experiments. FMU & AV provided PAP and coordinated the Lassa experiments with LR, AP and LT. FMU and HT wrote the manuscript. All authors read and approved manuscript.
Authors' contributions
FMU designed the research project. LR, AP & LT conducted the Lassa experiments. FMU & AV provided PAP and coordinated the Lassa experiments with LR, AP and LT. FMU and HT wrote the manuscript. All authors read and approved manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive.  TGFBR1 *6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that  TGFBR1 *6A may contribute to the development of prostate cancer.  TGFBR1 *6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in  TGFBR1 *6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that  TGFBR1 *6A does not contribute to the development of prostate cancer.TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1

Background Transforming Growth Factor Beta (TGF-ß) is one of the most potent inhibitor of cell growth [ 1 ]. Almost all cancer cells loose the ability to be growth inhibited by TGF-ß, which makes loss of TGF-ß growth inhibition a hallmark of cancer development [ 2 ].  TGFBR1 *6A is a common variant of the type I TGF-ß receptor,  TGFBR1  [ 3 ].  TGFBR1 *6A (*6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of  TGFBR1  (*9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates *6A from *9A is located within the predicted signal sequence cleavage region.  In vitro  studies have demonstrated that TGFBR1*6A responds less effectively than TGFBR1 to TGF-ß growth inhibitory signals [ 4 , 5 ]. The additional findings of an overrepresentation of  TGFBR1 *6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that  TGFBR1 *6A might act as a tumor susceptibility allele [ 5 ]. Two recent meta-analyses show that  TGFBR1 *6A carriers may have an increased risk of breast, colon and ovarian cancer [ 6 , 7 ]. To test the hypothesis that  TGFBR1 *6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls.
Background
Transforming Growth Factor Beta (TGF-ß) is one of the most potent inhibitor of cell growth [ 1 ]. Almost all cancer cells loose the ability to be growth inhibited by TGF-ß, which makes loss of TGF-ß growth inhibition a hallmark of cancer development [ 2 ].  TGFBR1 *6A is a common variant of the type I TGF-ß receptor,  TGFBR1  [ 3 ].  TGFBR1 *6A (*6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of  TGFBR1  (*9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates *6A from *9A is located within the predicted signal sequence cleavage region.  In vitro  studies have demonstrated that TGFBR1*6A responds less effectively than TGFBR1 to TGF-ß growth inhibitory signals [ 4 , 5 ]. The additional findings of an overrepresentation of  TGFBR1 *6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that  TGFBR1 *6A might act as a tumor susceptibility allele [ 5 ]. Two recent meta-analyses show that  TGFBR1 *6A carriers may have an increased risk of breast, colon and ovarian cancer [ 6 , 7 ]. To test the hypothesis that  TGFBR1 *6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls.TGFBR1TGFBR1TGFBR1TGFBR1In vitroTGFBR1TGFBR1TGFBR1TGFBR1Results A total of 907 cases and controls were genotyped for  TGFBR1 *6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59  TGFBR1 *6A heterozygotes and three  TGFBR1 *6A homozygotes among cases, 62  TGFBR1 *6A heterozygotes and 1  TGFBR1 *6A homozygote among controls.  TGFBR1 *6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in  TGFBR1 *6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67) (Table  1 ). Effect estimates from conditional logistic regression were similar (OR 1.01, 95% CI 0.29–3.52) to those from unconditional logistic regression (OR 0.96, 95% CI 0.56–1.64). Analyses restricted to subjects aged 40 years and above controlling age on a continuous scale produced essentially the same effect estimates (OR 0.94, 95% CI 0.56–1.60). To examine the possibility that  TGFBR1 *6A is associated with early onset prostate cancer, we determined the prostate cancer risk for individuals above and below the age of 55. Among younger patients with prostate cancer we found that 13 of 59 were  TGFBR1 *6A carriers yielding an allelic frequency of 0.119, one of the highest  TGFBR1 *6A allelic frequency ever reported. Only 45 of 367 controls in the same age range were  TGFBR1 *6A carriers yielding an allelic frequency of 0.063, which is similar to the  TGFBR1 *6A allelic frequency found among 3,451 healthy controls from Europe and the U.S. [ 7 ]. The association between carrier status of  TGFBR1 *6A and prostate cancer in younger age group was significant after adjustment for race (OR 2.13, 95% CI 1.06–4.27) but was not significant after adjustment for race and age strata within groups (OR 2.11, 95% CI 0.98–4.57) (Table  2 ). While in the older age group we did not observe a significant association either (OR 0.57, 95% CI 0.30–1.10), the test for multiplicative interaction between age and carrier status of  TGFBR1 *6A was significant (p = 0.01). Table 1 Distribution of Age, Ethnicity, and  TGFBR1  Genotypes and Adjusted Odds Ratios of Prostate Cancer by  TGFBR1  Genotype Status Cases (N = 442) Controls (N = 465) P-value  1 Adjusted OR (95% CI) 2 N % N % TGFBR1  genotype  9A/9A 380 86.0 402 86.5 0.67 1.00 (ref)  9A/6A 59 13.4 62 13.3 0.96 (0.56–1.64)  6A/6A 3 0.6 1 0.2 Age  20–40 1 0.2 205 44.1 <0.01  41–60 126 28.5 204 43.9  61–80 308 69.7 55 11.8  80+ 7 1.6 1 0.2 Race  White 396 89.6 415 89.3 1.00  Black 26 5.9 29 6.3  Hispanic 8 1.8 8 1.7  Asian 2 0.5 2 0.4  Other 1 0.2 2 0.4  Unknown 9 2.0 9 1.9 1 p -value for Chi-Square or Fisher's Exact Test (comparing proportions) 2  OR was adjusted for age strata and race, based on dominant model. Table 2 Adjusted Odds Ratios of prostate cancer by age groups (> 55, <= 55 years old) Age group/ Genotypes Cases N (% within age strata) Controls N (% within age strata) OR (95% CI) 1 OR (95% CI) 2 P for testing multiplicative interaction Age = 55  9A/9A 46 (78.0) 322 (87.7) 1.00 1.00  9A/6A or 6A/6A 13 (22.0) 45 (12.3) 2.13 (1.06–4.27) 2.11 (0.98–4.57) Age > 55 0.01  9A/9A 334 (87.2) 80 (81.6) 1.00 1.00  9A/6A or 6A/6A 49 (12.8) 18 (18.4) 0.64 (0.36–1.17) 0.57 (0.30–1.10) 1  OR was adjusted for race. 2  OR was adjusted for race and age strata within age groups
Results
A total of 907 cases and controls were genotyped for  TGFBR1 *6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59  TGFBR1 *6A heterozygotes and three  TGFBR1 *6A homozygotes among cases, 62  TGFBR1 *6A heterozygotes and 1  TGFBR1 *6A homozygote among controls.  TGFBR1 *6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in  TGFBR1 *6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67) (Table  1 ). Effect estimates from conditional logistic regression were similar (OR 1.01, 95% CI 0.29–3.52) to those from unconditional logistic regression (OR 0.96, 95% CI 0.56–1.64). Analyses restricted to subjects aged 40 years and above controlling age on a continuous scale produced essentially the same effect estimates (OR 0.94, 95% CI 0.56–1.60). To examine the possibility that  TGFBR1 *6A is associated with early onset prostate cancer, we determined the prostate cancer risk for individuals above and below the age of 55. Among younger patients with prostate cancer we found that 13 of 59 were  TGFBR1 *6A carriers yielding an allelic frequency of 0.119, one of the highest  TGFBR1 *6A allelic frequency ever reported. Only 45 of 367 controls in the same age range were  TGFBR1 *6A carriers yielding an allelic frequency of 0.063, which is similar to the  TGFBR1 *6A allelic frequency found among 3,451 healthy controls from Europe and the U.S. [ 7 ]. The association between carrier status of  TGFBR1 *6A and prostate cancer in younger age group was significant after adjustment for race (OR 2.13, 95% CI 1.06–4.27) but was not significant after adjustment for race and age strata within groups (OR 2.11, 95% CI 0.98–4.57) (Table  2 ). While in the older age group we did not observe a significant association either (OR 0.57, 95% CI 0.30–1.10), the test for multiplicative interaction between age and carrier status of  TGFBR1 *6A was significant (p = 0.01).TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1Distribution of Age, Ethnicity, and  TGFBR1  Genotypes and Adjusted Odds Ratios of Prostate Cancer by  TGFBR1  Genotype StatusTGFBR1TGFBR1TGFBR11 p -value for Chi-Square or Fisher's Exact Test (comparing proportions)p2  OR was adjusted for age strata and race, based on dominant model.Adjusted Odds Ratios of prostate cancer by age groups (> 55, <= 55 years old)1  OR was adjusted for race.2  OR was adjusted for race and age strata within age groupsDiscussion Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men [ 8 ]. A similar pattern is observed throughout the western world. There is strong epidemiologic evidence indicating that a large proportion of prostate cancers are caused by heritable factors. The most convincing data is a study of 44,788 Scandinavian twins showing that 42% of prostate cancer cancers may be caused by shared genes [ 9 ]. Despite intense efforts led by several research teams, the search for prostate cancer susceptibility genes has thus far remained elusive. Recent studies suggest that carriers of deleterious mutations of the  BRCA2  gene have an increased prostate cancer risk [ 10 ]. However, given the low prevalence of deleterious  BRCA2  mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of  TGFBR1 *6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that  TGFBR1 *6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that  TGFBR1 *6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between  TGFBR1 *6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high  TGFBR1 *6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the  TGFBR1 *6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high  TGFBR1 *6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that  TGFBR1 *6A is not associated with an increased risk of bladder cancer [ 6 ]. Our results suggest that the association between  TGFBR1 *6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer.
Discussion
Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men [ 8 ]. A similar pattern is observed throughout the western world. There is strong epidemiologic evidence indicating that a large proportion of prostate cancers are caused by heritable factors. The most convincing data is a study of 44,788 Scandinavian twins showing that 42% of prostate cancer cancers may be caused by shared genes [ 9 ]. Despite intense efforts led by several research teams, the search for prostate cancer susceptibility genes has thus far remained elusive. Recent studies suggest that carriers of deleterious mutations of the  BRCA2  gene have an increased prostate cancer risk [ 10 ]. However, given the low prevalence of deleterious  BRCA2  mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of  TGFBR1 *6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that  TGFBR1 *6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that  TGFBR1 *6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between  TGFBR1 *6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high  TGFBR1 *6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the  TGFBR1 *6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high  TGFBR1 *6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that  TGFBR1 *6A is not associated with an increased risk of bladder cancer [ 6 ]. Our results suggest that the association between  TGFBR1 *6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer.BRCA2BRCA2TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1TGFBR1Methods DNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board. A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons (predominantly pre-natal screening for non-cancer disease) constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls. DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech Advantage ®  GC rich kit (BD-Biosciences Clontech, Palo Alto, CA). The PCR reaction mixture included 20 ng of genomic DNA in a 10-µL reaction volume and the following concentration of other reagents: primers (0.25 µM each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg 2+ , 0.2 mM dNTPs and 0.16 µL of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94°C, then 35 cycles of denaturation for 30 seconds at 94°C and annealing/extension for 2 minutes at 72°C, followed by a final extension step of 5 minutes at 72°C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for data acquisition. A peak at 115 base pairs corresponded to  TGFBR1  allele, whereas a peak at 107 base pairs corresponded to the  TGFBR1 *6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%. Statistical analysis Distributions of  TGFBR1  genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic  TGFBR1 *6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of  TGFBR1 *6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of  TGFBR1 *6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level.
Methods
DNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board.A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons (predominantly pre-natal screening for non-cancer disease) constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls.DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech Advantage ®  GC rich kit (BD-Biosciences Clontech, Palo Alto, CA). The PCR reaction mixture included 20 ng of genomic DNA in a 10-µL reaction volume and the following concentration of other reagents: primers (0.25 µM each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg 2+ , 0.2 mM dNTPs and 0.16 µL of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94°C, then 35 cycles of denaturation for 30 seconds at 94°C and annealing/extension for 2 minutes at 72°C, followed by a final extension step of 5 minutes at 72°C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for data acquisition. A peak at 115 base pairs corresponded to  TGFBR1  allele, whereas a peak at 107 base pairs corresponded to the  TGFBR1 *6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%.TGFBR1TGFBR1Statistical analysis Distributions of  TGFBR1  genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic  TGFBR1 *6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of  TGFBR1 *6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of  TGFBR1 *6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level.
Statistical analysis
Distributions of  TGFBR1  genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic  TGFBR1 *6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of  TGFBR1 *6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of  TGFBR1 *6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level.TGFBR1TGFBR1TGFBR1TGFBR1Authors' contributions All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript.
Authors' contributions
All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript.