José A. Bonilla

Persistent Human Papillomavirus Infection Is Associated with a Generalized Decrease in Immune Responsiveness in Older Women

The development of cervical cancer and its precursors are linked to persistent infection with oncogenic types of human papillomavirus (HPV). Host immune responses seem to be determinants of risk for this disease. However, little is known about the immunologic determinants of HPV persistence. Here, we examined the association between lymphoproliferative responses to antigens/mitogens and persistent HPV infection in women older than 45 years. Women included in this study were participants in a 10,000-woman population-based cohort study of cervical neoplasia in Costa Rica. Women older than 45 years and HPV DNA positive at a screening visit were selected as cases (n = 283). We selected a comparably sized control group of HPV DNA-negative women, matched to cases on age and time since enrollment (n = 261). At an additional clinical visit, women were cytologically and virologically rescreened, and cervical and blood specimens were collected. Proliferative responses to phytohemagglutinin (PHA), influenza virus (Flu), and HPV16 virus-like particle (VLP) were lower among women with persistent HPV infection [median counts per minute (cpm): 72,849 for PHA, 1,241 for Flu, and 727 for VLP] than for the control group (median cpm: 107,049 for PHA, 2,111 for Flu, and 2,068 for VLP). The decreases were most profound in women with long-term persistence and were only observed for the oldest age group (>/=65 years). Our results indicate that an impairment in host immunologic responses is associated to persistent HPV infection. The fact that effects were evident for all studied stimuli is suggestive of a generalized effect.

Elevated Systemic Levels of Inflammatory Cytokines in Older Women with Persistent Cervical Human Papillomavirus Infection

Background: Defects in lymphoproliferative responses to mitogens/antigens in women >45 years old with a persistent type-specific human papillomavirus (HPV) infection have been reported. Methods: To determine whether these defects were associated with altered cytokine profiles, plasma and peripheral blood mononuclear cell (PBMC) culture supernatants from 50 cases (oversampled for their reduced lymphoproliferative ability) and 50 uninfected controls (oversampled for their robust lymphoproliferative ability) were examined for 24 cytokines using multiplexed bead–based immunoassays and ELISA. Results: The following plasma cytokines were significantly increased in cases relative to controls (cases versus controls; median pg/mL): interleukin (IL)-6, 393.1 versus 14.5; IL-8, 1,128.5 versus 43.9; tumor necrosis factor-α (TNF-α), 164.1 versus 9.2; macrophage inflammatory protein-1α (MIP-1α), 1,368.9 versus 25.5; granulocyte macrophage colony-stimulating factor (GM-CSF), 13.8 versus 7.3; IL-1β, 8.3 versus 1.6 (all P < 0.0001); and IL-1α, 218.2 versus 169.5 (P = 0.02). We focused our analysis on the cytokines IL-6, IL-8, TNF-α, and MIP-1α due to their high fold change (>10) and highly statistically significant difference between cases and controls. Length of persistence or type of infection (high risk and low risk) did not affect these differences. IL-6, TNF-α, and MIP-1α levels were also increased in unstimulated PBMC culture supernatants from cases compared with controls (P < 0.05), however, the cytokine levels from phytohemagglutinin-stimulated PBMC culture supernatants were significantly lower in the cases (P < 0.0001). Conclusions: Persistent HPV infection in older women with evidence of immune deficit is associated with an increase in systemic inflammatory cytokines. Impact: Future studies are needed to determine whether the inflammatory profile is age dependent and to examine the role that inflammatory cytokines play in HPV-induced progression from infection to cervical cancer. Cancer Epidemiol Biomarkers Prev; 19(8); 1954–9. ©2010 AACR.

Prevalence of and Risk Factors for Oral Human Papillomavirus Among Young Women in Costa Rica

BACKGROUND Little is known about the epidemiology of oral human papillomavirus (HPV) in Latin America. METHODS Women (N = 5838) aged 22-29 in the control and vaccine arms of an HPV-16/18 vaccine trial in Costa Rica had oral, cervical, and anal specimens collected. Samples were tested for alpha mucosal HPV types (SPF10/LiPA25 version 1); a subset of oral samples (n = 500) was tested for cutaneous HPV types in the genera alpha, beta, gamma, mu, and nu. RESULTS In the control arm (n = 2926), 1.9% of women had an oral alpha mucosal HPV detected, 1.3% had carcinogenic HPV, and 0.4% had HPV-16; similar patterns for non-16/18 HPV types were observed in the vaccine arm. Independent risk factors for any oral alpha mucosal HPV among women in the control arm included marital status (adjusted odds ratio [AOR], 3.2; 95% confidence interval [CI], 1.8-5.7 for single compared to married/living as married), number of sexual partners (AOR, 2.4; 95% CI, 1.0-6.1 for ≥4 partners compared to 0-1 partners), chronic sinusitis (AOR, 3.1; 95% CI, 1.5-6.7), and cervical HPV infection (AOR, 2.6; 95% CI, 1.4-4.6). Detection of beta HPV was common (18.6%) and not associated with sexual activity. CONCLUSIONS Unlike cutaneous HPV types, alpha mucosal HPV types were uncommon in the oral region and were predominately associated with sexual behavior. Clinical Trials Registration. NCT00128661.

Anticancer activities of two sesquiterpene lactones, millerenolide and thieleanin isolated from Viguiera sylvatica and Decachaeta thieleana

The present study was carried out in order to examine the anticancer properties of two sesquiterpene lactones, millerenolide and thieleanin, isolated from Viguiera sylvatica and Decachaeta thieleana, against cell lines in vitro, and on the growth B16/BL6 melanoma tumors in C57BL/6 mice. Millerenolide and thieleanin showed a similar pattern of cytotoxicity with the greatest effect on viability being evident with A549 human lung cancer cells (IC(50) - 40 and 32 microM respectively), and with the 3T3/HER2 cell line which are 3T3 mouse fibroblasts transfected with the HER2 oncogene (IC(50) - 16 and 28 microM respectively). The parent 3T3 cells and the B16/BL6 mouse melanoma cells were less sensitive to these compounds, with thieleanin showing an IC(50) with B16/BL6 greater than the highest dose tested (203 microM). Treatment with millerenolide (8 mg/kg, i.p. on days 0, 2 and 4 post-inoculation) significantly inhibited the growth of subcutaneous B16/BL6 tumors in C57BL/6 mice, (50% inhibition at day 25, P=0.015), as well as retarding the appearance of detectable tumor (millerenolide - day 15.2+/-0.4 vs control - day 12.8+/-0.5, mean+/-SEM, P=0.011). In contrast, treatment with thieleanin (8 mg/kg every other day up to the day of kill) neither retarded the appearance of the tumor nor its growth.

Efecto in vitro de los terpenos lupeol y casearina G sobre células sanguíneas y tumorales

Los bosques tropicales constituyen una fuente importante de compuestos naturales con propiedades terapeuticas. Aunque existen diversos antiinflamatorios y antineoplasicos, se requieren nuevas drogas que ejerzan su efecto terapeutico con la menor cantidad de efectos adversos. Este estudio evaluo el efecto in vitro de dos compuestos naturales lupeol (triterpeno) y casearina G (diterpeno) sobre la funcion fagocitica de monocitos/ macrofagos humanos determinando el porcentaje de celulas fagociticas y el indice fagocitico. El efecto de estos compuestos sobre la produccion de oxido nitrico (NO) fue medido por medio de la reaccion de Griess. Ademas, el efecto citotoxico de estos compuestos sobre las lineas celulares MCF-7 (adenocarcinoma de mama humano) y PC-3 (cancer de prostata humano) fue medido con un ensayo cromogenico (MTS/PMS) y por el ensayo de sulforodamina B, el cual permite distinguir entre un efecto citostatico y citotoxico. Los resultados muestran que la exposicion a lupeol (?1mg/ml) causo la disminucion del porcentaje de celulas fagociticas y el indice fagocitico. Se observo que los macrofagos tratados con lupeol (30?g/ml) y casearina G (2,5?g/ml) disminuyeron la produccion de NO. Lupeol ( celulas mononucleares de sangre periferica . C asearina G mostro efecto citotoxico (IC 50 , LC 50 ) y citostatico (GI 50 ) contra las celulas tumorales PC-3 (IC 50 =12,5?M; GI 50 =13,3?M; LC 50 =51,9?M) y MCF-7 (IC 50 =112,8?M; GI 50 =11,8?M; LC 50 =49,4?M) y efecto hemolitico (? 100 ?g/ml) sobre eritrocitos humanos. Estos resultados sugieren que lupeol y casearina G podrian ser utiles en la elaboracion de drogas antiinflamatorias mientras que casearina G podria ser vir para el desarrollo de farmacos antitumoral es .

Alterations of T-cell surface markers in older women with persistent human papillomavirus infection.

We previously reported decreased lymphocyte proliferative responses among older women with persistent human papillomavirus (HPV) infection. To characterize the phenotype of peripheral lymphocytes associated with persistent HPV infection, we evaluated the expression of different cell surface markers in peripheral blood mononuclear cells (PBMCs) from a case–control study within a 10,049 woman population‐based cohort study in Guanacaste, Costa Rica. Women in the cohort aged 46–74 and with HPV results at their 5th year anniversary visit were considered, and all women (n = 87) with persistent HPV infections, all women (n = 196) with transient HPV infections and a random sample of HPV DNA‐negative women (n = 261) frequency‐matched to cases on age were selected for this study. A median of 3 years after the case–control matching visit, cervical cells were collected for liquid‐based cytology and repeat HPV DNA genotyping. Blood was obtained from which PBMCs were extracted and cryopreserved for immunological phenotyping via flow cytometry. Significant increases in risk of HPV persistence were observed for 3 marker subsets indicative of immune cell activation/differentiation. Relative risk estimates were 5.4 (95% CI = 2.2–13.3) for CD69+CD4+, 2.6 (95% CI = 1.2–5.9) for HLADR+CD3+CD4+ and 2.3 (95% CI = 1.1–4.7) for CD45RO+CD27−CD8+. A significant decrease in HPV persistence was observed for a subset marker indicative of an immature, undifferentiated memory state CD45RO+CD27+CD4+ (OR = 0.36; 95% CI = 0.17–0.76). Adjustment for these markers only partially explained the previously reported association between decreased lymphoproliferative responses and persistent HPV infection. Whether phenotypic alterations observed predispose to HPV persistence or result from it should be the focus of future studies.

Actividad supresora del millerenólido sobre células mononucleares de sangre periférica humana

BACKGROUND Natural products are used in the production of therapeutic drugs due to their wide diversity and excellent adaptability to biological structures. Sesquiterpene lactones are the active constituents of several plants from the Asteraceae family. AIM To assess the in vitro effect of a sesquiterpene lactone (millerenolide). MATERIAL AND METHODS The drug effect was assessed measuring the proliferation of lymphocytes using the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonylJ-2H-tetrazolium hydroxide (XTT) technique. Changes on the cell cycle were analyzed on a FACSort flow cytometer The effect of millerenolide on the production of nitric oxide (NO) by macrophages was evaluated using the Griess reagent. Additionally, phagocytosis of latex particles and nitroblue tetrazolium (NBT) reduction by macrophages were evaluated microscopically. RESULTS Treatment of human peripheral blood mononuclear cells (PBMC) with millerenolide decreases the proliferation of lymphocytes, decreases the percentage of cells in S, and G2/M phases, and increases the proportion of cells in GO/Gl phase. Treatment of macrophages with millerenolide, reduces the production of NO, the phagocytic capacity and the number of cells able to reduce NBT. Cytotoxic effects of the lactone on human PBMC were only observed when the concentration was increased to 6 microg/ml. CONCLUSIONS Millerenolide could be considered as a potential therapeutic agent with immunosuppressive properties.

Low prevalence of hepatitis A virus infection among autochthonous populations of New World non‐human primates

Hepatitis A virus (HAV) belongs to the genus hepatovirus, family Picornaviridae (Cristina and Costa-Mattioli 2007) [3]. HAV infection is highly prevalent in developing countries. Latin America exhibits an intermediate to high prevalence of HAV infection, with a tendency to lower incidence associated with sanitary improvement in each country (Jacobsen 2004) [4, 8]. Six genotypes of HAV have been described in the world, three of them infecting humans and other primates and three additional found only in non-human primates (Cristina and Costa-Mattioli 2007) [3, 8]. It is known that HAV circulates among non-human primates from the Old World [9] (Cristina and CostaMattioli 2007) and that many species non-human primates from both worlds can get infected by this virus [1]. It is not known, however, whether HAV circulates among New World non-human primates in the wild. The aim of this study was to evaluate the prevalence of antibodies among non-human primates in different ecological contexts in Latin America.