Plasmatic MicroRNAs and Treatment Outcomes of Patients with Metastatic Castration-Resistant Prostate Cancer: A Hospital-Based Cohort Study and In Silico Analysis.

Prostate cancer (PCa) is one of the most common malignancies among men worldwide. Inevitably, all advanced PCa patients develop metastatic castration-resistant prostate cancer (mCRPC), an aggressive phase of the disease. Treating mCRPC is challenging, and prognostic tools are needed for disease management. MicroRNA (miRNA) deregulation has been reported in PCa, constituting potential non-invasive prognostic biomarkers. As such, this study aimed to evaluate the prognostic potential of nine miRNAs in the liquid biopsies (plasma) of mCRPC patients treated with second-generation androgen receptor axis-targeted (ARAT) agents, abiraterone acetate (AbA) and enzalutamide (ENZ). Low expression levels of miR-16-5p and miR-145-5p in mCRPC patients treated with AbA were significantly associated with lower progression-free survival (PFS). The two miRNAs were the only predictors of the risk of disease progression in AbA-stratified analyses. Low miR-20a-5p levels in mCRPC patients with Gleason scores of <8 were associated with worse overall survival (OS). The transcript seems to predict the risk of death regardless of the ARAT agent. According to the in silico analyses, miR-16-5p, miR-145-5p, and miR-20a-5p seem to be implicated in several processes, namely, cell cycle, proliferation, migration, survival, metabolism, and angiogenesis, suggesting an epigenetic mechanism related to treatment outcome. These miRNAs may represent attractive prognostic tools to be used in mCRPC management, as well as a step further in the identification of new potential therapeutic targets, to use in combination with ARAT for an improved treatment outcome. Despite the promising results, real-world validation is necessary.

Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions.

Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, the effect of chalcones 1-18 against the metabolic viability of cervical (HeLa) and prostate (PC-3 and LNCaP) tumor cell lines was tested, to compare the activity against solid and liquid tumor cells. Their effect was also evaluated on the Jurkat cell line. Chalcone 16 showed the highest inhibitory effect on the metabolic viability of the tested tumor cells and was selected for further studies. Recent antitumor therapies include compounds with the ability to influence immune cells on the tumor microenvironment, with immunotherapy being one actual goal in cancer treatment. Therefore, the effect of chalcone 16 on the expression of mTOR, HIF-1α, IL-1ß, TNF-α, IL-10, and TGF-ß, after THP-1 macrophage stimulation (none, LPS or IL-4), was evaluated. Chalcone 16 significantly increased the expression of mTORC1, IL-1ß, TNF-α, and IL-10 of IL-4 stimulated macrophages (that induces an M2 phenotype). HIF-1α and TGF-ß were not significantly affected. Chalcone 16 also decreased nitric oxide production by the RAW 264.7 murine macrophage cell line, this effect probably being due to an inhibition of iNOS expression. These results suggest that chalcone 16 may influence macrophage polarization, inducing the pro-tumoral M2 macrophages (IL-4 stimulated) to adopt a profile closer to the antitumor M1 profile.

Effect of 1-Carbaldehyde-3,4-dimethoxyxanthone on Prostate and HPV-18 Positive Cervical Cancer Cell Lines and on Human THP-1 Macrophages.

Xanthone derivatives have shown promising antitumor properties, and 1-carbaldehyde-3,4-dimethoxyxanthone (1) has recently emerged as a potent tumor cell growth inhibitor. In this study, its effect was evaluated (MTT viability assay) against a new panel of cancer cells, namely cervical cancer (HeLa), androgen-sensitive (LNCaP) and androgen-independent (PC-3) prostate cancer, and nonsolid tumor derived cancer (Jurkat) cell lines. The effect of xanthone 1 on macrophage functions was also evaluated. The effect of xanthone 1-conditioned THP-1 human macrophage supernatants on the metabolic viability of cervical and prostate cancer cell lines was determined along with its interference with cytokine expression characteristic of M1 profile (IL-1 ≤ ß; TNF-α) or M2 profile (IL-10; TGF-ß) (PCR and ELISA). Nitric oxide (NO) production by murine RAW264.7 macrophages was quantified by Griess reaction. Xanthone 1 (20 µM) strongly inhibited the metabolic activity of the cell lines and was significantly more active against prostate cell lines compared to HeLa (p < 0.05). Jurkat was the cell most sensitive to the effect of xanthone 1. Compound 1-conditioned IL-4-stimulated THP-1 macrophage supernatants significantly (p < 0.05) inhibited the metabolic activity of HeLa, LNCaP, and PC-3. Xanthone 1 did not significantly affect the expression of cytokines by THP-1 macrophages. The inhibiting effect of compound 1 observed on the production of NO by RAW 264.7 macrophages was moderate. In conclusion, 1-carbaldehyde-3,4-dimethoxyxanthone (1) decreases the metabolic activity of cancer cells and seems to be able to modulate macrophage functions.

Prevalence of Neisseria gonorrhoeae and Trichomonas vaginalis in Portuguese women of childbearing age.

The purpose of this study was to evaluate the prevalence of Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) in Portuguese women of childbearing age. Cervicovaginal self-collected samples of 680 childbearing-age women (15-44 years) were tested for NG and TV by polymerase chain reaction. Sociodemographic, clinical and behavioural data were assessed through an anonymous self-administered questionnaire. NG and TV prevalence was 1.3% (95% confidence interval (CI) 0.7-2.5%) and 1.0% (95% CI 0.5-2.1%), respectively. The prevalence of TV was significantly higher in women aged >22 years (p = .003), with >6 years after sexual intercourse (p = .003), and who reported previous pregnancy (p = .004). Our study suggests that NG and TV are rare in Portuguese women of childbearing age. However, larger epidemiological studies with a nationally representative sample of female subjects are warranted, to clarify the need for screening of these microorganisms in Portuguese women, since its prevalence is probably underestimated.IMPACT STATEMENTWhat is already known on this subject? Studies on the prevalence of NG and TV have been performed in several developed and developing countries. However, limited data is available in Portuguese women. The detection of NG and TV is necessary because, beside the risk of transmission to sex partners, these STIs may be associated with an increased risk of HIV acquisition and transmission, and ultimately with reproductive, pregnancy and perinatal complications.What do the results of this study add? Our study adds new findings to the body of knowledge on NG and TV prevalence in Portuguese women of reproductive age. As so, we found a low prevalence of both NG (1.3%) and TV (1.0%) in the studied population.What are the implications of these findings for clinical practice and/or further research? Our results may be a step ahead to encourage future nationally representative studies evaluating the prevalence of NG and TV genital infection and, consequently, to clarify the need for screening of these microorganisms. In clinical practice, it should be highlighted the appropriate management of NG and TV infection in specific situations, such as pregnancy. Also, sexual partners must be treated to prevent the recurrences in the index cases and reduce transmission to other partners.

Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

Chlamydia trachomatis infection: implications for HPV status and cervical cancer.

Genital Chlamydia trachomatis (CT) infections have been identified as a major health problem concern. CT is associated with adverse effect on women reproduction and also associated with cervical hypertrophy and induction of squamous metaplasia, providing a possible relationship with human papillomavirus (HPV) infection. Infection by high-risk HPV types is crucial to the pathogenesis of invasive cervical cancer (ICC), but other co-variants/cofactors must be present for the development of malignancy. CT biological effect may damage the mucosal barrier, improving HPV infection, or may interfere in immune response and viral clearance supporting the persistence of HPV infection. Moreover, CT-related chronic cervical inflammation, decrease of lower genital tract antigen-presenting cells, inhibition of cell-mediated immunity, and anti-apoptotic capacity may influence the natural history of HPV infection, namely persistence progression or resolution. Although several epidemiological studies have stated a positive association involving CT and HPV-related cervical neoplastic lesions and/or cervical cancer (CC), the specific role of this bacterium in the pathogenesis of cervical neoplasia has not been completely clarified. The present review summarizes several studies on CT role in cervical cancer and suggests future research directions on HPV and CT interaction.

The most dreadful mushroom toxins: a review of their toxicological mechanisms, chemical structural characteristics, and treatment.

Mushroom consumption is a worldwide custom that continues to grow in popularity. On the other hand, foraging for wild mushrooms can lead to serious disease and even death if deadly mushrooms are accidentally consumed. Mushroom poisoning is difficult to diagnose and treat since the symptoms are similar to those of other disorders. In terms of chemistry, mushroom poisoning is associated with extraordinarily strong toxins, meaning that isolating and identifying toxins has substantial scientific relevance, especially in understanding the lethal components of toxic mushrooms. Most of these toxins exhibit exceptional physiological features that might help enhance chemistry, biochemistry, physiology, and pharmacology research. Despite the discovery of more than 100 poisons, several dangerous mushrooms remain unexplored. This review covers the chemistry (including chemical structures, complete synthesis, and biosynthesis), as well as the toxicology, namely the toxicokinetics, mechanisms of toxicology, and clinical toxicology of these poisons, in addition to the discussion of the development of their most effective diagnostic and therapeutic strategies with the hopes of spurring additional studies, focusing on individual classes of toxins found in poisonous mushrooms such as amatoxins, gyromitrin, orellanine, and phallatoxins. See also the graphical abstract(Fig. 1).

Is Chlamydia trachomatis related to human papillomavirus infection in young women of southern European population? A self-sampling study.

BACKGROUND: It is well established the role of human papillomavirus (HPV) in cervical cancer (CC) etiology. Chlamydia trachomatis (CT) infection seems to synergize with HPV in CC multistage process. The aim of this study was to assess the correlation of HPV and CT infection in young student women. MATERIAL AND METHODS: Cervicovaginal samples were obtained by self-sampling method from young women (n = 432; mean age 18.0 ± 2.46 years) recruited among the student community and tested for HPV and CT DNA by polymerase chain reaction. RESULTS: In CT positive cases, there is an increased risk of HPV detection [odds ratio (OR) 2.4, 95 % confidence interval (CI) 1.03-6.27, p < 0.037]. The highest rate of coinfection with HPV and CT were observed in the mean age of 20.6 years, and all coinfection cases were found in young women who referred no previous history or symptoms of sexually transmitted infections. Within HPV-positive young women, there was no significant difference between CT and high-risk HPV. CONCLUSIONS: Our results suggest a causal association between HPV and CT infection in young women; infection with this bacterium may be a predisposing factor for subsequent infection with HPV, or vice versa, due to similar mode of sexual transmission, inferring the promising role of CT in CC development. However, the specific question on multistage process of HPV-associated carcinogenesis, which may be affected by CT, remains unclear.

A Novel Approach to Assess Balneotherapy Effects on Musculoskeletal Diseases-An Open Interventional Trial Combining Physiological Indicators, Biomarkers, and Patients' Health Perception.

The present study aimed to evaluate whether a 14-day period of balneotherapy influences the inflammatory status, health-related quality of life (QoL) and quality of sleep, underlying overall health state, and clinically relevant benefits of patients with musculoskeletal diseases (MD). The health-related QoL was evaluated using the following instruments: 5Q-5D-5L, EQ-VAS, EUROHIS-QOL, B-IPQ, and HAQ-DI. The quality of sleep was evaluated by a BaSIQS instrument. Circulating levels of IL-6 and C-reactive protein (CRP) were measured by ELISA and chemiluminescent microparticle immunoassay, respectively. The smartband, Xiaomi MI Band 4, was used for real-time sensing of physical activity and sleep quality. MD patients improved the health-related QoL measured by 5Q-5D-5L (p < 0.001), EQ-VAS (p < 0.001), EUROHIS-QOL (p = 0.017), B-IPQ (p < 0.001), and HAQ-DI (p = 0.019) after balneotherapy; the sleep quality was also improved (BaSIQS, p = 0.019). Serum concentrations of IL-6 were markedly decreased after the 14-day balneotherapy (p < 0.001). No statistically significant differences were observed regarding the physical activity and sleep quality data recorded by the smartband. Balneotherapy may be an effective alternative treatment in managing the health status of MD patients, with a decrease in inflammatory states, along with positive effects on pain reduction, patient's functionality, QoL, quality of sleep, and disability perception status.

Antibiofilm Potential of Medicinal Plants against Candida spp. Oral Biofilms: A Review.

The use of natural products to promote health is as old as human civilization. In recent years, the perception of natural products derived from plants as abundant sources of biologically active compounds has driven their exploitation towards the search for new chemical products that can lead to further pharmaceutical formulations. Candida fungi, being opportunistic pathogens, increase their virulence by acquiring resistance to conventional antimicrobials, triggering diseases, especially in immunosuppressed hosts. They are also pointed to as the main pathogens responsible for most fungal infections of the oral cavity. This increased resistance to conventional synthetic antimicrobials has driven the search for new molecules present in plant extracts, which have been widely explored as alternative agents in the prevention and treatment of infections. This review aims to provide a critical view and scope of the in vitro antimicrobial and antibiofilm activity of several medicinal plants, revealing species with inhibition/reduction effects on the biofilm formed by Candida spp. in the oral cavity. The most promising plant extracts in fighting oral biofilm, given their high capacity to reduce it to low concentrations were the essential oils extracted from Allium sativum L., Cinnamomum zeylanicum Blume. and Cymbopogon citratus (DC) Stapf.

The prevalence and risk-factors of oral HPV DNA detection among HIV-infected men between men who have sex with men and heterosexual men.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal carcinomas are becoming more common with epidemiological impact on human immunodeficiency virus (HIV)- positive individuals. Objective: We evaluated prevalence and risk factors for oral HPV DNA among HIV-infected men who have sex with men (MSM) or heterosexual men. Methods: This cross-sectional hospital-based study included 255 HIV-infected men with different sexual orientation 142 MSM and 113 heterosexual men, who answered a self-administered questionnaire on sociodemographic, clinical and behavioural data. Oral swab and mouthwash samples were analysed by polymerase chain reaction and genotyped by AnyplexTM II 28 (Seegene®). Results: Oral HPV was detected in 17.6% (95% Confidence Interval (CI) 13.5-22.8%), 17.6% in MSM and 17.7% in heterosexual men (p = .984). Multiple HPV infections were detected in 86.7% of HPV-positive men. HPV 56 (13.7%) was the most prevalent high-risk genotype, HPV 66 (7.8%) and HPV 70 (12.3%) were the most prevalent probable HR and low-risk HPV genotypes (12.3% and 7.1%, respectively). At multivariable analysis models, oral HPV was associated with >100 lifetime sexual partners (Odds Ratio (OR) 3.73; 95% CI 1.42-9.77) or lifetime tongue-kissing partners (OR 3.20; 95% CI 1.22-8.39) and lower education level (OR 2.90; 95% CI 1.08-7.78 and 2.74; 95% CI 1.04-7.27, respectively). Conclusions: Oral HPV prevalence was similar between HIV-infected MSM and heterosexual men. Oral HPV was associated with lifetime sexual partners, lifetime tongue-kissing partners and being undergraduate, independently of sexual orientation.

Aggressive prostate cancer phenotype and genome-wide association studies: where are we now?

The majority of prostate cancer (PCa) is indolent, however, a percentage of patients are initially diagnosed with metastatic disease, for which there is a worse prognosis. There is a lack of biomarkers to identify men at greater risk for developing aggressive PCa. Genome-wide association studies (GWAS) scan the genome to search associations of SNPs with specific traits, like cancer. To date, eight GWAS have resulted in the reporting of 16 SNPs associated with aggressive PCa (p < 5.00 × 10-2). Still, validation studies need to be conducted to confirm the obtained results as GWAS can generate false-positive results. Furthermore, post-GWAS studies provide a better understanding of the functional consequences.

Human papillomavirus prevalence and distribution in self-collected samples from female university students in Maputo.

OBJECTIVE: To characterize human papillomavirus (HPV) prevalence and distribution among female university students in Maputo, Mozambique, and evaluate the determinants of HPV infection. METHODS: A cross-sectional study among 504 female university students between February and April 2017. Cervicovaginal self-collected samples were analyzed for HPV genotypes by polymerase chain reaction-restriction fragment length polymorphism and AnyplexTM II HPV28 Detection kit (Seegene® ). RESULTS: The prevalence of any HPV genotype was 28.6% (144/504). Single and multiple HPV infections were detected in 76 (15.1%) and 68 (13.5%) participants, respectively. Prevalence of high-risk HPV was significantly higher than that of low-risk HPV (P<0.001). HPV16 was the most frequent genotype, followed by HPV58, HPV66, HPV52, HPV18, HPV56, HPV61, and HPV70. The prevalence of genotypes covered by the bivalent, quadrivalent, and nonavalent vaccine was 14.3%, 15.9%, and 23.4%, respectively. Number of sexual partners over lifetime and in the past 12 months was associated with HPV infection (P<0.001 and P=0.039, respectively). CONCLUSIONS: Knowledge of HPV genotype-specific prevalence among young women is important to set up strategies for HPV vaccination. The findings suggest that introduction of the nonavalent HPV vaccine might be the way forward in the present low-resource setting. In addition, self-sampling was useful for HPV detection and genotyping.

YB-1 variant and androgen receptor axis-targeted agents in metastatic castration-resistant prostate cancer patients.

Aim: To evaluate the influence of YB-1 rs10493112 variant as a genetic marker for response to second-generation androgen receptor axis-target agents. Methods: A hospital-based cohort study of 78 patients with metastatic castration-resistant prostate cancer was conducted. Genotyping was performed by TaqMan® allelic discrimination technology. Main results: In abiraterone-treated and high-risk patients, YB-1 rs10493112 AA genotype carriers showed lower progression-free survival than C allele genotype patients (4 vs 17 months; p = 0.009). For carriers of AA genotype, multivariate Cox regression analysis revealed a fivefold increased risk of progression (p = 0.035). Conclusion: The study findings suggest that, for metastatic and castration-resistant prostate cancer patients, this polymorphism might be a putative marker for the clinical outcome.

Genital mycoplasmas and ureaplasmas in cervicovaginal self-collected samples of reproductive-age women: prevalence and risk factors.

The purpose of this study was to characterise the prevalence and risk factors associated with genital mycoplasmas ( Mycoplasma hominis [MH], M. genitalium [MG]) and ureaplasmas ( Ureaplasma urealyticum [UU], U. parvum [UP]) in Portuguese women of reproductive age. The cross-sectional study included 612 cervicovaginal self-collected samples from women aged 15-44 years, tested for MH, MG, UU, UP by polymerase chain reaction. Y chromosome (Yc) DNA was detected as a biomarker of recent unprotected sexual intercourse. The prevalences of UU, UP, MH and MG were 28.4% (95% confidence interval [CI] 25.0-32.1), 22.4% (95% CI 19.3-25.9), 8.5% (95% CI 6.5-11.0) and 0.8% (95% CI 0.4-1.9), respectively. Overall, women aged 20-29 years (odds ratio [OR] 1.78; P = 0.010) and the presence of Yc-DNA (OR 2.33; P = 0.038) were associated with an increased risk of UU. Lifetime number of sexual partners was a predictor of UU, UP and MH (OR 2.46; P < 0.001, OR 2.78; P < 0.001 and OR 1.55; P < 0.001, respectively, for more than one versus one partner). The prevalence of MG was low, while UU, UP and MH were common in Portuguese women of reproductive age. The presence of UU, UP and MH was associated with sexual activity (number of sexual partners), although the consequences of its prevalence are not fully understood and should be further investigated.

Acceptability of self-sampling in Portuguese women: the good, the bad or the ugly?

BACKGROUND: Self-sampling is a less costly approach that has been used for human papillomavirus (HPV) testing. METHODS: A cross-sectional study involving 313 Portuguese women assessed the acceptability of cervicovaginal self-sampling. RESULTS: Self-sampling was a well-accepted method [75.7%; 95% confidence interval (CI) 70.5-80.2], and the majority of women felt no pain (67.4%; 95% CI 61.9-72.5), no discomfort (70.9%; 95% CI 65.5-75.8) and no complexity (76.4%; 95% CI 71.2-80.9). The willingness to repeat self-sampling was high (89.5%; 95% CI 85.4-92.5). Compared to physician-sampling, women reported a preference for self-sampling (58.1%; 95% CI 52.5-63.6), as it was more comfortable (67.1%; 95% CI 61.5-72.2) and caused less pain (16.3%; 95% CI 12.5-20.9) and embarrassment (13.4%; 95% CI 9.9-17.8). CONCLUSION: Offering self-sampling for HPV testing may improve screening participation rates and overcome women's embarrassment regarding physician examination.

Y chromosome DNA in cervicovaginal self-collected samples of childbearing age women: Implications for epitheliotropic sexually transmitted infections?

AIMS: Assuming a possible association between Y chromosome (Yc)-DNA and sexually transmitted infection (STI) transmission rate, could Yc-DNA be related to an increased prevalence of Human Papillomavirus (HPV), Herpes Simplex Virus (HSV-1/2) and Chlamydia trachomatis (CT)? Could Yc-DNA be used to validate self-reported condom use and sexual behaviors? MAIN METHODS: Cervicovaginal (CV) self-collected samples of 612 Portuguese women at childbearing age were tested for Yc, HPV, HSV-1/2 and CT by polymerase chain reaction (PCR). KEY FINDINGS: The prevalence of Yc, HPV, CT and HSV-2 was 4.9%, 17.6%, 11.6% and 2.8%, respectively. There was a statistically significant trend for increased Yc-DNA prevalence in HPV positive samples [odds ratio (OR) 2.35, 95% confidence interval (CI) 1.03-5.31] and oral contraceptive (OC) use (OR 4.73, 95% CI 1.09-20.44). A protective effect of condom use was observed in Yc-DNA detection (OR 0.40, 95% CI 0.18-0.89). No statistically significant difference was found between Yc-DNA, CT and HSV-2 infection. HPV infection risk increased with age (>20 years), young age at first sexual intercourse (FSI) (≤18 years), >1 lifetime sexual partner (LSP) and OC use. Risk factors for CT infection were young age (≤20 years) and young age at FSI (≤18 years). HSV-2 infection risk increased with age (>20 years) and >1 LSP. SIGNIFICANCE: Considering the prevalence of HPV and CT in Yc positive samples, we hypothesize a current infection due to recent sexual activity. The study of Yc PCR may add information as (i) a predictor of STI transmission and (ii) an indicative biomarker to validate self-reported condom use.

Oncogenic HPV Types Infection in Adolescents and University Women from North Portugal: From Self-Sampling to Cancer Prevention.

This study aimed to characterize the HPV infection status in adolescents and young university women in Portugal. The distribution of HPV genotypes was evaluated by PCR DNA genotyping after self-sampling collection from 435 women of exfoliated cervical cells using a commercial kit. We observed an overall frequency of HPV infection of 11.5%. Furthermore, HPV DNA prevalence was 16.6% in those young women that self-declared as sexually active. The more frequently detected HPV types were 31, 16, 53, and 61. Statistical analysis identified median age (OR = 3.56; P = 0.001), the number of lifetime sexual partners (OR = 4.50; P < 0.001), and years of sexual activity (OR = 2.36; P = 0.008) as risk factors for HPV acquisition. Hence, our study revealed that oncogenic HPV infection is common in young asymptomatic women Portuguese women, with a history of 2-5 sexual partners and over 2 year of sexual activity. Moreover, these results demonstrate that HPV detection performed in self-collected samples may be important to appraise better preventive strategies and to monitorize the influence of vaccination programmes within different populations.