Antonio Carlos de Freitas

Susceptibility to cervical cancer: an overview.

Cervical cancer is the second most common cancer in females worldwide. It is well-established that Human Papillomavirus (HPV) infections play a critical role in the development of cervical cancer. However, a large number of women infected with oncogenic HPV types will never develop cervical cancer. Thus, there are several external environment and genetic factors involved in the progression of a precancerous lesion to invasive cancer. In this review article, we addressed possible susceptible phenotypes to cervical cancer, focusing on host genome and HPV DNA variability, multiple HPV infections, co-infection with other agents, circulating HPV DNA and lifestyle.

Detection of Bovine Papillomavirus Types, Co-Infection and a Putative New BPV11 Subtype in Cattle

The aim of this study was to evaluate the presence of different types of Bovine papillomavirus (BPV) in cattle skin lesions and to identify new viral types in Brazil. A total of 72 skin lesions were analysed from 66 different bovines by PCR using degenerate and specific primers, and subsequent sequencing. Sequencing quality was determined using Staden package with Phred 30. Similarity analysis was performed with BioEdit and BLAST programs to verify the identity with known BPV types. Phylogenetic analysis was carried out using Maximum Likelihood method with TIM3 + G as nucleotide substitution model in PAUP*, and 1000 non-parametric bootstrap replicates. Analyses revealed the presence of ten different types of BPV in the samples, with the exception of BPV7. The presence of co-infections was very high as almost all samples (89%) were co-infected. A putative new BPV11 subtype was also found in lesions from different animals. These results add significant knowledge about the prevalence and diversity of BPV infection in Brazilian cattle, which could be used in future studies aiming at the development of more specific treatment and diagnostic methods.

Molecular targets of HPV oncoproteins: potential biomarkers for cervical carcinogenesis.

Cervical cancer is the second most common cancer among women worldwide and is responsible for 275,000 deaths each year. Persistent infection with high-risk human papillomavirus (HR-HPV) is an essential factor for the development of cervical cancer. Although the process is not fully understood, molecular mechanisms caused by HPV infection are necessary for its development and reveal a large number of potential biomarkers for diagnosis and prognosis. These molecules are host genes and/or proteins, and cellular microRNAs involved in cell cycle regulation that result from disturbed expression of HR-HPV E5, E6 and E7 oncoproteins. One of the current challenges in medicine is to discover potent biomarkers that can correctly diagnose cervical premalignant lesions and standardize clinical management. Currently, studies are showing that some of these molecules are potential biomarkers of cervical carcinogenesis, and it is possible to carry out a more accurate diagnosis and provide more appropriate follow-up treatment for women with cervical dysplasia. In this paper, we review recent research studies on cell cycle molecules deregulated by HPV infections, as well as their potential use for cervical cancer screening.

Circulating cell-free DNA in serum as a biomarker of colorectal cancer.

Aims The aim of this study was to report circulating cell-free DNA using ALU247 and ALU247/ALU115 biomarkers in serum of operated and non-operated patients with colorectal cancer (CRC). Methods To undertake this, 90 blood samples were collected, including 30 samples from healthy volunteers; 27 samples from CRC non-operated patients and 33 samples from CRC-operated patients. Circulating cell-free DNA was verified through quantitative real-time PCR (qPCR) using ALU115 and ALU247 primers. Results With regard to the ALU115-qPCR biomarker, the increased levels of circulating cell-free DNA in serum of non-operated patients were significant when compared with control (p<0.05). Moreover, levels of ALU247-qPCR biomarker were statistically significant between non-operated versus operated and non-operated versus control groups (p=0.000). With regard to the ALU247/115-qPCR biomarker, significant differences were observed between control versus non-operated patients (p=0.019), operated versus non-operated patients (p=0.005) and control versus operated patients (p=0.043). Conclusions Thus, the ALU247 and ALU247/ALU115-qPCR biomarkers may be important in detecting and monitoring CRC patients in both early and late stages.

Experimental infection by Toxoplasma gondii using contaminated semen containing different doses of tachyzoites in sheep

Toxoplasmosis is a parasitic disease caused by Toxoplasma gondii that affects reproductive performance in small ruminants. Although the T. gondii life cycle is well understood since 1960s, several aspects related to its infection remain unclear. In the present study we hypothesized that sheep inseminated with T. gondii-contaminated semen would develop toxoplasmosis. In order to test that hypothesis, 41 sheep were experimentally infected with semen spiked with the organism. Females were divided in three groups (G1-G3): (a) females in G1 group were inseminated with semen containing 6.5 x 10(4) tachyzoites; (b) females in G2 group with semen containing 4 x 10(7) tachyzoites; and (c) females in G3 group with tachyzoite-free semen (control group). To confirm T. gondii infection via semen, serological tests were performed using indirect immunofluorescence reaction and the detection of parasite DNA in the blood stream using the nested PCR test. While in G1 group only 5/15 (33.3%) of the females presented seroconversion, all sheep in G2 15/15 (100%) seroconverted. The nested PCR test showed that 14/15 (93.3%) of the females in the G1 and 14/15 (93.3%) in the G2 group were positive for T. gondii while in the G3 group all samples were negative. In addition, ultra-sound test evidenced that in sheep presented embryonic reabsorption in animals from the infected groups. In conclusion, insemination using fresh semen experimentally contaminated with different infectant doses of T. gondii tachyzoites was able to infect sheep, leading to the possibility of toxoplasmosis transmission via semen.

Human Papillomavirus Vertical Transmission: Review of Current Data

Despite the increasing evidence of human papillomavirus (HPV) vertical transmission, this route is regarded as less clinically important because of the detections of transient HPV DNA. However, recent studies have provided clear evidence of papillomavirus productive infection in lymphocytes, placenta, and bovine fetal tissue. Furthermore, a model of papillomavirus latency has been recently proposed that could explain the failure or transience in HPV detection observed in some infected infants. This new evidence of hematogeneous and vertical spread of HPV suggests that these modes of transmission should be investigated in greater detail to obtain a better understanding of the infection and a fuller awareness of the preventive measures that can be taken against HPV-related diseases.

HPV31 and HPV33 incidence in cervical samples from women in Recife, Brazil.

Human papillomavirus (HPV) has been extensively studied concerning genomic structure, infection mechanisms, and diversity of types, as well as disease progression stages and development of vaccines. HPV type prevalence can differ in specific populations in different countries, according to ethnicity. This is the first report of an integrated project to evaluate the incidence of HPV types in different regions in Brazil in order to obtain data for vaccine development. Cervical samples were collected from women seen at a public hospital in Pernambuco, Northeast Brazil, for routine evaluation of genital alterations. Selection of the patients was random. There was a strong prevalence of HPV16 and a high incidence of HPV types 31 and 33. These data foster the discussion about the need to evaluate viral prevalence in each geographic region in order to develop targeted vaccine programs.

Association between MBL2 gene functional polymorphisms and high-risk human papillomavirus infection in Brazilian women

We studied the association between high-risk human papillomavirus (HPV) infection and MBL2 functional polymorphisms in a group of 180 high-risk HPV-infected women and 180 healthy control subjects. The most frequent high-risk HPV genotypes were 16 (47.2%), 31 (11.7%), 33 (5%), and 18 (2.2%), respectively. Of the 180 HPV-infected women, 99 presented with uterine cervical cancer and 81 did not. No differences in MBL2 genotype or in allelic or haplotype frequencies were found between HPV patients who developed cervical uterine cancer and those who did not. When considering combined genotypes grouped according to MBL production (designated as high, low, and deficient producers), we detected a significant difference between healthy controls and high-risk HPV-positive patients, the latter group showing increased frequencies of deficient-producer genotypes (14.4% vs 9.4% in the healthy control group, corrected p = 0.04). In conclusion, a correlation between MBL2 polymorphisms and high-risk HPV infection was found in this study.

Molecular epidemiology of bovine papillomatosis and the identification of a putative new virus type in Brazilian cattle

Bovine papillomaviruses (BPVs) are a diverse group of double-stranded DNA viruses, of which 12 viral types have been detected and characterized so far. However, there is still a limited understanding of the diversity of BPV. Several putative new BPVs have been detected and some of these have been recently characterized as new viral types. However, only a very limited amount of information is available on the pathology associated with these novel viral types yet this information could be of significant value in improving our understanding of the biology of BPV. The objective of this study was to examine some of the epidemiological features of cutaneous bovine papillomatosis in Brazilian cattle, in particular to establish the relationship between BPV types isolated from beef and dairy cattle herds and the lesions they cause. Seventy-two cutaneous lesions were collected from 60 animals. Histopathological, PCR and sequencing assays were conducted to characterize the lesions and detect the BPV types responsible. Phylogenetic analysis was carried out using the maximum likelihood method. BPV types 1-6 and 8-10 were found, as well as a putative new BPV type that belongs to the Deltapapillomavirus genus. The tumors were all classified as fibropapillomas. This is believed to be the first record of BPV types 3 and 10 associated with fibropapillomas. These results confirm that there is a wide range of BPV types that infect cattle, and that an understanding of this diversity is necessary for improved methods of therapeutic treatment.

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.