Gilda Alves

Comparative proteomic analysis of whole saliva from chronic periodontitis patients

Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.

Genetic imbalances in 26 cases of penile squamous cell carcinoma

To obtain more information on chromosomal changes in the up‐to‐now poorly studied tumor class of penile squamous cell carcinoma (SCC), we performed a comparative genomic hybridization study of 26 cases of this rare tumor. DNA sequence copy number alterations (CNAs) very similar to those detected in other SCC types, such as oral and esophageal SCC, were noted. The most common copy number gains were found in 8q24, 16p11–12, 20q11–13, 22q, 19q13, and 5p15, and the most common deletions were detected in 13q21–22, 4q21–32, and along the X chromosome. Classifying the patients according to the number of CNAs showed a possible correlation with clinical outcome.

Analysis of the salivary proteome in gingivitis patients

BACKGROUND AND OBJECTIVE Gingivitis is a disease that is characterized by inflammation of the gingival tissue, which can progress to periodontitis and tooth loss. Although many studies have attempted to identify salivary proteins that are associated with the disease, this is the first study to use a proteomic approach to analyze and compare the proteomic profile of whole saliva from gingivitis patients and healthy controls. MATERIAL AND METHOD To analyze the saliva proteome, two-dimensional gel electrophoresis and liquid chromatography were used, followed by mass spectrometry. RESULTS The analyses showed that gingival inflammation was associated with increased amounts of blood proteins (serum albumin and hemoglobin), immunoglobulin peptides and keratins. In the control group, salivary cystatins, which were detected using capillary Liquid Chromatography on line to electrospray ionization Quadrupole Time-of-flight mass spectrometry, appeared to be more abundant. CONCLUSION This approach provides novel insight into profiles of the salivary proteome during gingival inflammation, which may contribute to improvements in diagnosis.

The prevalence of human cytomegalovirus DNA in gliomas of Brazilian patients

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Renal Cell Carcinoma and Proteomics

Renal cell carcinoma (RCC) represents 3% of adult malignancies. About 30% of RCC patients develop metastatic disease. So far, drugs cannot significantly increase the survival of these patients. We present a recent review of proteomics and RCC. Proteomic technologies have been used in the research to discover new markers of RCC that might increase survival. Furthermore, newly discovered markers cannot increase patient survival, rather their prognostic value supporting therapeutic decisions or new agents targeted at these new markers. More research is required to develop proteomic technologies and biomarkers for identification and validation.

Urine screening by Seldi-Tof, followed by biomarker identification, in a Brazilian cohort of patients with Renal Cell Carcinoma (RCC)

PURPOSE To screen proteins/peptides in urine of Renal Cell Carcinoma (RCC) patients by SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) in search of possible biomarkers. MATERIAL AND METHODS Sixty-one urines samples from Clear Cell RCC and Papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a ProteinChip Reader PCS 4,000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (Clear Cell and Papillary Carcinoma). For identification urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound. RESULTS Proteins that were identified from urine of controls included immunoglobulin light chains, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin are quite common by proteomics and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while vitellite membrane outer layer 1 role is unknown since it was never purified in humans. CONCLUSIONS The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine marker and should be validated in a bigger cohort including the other less common histological RCC subtypes.

DNA Release by Line-1 (L1) Retrotransposon Could It Be Possible?

Abstract: We have verified the presence of line‐1 retrotransposon (L1) in plasma DNA in 15/17 brain tumor (glioma) patients and in 6/6 healthy people by applying PCR amplification of part of the L1 5′ end. The same samples were separately amplified for K‐ras. Results suggested that L1 sequences are circulating throughout the body. We hypothesized the participation of transposable elements such as L1 in a putative DNA release mechanism.

Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma

OBJECTIVE The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry-based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. METHODS AND MATERIALS Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry-based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MS(E) (1D LC-MS(E)), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. RESULTS All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MS(E) quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. CONCLUSIONS The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MS(E) and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.

Lack of association between glutathione S-transferase polymorphisms and primary glioma in a case-control study in Rio de Janeiro.

The glutathione S-transferases (GSTs), a family of phase II isozymes, detoxify several carcinogens. Genetic variations in GSTs have been associated with increased risk for cancer due to a heritable deficiency in detoxification pathways for environmental carcinogens. Conflicting findings have been reported about the association between constitutive GST polymorphisms and gliomas in different populations. The present case-control study examined 78 patients with primary glioma and 347 controls from Rio de Janeiro. DNA was isolated from whole blood, and four genetic polymorphisms (GSTM1, GSTM3, GSTT1, and GSTP1) were determined by PCR-RFLP. The distributions of the genotypic frequencies of these polymorphisms did not differ significantly between cases and controls and were as expected by Hardy-Weinberg equilibrium (P > 0.05). Risk analysis did not show an association between GSTs and primary glioma, suggesting that these polymorphisms do not influence the risk of primary glioma, at least in this population in Rio de Janeiro, Brazil.

Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML) which harbored del(11q23) in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT) from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14) and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23). Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donors, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity.In summary, we report the first case of AML involving XPD and XRCC3 polymorphisms from donor origin following allogeneic stem cell transplantation and highlight the potential need for careful analysis of DNA repair gene polymorphisms in selecting candidate donors prior to allogeneic stem cell transplantation.