Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2.

The occurrence of dengue disease has increased radically in recent decades. Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1/E/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1/E/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1/E/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1/E/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.

Supratracheal laryngectomy: a multi-institutional study / Laringectomia supratraqueal: um estudo multi-institucional

Abstract Introduction: Supratracheal laryngectomy has been described as a surgical procedure for glottic or supraglottic cancer extending to the subglottic region and/or involving the cricoarytenoid joint, aiming to preserve laryngeal function (breathing, phonation and swallowing), without diminishing locoregional cancer control. The choice of supracricoid laryngectomy in these cases could result in a high risk of compromised resection margins. Objective: To determine the safety, viability, adequacy of surgical margins and the supratracheal laryngectomy results for intermediate and advanced laryngeal cancer by reviewing the results at three different institutions in Brazil. Methods: This is a retrospective study that analyzed the charts of 29 patients submitted to supratracheal laryngectomy from October 1997 to June 2017. The type of laryngectomy performed was classified according to the European Laryngological Society classification for horizontal laryngectomies. Early and late results were evaluated. Survival rates (overall, specific, disease-free and total laryngectomy-free survival) were calculated. The mean follow-up time was 44 months. Results: Of the 29 patients submitted to supratracheal laryngectomy, 25 had no previous treatment. One patient (3.4%) had compromised margins. Four patients (13.8%) had recurrence. Of these, three had local recurrence and one had regional recurrence. Five patients (17.2%) required a total laryngectomy, two due to ruptured pexy and three due to local recurrence. Four of these patients (80%) achieved a successful total procedure. Four patients (13.8%) died, two due to postoperative complications and two due to recurrence. Overall, specific, disease-free and total laryngectomy-free survival at 5 years were, respectively, 82.1%; 88.2%; 83.0% and 80.2%. Conclusion: Selected patients with intermediate and advanced laryngeal cancer may benefit from supratracheal laryngectomy, that resulted in total laryngectomy-free survival and specific survival of 80.2% and 88.2%, respectively.

Resumo Introdução: A laringectomia supratraqueal tem sido descrita como um procedimento cirúrgico com objetivo de preservar a função da laringe (respiração, fonação e deglutição), sem prejuízo no controle oncológico locorregional, para câncer glótico ou supraglótico com extensão à subglote e/ou envolvimento da articulação cricoaritenóidea. A opção pela laringectomia supracricoide nesses casos poderia resultar em grande risco para margens de ressecção comprometidas. Objetivo: Determinar a segurança, viabilidade, adequação das margens cirúrgicas e os resultados da laringectomia supratraqueal para o câncer de laringe intermediário e avançado através da revisão dos resultados de três instituições distintas no Brasil. Método: Estudo retrospectivo, com análise dos prontuários de 29 pacientes submetidos à laringectomia supratraqueal, de outubro de 1997 a junho de 2017. O tipo de laringectomia realizada foi classificado de acordo com a classificação da Sociedade Laringológica Europeia para laringectomias horizontais. Foram avaliados os resultados precoces e tardios. As taxas de sobrevida (global, específica, livre de doença e livre de laringectomia total) foram calculadas. O tempo médio de seguimento foi 44 meses. Resultados: Dos 29 pacientes submetidos à laringectomia supratraqueal, 25 não tinham tratamento prévio. Um paciente (3,4%) teve margens comprometidas. Quatro pacientes (13,8%) recidivaram. Desses, três tiveram recidiva local e um apresentou recidiva regional. Cinco pacientes (17,2%) necessitaram de totalização da laringectomia, duas por ruptura da pexia e três por recidiva local. Quatro desses pacientes (80%) obtiveram sucesso na totalização. Quatro pacientes (13,8%) foram a óbito, dois por complicações pós-cirúrgicas e dois por recidiva. As sobrevidas global, específica, livre de doença e livre de laringectomia total em 5 anos foram, respectivamente, 82,1%; 88,2%; 83,0% e 80,2%. Conclusão: Pacientes selecionados com câncer intermediário e avançado de laringe podem ser beneficiados com laringectomia supratraqueal, que ofereceu sobrevida livre de laringectomia total e sobrevida específica de 80,2% e 88,2%, respectivamente.

Supratracheal laryngectomy: a multi-institutional study.

INTRODUCTION: Supratracheal laryngectomy has been described as a surgical procedure for glottic or supraglottic cancer extending to the subglottic region and/or involving the cricoarytenoid joint, aiming to preserve laryngeal function (breathing, phonation and swallowing), without diminishing locoregional cancer control. The choice of supracricoid laryngectomy in these cases could result in a high risk of compromised resection margins. OBJECTIVE: To determine the safety, viability, adequacy of surgical margins and the supratracheal laryngectomy results for intermediate and advanced laryngeal cancer by reviewing the results at three different institutions in Brazil. METHODS: This is a retrospective study that analyzed the charts of 29 patients submitted to supratracheal laryngectomy from October 1997 to June 2017. The type of laryngectomy performed was classified according to the European Laryngological Society classification for horizontal laryngectomies. Early and late results were evaluated. Survival rates (overall, specific, disease-free and total laryngectomy-free survival) were calculated. The mean follow-up time was 44 months. RESULTS: Of the 29 patients submitted to supratracheal laryngectomy, 25 had no previous treatment. One patient (3.4%) had compromised margins. Four patients (13.8%) had recurrence. Of these, three had local recurrence and one had regional recurrence. Five patients (17.2%) required a total laryngectomy, two due to ruptured pexy and three due to local recurrence. Four of these patients (80%) achieved a successful total procedure. Four patients (13.8%) died, two due to postoperative complications and two due to recurrence. Overall, specific, disease-free and total laryngectomy-free survival at 5 years were, respectively, 82.1%; 88.2%; 83.0% and 80.2%. CONCLUSION: Selected patients with intermediate and advanced laryngeal cancer may benefit from supratracheal laryngectomy, that resulted in total laryngectomy-free survival and specific survival of 80.2% and 88.2%, respectively.

Predictive factors for late cervical metastasis in stage I and II squamous cell carcinoma of the lip.

PURPOSE: Many authors have described clinicopathologic parameters as factors related to cervical lymph node metastasis development in CN0 stage lip cancer. However, predictive factors for occult lymph node metastasis and criteria for elective neck dissection, especially for early tumour, remain undefined. METHODS: A multi-institutional study with 193 consecutive patients with early lip SCC treated from January 1990 to March 2006 was carried out retrospectively to determine factors predicting occult metastasis. RESULTS: The overall late LNM rate was 13% (25/193). In the multivariate logistic regression study, tumour size and pattern of tumour invasion were factors related to the occurrence of late LNM with rates of sensitivity, specifity and accuracy for occult LNM prediction of 50%, 89.5% and 87%, respectively. CONCLUSION: Our results indicate that patients with stage I and II SCC of the lip with tumour size greater than 18 mm and more aggressive pattern of invasion must be considered a high-risk group for LNM and an END should be performed.

BALB/c mice infected with DENV-2 strain 66985 by the intravenous route display injury in the central nervous system.

Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.

A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue.

OBJECTIVE: This study aimed to compare a new vitrification protocol with reduced cryoprotectant exposure to the slow freezing method in the cryopreservation of prepubertal rat testicular tissue. METHODS: Five sexually immature male Wistar rats were submitted to bilateral orchiectomy. Tissue samples from each testicle were fragmented into small pieces and randomly assigned to three groups: Group A, fresh tissue (control); Group B, slow programmable freezing (SPF); and Group C (vitrification). Frozen/thawed, vitrified/warmed, and fresh testicular tissue were histologically compared. A pathologist blinded to the procedures assessed the morphology (cell differentiation, nuclei, and epithelium) of 10 seminiferous tubules from each testicle (100 tubules per Group). RESULTS: Sertoli and spermatogonial stem cells were easily differentiated, and the nucleoli were easily viewed in the tubules assessed in all three groups. Small alterations in tissue architecture were observed in the control group as a result of tissue handling. Moderate alterations of the epithelium with the formation of small gaps and cell detachment from the basement membrane were observed in 28% of the frozen and 9% of the vitrified tubules. Condensed nuclei involving a small proportion of cells were observed in six and three tubules of the frozen and vitrified group, respectively. Despite the alterations, 97% of the frozen and 99% of the vitrified tubules were considered well preserved. CONCLUSIONS: The findings indicate that the vitrification protocol tested in this study adequately preserved the morphological integrity of prepubertal testicular tissue in a rat model. Further studies are required to confirm testicular tissue function after grafting.

Viability of homologous and heterologous subcutaneous transplantation of fresh germ cells in rabbits.

OBJECTIVE: This study aimed to compare heterologous to homologous transplantation of fresh ovarian germ cells in rabbits. METHODS: Twelve female white New Zealand rabbits (Oryctolagus cuniculus) were randomly numbered and submitted to bilateral oophorectomies. The ovaries from the six odd-numbered rabbits were dissected and cortical germinal tissue was digested in collagenase type 1 to obtain six solutions containing stromal and germ cells, which were injected in the abdominal region of the odd-numbered rabbits themselves (homologous transplantation) and of the even-numbered rabbits (heterologous transplantation) off immunosuppression. Sixty days after transplantation, the tissue around the transplanted region was excised, processed and sent to histological analysis with hematoxylin-eosin staining and Bcl-2 immunohistochemistry to verify the presence and viability of the transplanted cells. RESULTS: The analyzed specimens contained ovarian stroma, while follicular cells were found in 66.6% of the homologous and in 60% of the heterologous transplant specimens. Mild inflammatory reaction was observed in all heterologous specimens, and in only one (16.7%) of the homologous specimens. However, this inflammatory reaction was not so intense as to cause the death of the implanted cells. Except for the specimens from rabbits 7 and 8, all specimens were stained for Bcl-2, indicating that most of them were viable. CONCLUSIONS: The results of this study supported the viability of heterologous transplantation of fresh ovarian germ cells. However, more studies are required to further our understanding and improve the germ cell separation technique.

Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route.

Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.

Prevalence of human papillomavirus types and variants and p16(INK4a) expression in head and neck squamous cells carcinomas in São Paulo, Brazil.

BACKGROUND: Human papillomavirus (HPV) prevalence in head and neck squamous cell carcinomas (HNSCC) diverges geographically. The reliability of using p16(INK4a) expression as a marker of viral infection is controversial in HNSCC. We evaluated HPV types and HPV-16 variants prevalence, and p16(INK4a) expression in HNSCC specimens provided by two different Institutions in São Paulo. METHODS: HPV DNA from formalin-fixed specimens was accessed by Inno-LiPA, HPV-16 variants by PCR-sequencing, and p16(INK4a) protein levels by immunohistochemistry. RESULTS: Overall, HPV DNA was detected among 19.4 % of the specimens (36/186). Viral prevalence was higher in the oral cavity (25.0 %, 23/92) then in other anatomical sites (oropharynx 14,3 %, larynx 13.7 %) when samples from both Institutions were analyzed together. HPV prevalence was also higher in the oral cavity when samples from both Institutions were analyzed separately. HPV-16 was the most prevalent type identified in 69.5 % of the HPV positive smaples and specimens were assigned into Asian-American (57.2 %) or European (42.8 %) phylogenetic branches. High expression of p16(INK4a) was more common among HPV positive tumors. CONCLUSION: Our results support a role for HPV-16 in a subset of HNSCC.

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge.

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant.

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LT(G33D)), originally produced by some enterotoxigenic Escherichia coli (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LT(G33D). Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LT(G33D) elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LT(G33D.) Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LT(G33D). Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine.

Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

Design of a complex bioimpedance spectrometer using DFT and undersampling for neural networks diagnostics.

Electrical impedance spectroscopy offers many applications in the medical field due the fast response, non-invasiveness and low cost. One promising area is the use of this method for diagnostics. This paper describes the design and experimental evaluation of a multifrequencial complex bioimpedance analyzer. Impedance amplitude and phase were calculated using Discrete Fourier Transform (DFT) and high frequency signals were measured with undersampling. The prototype was able to measure values from 1 Ω to 50 kΩ (frequency range from 50 Hz to 500 kHz). The accuracy of the technique was compared with a commercial equipment. The analysis of passive components resulted in a mean error of 2.9% for the magnitude and 0.69 degrees for the phase. Besides, an initial study for head and neck cancer detection through neural networks is shown. One used bioimpedance values as well as gender, age and body mass index as inputs. The network used 120 training and 40 validation data and was able to simulate 77.5% of the two types of diagnostic correctly.

Epidemiological and histopathological data and E-cadherin-like prognostic factors in early carcinomas of the tongue and floor of mouth.

Despite recent advances in diagnosis and treatment, overall survival of people with squamous cell carcinomas of the tongue and mouth floor continues to be low. Prognostic information is essential for improved evaluation and treatment. A molecular approach is necessary for a better understanding of cancer. We analyzed 50 patients with early squamous cell carcinoma (stages I and II - AJCC, 1997), who were surgically treated in head and neck departments at Heliopolis Hospital and Santa Casa in São Paulo, Brazil. We searched for epidemiological and pathological behaviors that could be involved in tumor recurrence and the influence of cellular adhesion loss in patient outcome. We established two groups: patients with disease recurrence within 3 years of follow-up and patients with no cancer recurrence in this period. Significant prognostic variables included age, daily alcohol intake, neural invasion and maximum tumor thickness. Our findings are similar to other published data in which younger patients have a better prognosis. The odds ratio for neural invasion was low but was significant for thickness. No significant difference was observed among groups in E-cadherin expression. Our data suggest that 3 and 5mm are the prognostic determinants of thickness. E-cadherin mutation was not related to recurrences but was associated with locally aggressive invasion patterns.

New administration model of trans-chalcone biodegradable polymers for the treatment of experimental leishmaniasis.

The present study was designed to investigate a new administration model and the antileishmanial activity of a semi-synthetic chalcone, benzylideneacetophenone (trans-chalcone). The antileishmanial activity of this product was first tested in vitro against promastigotes of L. braziliensis, L. tropica, L. infantum and L. amazonensis. An in vivo experiment was carried out using subcutaneous administration of trans-chalcone and implants of synthetic biodegradable polymers, polylactic acid (PLA) and polylactic/glycolic acid (PLGA). This compound showed potent inhibitory effects on the growth of all Leishmania strains examinated. Subcutaneous administration of trans-chalcone at a single dose of 4 mg/kg of body weight reduced lesion development in mice infected with L. amazonensis. A similar inhibition of the lesion growth in mice treated with trans-chalcone and pentamidine was observed. PLA and PGLA implants of trans-chalcone at 4 mg/kg were administered to mice infected with L. amazonensis. PLGA implants induced a highest reduction in the lesion size (31.25%) than PLA implants (10.75%). Treatment in vitro with trans-chalcone at IC50, completely inhibited the pathogenicity of this parasite in vivo. The development of this model provides a new practical technique for delivering drugs and can be useful for experimental leishmaniasis treatment.

Polymerase chain reaction screening for fungemia and/or invasive fungal infections in patients with hematologic malignancies.

INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

A nine-year institutional experience with near-total laryngectomy.

BACKGROUND: Pearson's near-total laryngectomy (NTL) is an alternative procedure to total laryngectomy in selected patients with advanced laryngeal cancer. Based on our experience with NTL for >9 years, we present here the functional results, complications, and survival rates. METHODS: A retrospective study was carried out from January 1993 to May 2002. We studied 15 patients with advanced laryngeal, oropharyngeal, and hypopharyngeal cancer who underwent NTL. Survival rates were calculated using the Kaplan-Meier method. RESULTS: The most common complication was fistula (8 of 15) followed by minor aspiration (4 of 15 patients). Eleven patients (73.5%) attained a good voice; 3 patients (19.9%) obtained a bad voice; and 1 did not achieved vocal ability. Three patients (19.9%) had local recurrence; no patients had neck recurrence; and 2 patients (13.3%) had distant metastasis. Six patients (40%) died from their disease, and 2 (13.3%) patients died from other causes. The 3-year actuarial survival rate was 81.6%. CONCLUSIONS: NTL is useful in the treatment of selected patients with advanced laryngeal, oropharyngeal, and hypopharyngeal cancer and results in good control and survival rates. Satisfactory functional results can be attained in the majority of patients. When the surgical margins are positive or close, TL must be carried out.

Effects of amidine derivatives on parasite-macrophage interaction and evaluation of toxicity.

In this work, the effect of amidine derivatives (with bromine and methoxy as substituents) in the "in vitro" parasite-macrophage interaction was evaluated. The potential toxicity was also analyzed. The results show that the methoxy-derivative was able to decrease the percentage of "in vitro" infection, being not hazardous to the host cell. Furthermore, experiments using Balb/c mice showed that this compound was very effective in avoiding infection in these animals. On the other hand, the compound with bromine as substituent was toxic to macrophages and unable to prevent infection in Balb/c mice. Pentamidine isethionate, used as reference drug, was not efficient in both experiments.