Long-term impact of hypothyroidism during gestation and lactation on the mammary gland.

The functional differentiation of the mammary gland (MG) is fundamental for the prevention of mammary pathologies. This process occurs throughout pregnancy and lactation, making these stages key events for the study of pathologies associated with development and differentiation. Many studies have investigated the link between mammary pathologies and thyroid diseases, but most have ignored the role of thyroid hormone (TH) in the functional differentiation of the MG. In this work, we show the long-term impact of hypothyroidism in an animal model whose lactogenic differentiation occurred at low TH levels. We evaluated the ability of the MG to respond to hormonal control and regulate cell cycle progression. We found that a deficit in TH throughout pregnancy and lactation induces a long-term decrease in Rb phosphorylation, increases p53, p21, Cyclin D1 and Ki67 expression, reduces progesterone receptor expression, and induces nonmalignant lesions in mammary tissue. This paper shows the importance of TH level control during mammary differentiation and its long-term impact on mammary function.

Identification of Immunodominant Antigens From a First-Generation Vaccine Against Cutaneous Leishmaniasis.

Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.

Identification of immunodominant antigens from a first-generation vaccine against cutaneous Leishmaniasis

Leishmaniasis is a neglected tropical disease (NTD) caused by parasites belonging to the Leishmania genus for which there is no vaccine available for human use. Thus, the aims of this study are to evaluate the immunoprotective effect of a first-generation vaccine against L. amazonensis and to identify its immunodominant antigens. BALB/c mice were inoculated with phosphate buffer sodium (PBS), total L. amazonensis antigens (TLAs), or TLA with Poly (I:C) and Montanide ISA 763. The humoral and cellular immune response was evaluated before infection. IgG, IgG1, and IgG2a were measured on serum, and IFN-γ, IL-4, and IL-10 cytokines as well as cell proliferation were measured on a splenocyte culture from vaccinated mice. Immunized mice were challenged with 104 infective parasites of L. amazonensis on the footpad. After infection, the protection provided by the vaccine was analyzed by measuring lesion size, splenic index, and parasite load on the footpad and spleen. To identify immunodominant antigens, total proteins of L. amazonensis were separated on 2D electrophoresis gel and transferred to a membrane that was incubated with serum from immunoprotected mice. The antigens recognized by the serum were analyzed through a mass spectrometric assay (LC-MS/MS-IT-TOF) to identify their protein sequence, which was subjected to bioinformatic analysis. The first-generation vaccine induced higher levels of antibodies, cytokines, and cell proliferation than the controls after the second dose. Mice vaccinated with TLA + Poly (I:C) + Montanide ISA 763 showed less footpad swelling, a lower splenic index, and a lower parasite load than the control groups (PBS and TLA). Four immunodominant proteins were identified by mass spectrometry: cytosolic tryparedoxin peroxidase, an uncharacterized protein, a kinetoplast-associated protein-like protein, and a putative heat-shock protein DNAJ. The identified proteins showed high levels of conserved sequence among species belonging to the Leishmania genus and the Trypanosomatidae family. These proteins also proved to be phylogenetically divergent to human and canine proteins. TLA + Poly (I:C) + Montanide ISA 763 could be used as a first-generation vaccine against leishmaniasis. The four proteins identified from the whole-protein vaccine could be good antigen candidates to develop a new-generation vaccine against leishmaniasis.

Effects of thyroxine on apoptosis and proliferation of mammary tumors.

Hypothyroidism is a protective factor against breast cancer but long-term exposure or overdoses of thyroid replacement therapy with thyroxine (T4) may increase breast cancer risk. OBJECTIVE: to study, in vivo and in vitro, the effects of T4 on the proliferation and apoptosis of mammary tumors of hypo- and euthyroid rats, and the possible mechanisms involved in these effects. MATERIAL AND METHODS: Female Sprague-Dawley rats were treated with a single dose of dimethylbenzathracene (15 mg/rat) at 55 days of age and were divided into three groups: hypothyroidism (HypoT; 0.01% 6-N-propyl-2-thiouracil -PTU- in drinking water, n = 20), hypothyroidism treated with T4 (HypoT + T4; 0.01% PTU in drinking water and 0.25 mg/kg/day T4 via sc; n = 20) and EUT (untreated control, n = 20). At sacrifice, tumor explants from HypoT and EUT rats were obtained and treated either with 10-10 M T4 in DMEM/F12 without phenol red with 1% Charcoalized Fetal Bovine Serum or DMEM/F12 only for 15 min to evaluate intracellular signaling pathways associated with T4, and 24 h to evaluate changes in the expression of hormone receptors and proteins related to apoptosis and proliferation by immunohistochemistry and Western Blot. RESULTS: In vivo, hypothyroidism retards mammary carcinogenesis but its treatment with T4 reverted the protective effects. In vitro, the proliferative and anti-apoptosis mechanisms of T4 were different regarding the thyroid status. In EUT tumors, the main signaling pathway involved was the cross-talk with other receptors, such as ERα, PgR, and HER2. In HypoT tumors, the non-genomic signaling pathway of T4 was the chief mechanism involved since αvß3 integrin, HER2, ß-catenin and, downstream, PI3K/AKT and ERK signaling pathways were activated. CONCLUSION: T4 can regulate mammary carcinogenesis by mainly activating its non-genomic signaling pathway and by interacting with other hormone or growth factor pathways endorsing that overdoses of thyroid replacement therapy with T4 can increase the risk of breast cancer.

Pro-Tumorigenic Macrophage Infiltration in Oral Squamous Cell Carcinoma and Possible Macrophage-Aimed Therapeutic Interventions.

In Oral Squamous Cell Carcinomas (OSCC), as in other solid tumors, stromal cells strongly support the spread and growth of the tumor. Macrophages in tumors (tumor-associated macrophages or "TAMs"), can swing between a pro-inflammatory and anti-tumorigenic (M1-like TAMs) state or an anti-inflammatory and pro-tumorigenic (M2-like TAMs) profile depending on the tumor microenvironment cues. Numerous clinical and preclinical studies have demonstrated the importance of macrophages in the prognosis of patients with different types of cancer. Here, our aim was to review the role of M2-like TAMs in the prognosis of patients with OSCC and provide a state of the art on strategies for depleting or reprogramming M2-like TAMs as a possible therapeutic solution for OSCC. The Clinical studies reviewed showed that higher density of CD163+ M2-like TAMs associated with worse survival and that CD206+ M2-TAMs are involved in OSCC progression through epidermal growth factor (EGF) secretion, underlining the important role of CD206 as a marker of OSCC progression and as a therapeutic target. Here, we provide the reader with the current tools, in preclinical and clinical stage, for depleting M2-like TAMs, re-educating them towards M1-like TAMs, and exploiting TAMs as drug delivery vectors.

SPINK7 expression changes accompanied by HER2, P53 and RB1 can be relevant in predicting oral squamous cell carcinoma at a molecular level.

The oral squamous cell carcinoma (OSCC), which has a high morbidity rate, affects patients worldwide. Changes in SPINK7 in precancerous lesions could promote oncogenesis. Our aim was to evaluate SPINK7 as a potential molecular biomarker which predicts OSCC stages, compared to: HER2, TP53, RB1, NFKB and CYP4B1. This study used oral biopsies from three patient groups: dysplasia (n = 33), less invasive (n = 28) and highly invasive OSCC (n = 18). The control group consisted of clinically suspicious cases later to be confirmed as normal mucosa (n = 20). Gene levels of SPINK7, P53, RB, NFKB and CYP4B1 were quantified by qPCR. SPINK7 levels were correlated with a cohort of 330 patients from the TCGA. Also, SPINK7, HER2, TP53, and RB1, were evaluated by immunohistofluorescence. One-way Kruskal-Wallis test and Dunn's post-hoc with a p < 0.05 significance was used to analyze data. In OSCC, the SPINK7 expression had down regulated while P53, RB, NFKB and CYP4B1 had up regulated (p < 0.001). SPINK7 had also diminished in TCGA patients (p = 2.10e-6). In less invasive OSCC, SPINK7 and HER2 proteins had decreased while TP53 and RB1 had increased with respect to the other groups (p < 0.05). The changes of SPINK7 accompanied by HER2, P53 and RB1 can be used to classify the molecular stage of OSCC lesions allowing a diagnosis at molecular and histopathological levels.

The epigenetic role of breastfeeding in mammary differentiation.

Maternal milk consumption can cause changes in the mammary epithelium of the offspring that result in the expression of molecules involved in the induction of differentiation, reducing the risk of developing mammary cancer later in life. We previously showed that animals that maintained a higher intake of maternal milk had a lower incidence of mammary cancer. In the present study, we evaluated one of the possible mechanisms by which the consumption of maternal milk could modify the susceptibility to mammary carcinogenesis. We used Sprague Dawley rats reared in litters of 3 (L3), 8 (L8), or 12 (L12) pups per mother in order to generate a differential consumption of milk. Whole mounts of mammary glands were performed to analyze the changes in morphology. Using real-time polymerase chain reaction (PCR), we analyzed the expression of mammary Pinc, Tbx3, Stat6, and Gata3 genes. We use the real-time methylation-specific polymerase chain reaction method to assess the methylation status of Stat6 and Gata3 CpG sites. Our findings show an increase in the size of the epithelial tree and a smaller number of ducts called terminal end buds in L3 vs. L12. We observed an increased expression of mRNA of Stat6, Gata3, Tbx3, and a lower expression of Pinc in L3 with respect to L12. Stat6 and Gata3 are more methylated in the CpG islands of the promoter analyzed in L12 vs. L3. In conclusion, the increased consumption of maternal milk during the postnatal stage generates epigenetic and morphological changes associated with the differentiation of the mammary gland.

The Chemical Compositions of Essential Oils Derived from Cryptocarya alba and Laurelia sempervirens Possess Antioxidant, Antibacterial and Antitumoral Activity Potential.

Cryptocarya alba (Peumo; CA) and Laurelia sempervirens (Laurel; LS) are herbs native to the Chilean highlands and have historically been used for medicinal purposes by the Huilliches people. In this work, the essential oils were extracted using hydrodistillation in Clevenger apparatus and analyzed by GC-MS to determine their composition. The antioxidant capacity (AC) was evaluated in vitro. The cytotoxicity was determined using cell line cultures both non tumoral and tumoral. The toxicity was determined using the nematode Caenorhabditis elegans. The antimicrobial activity was evaluated against 52 bacteria using the agar disc diffusion method and the minimum inhibitory concentrations (MICs) were determined. The principal compounds found in C. alba essential oil (CA_EO) were α-terpineol (24.96%) and eucalyptol (21.63%) and were isazafrol (91.9%) in L. sempervirens essential oil (LS_EO). Both EOs showed antioxidant capacity in vitro. Both EO showed antibacterial activity against bacteria using. LS_EO showed more inhibitory effect on these cell lines respect to CA_EO. Both EOs showed toxicity against the nematode C.elegans at 3.12-50 mg/mL. The essential oils of CA and LS have an important bioactive potential in their antioxidant, antibacterial and cytotoxicity activity. Both essential oils could possibly be used in the field of natural medicine, natural food preservation, cosmetics, sanitation and plaguicides among others.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis.

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Validação do diagnóstico autodeclarado de depressão em participantes da Coorte de Universidades Mineiras (Projeto CUME) / Validation of the self-reported diagnosis of depression in participants of the Cohort of Universities of Minas Gerais (CUME Project)

Introdução: A depressão é um importante problema de saúde pública, sendo considerada a principal causa de incapacidade globalmente. A prevalência desta doença, em 2015, foi estimada em 4,4%, correspondendo a 322 milhões de casos em todo mundo. No Brasil, estima-se que 5,8% da população tenha depressão. Além da incapacidade provocada pela doença, ela está associada à maior ocorrência de suicídio e também é fator de risco para o desenvolvimento de doenças crônicas não transmissíveis. Nesse contexto, a Coorte de Universidades Mineiras (CUME) tem, dentre um dos seus objetivos, avaliar a relação da depressão com hábitos de vida e desfechos crônicos de saúde em egressos de universidades federais do Estado de Minas Gerais. Entretanto, o diagnóstico de depressão tem sido autodeclarado pelos participantes, necessitando ser validado para melhoria da qualidade das evidências científicas produzidas pelo referido projeto. Objetivo: Validar o diagnóstico autodeclarado de depressão de participantes do projeto CUME. Métodos: Participaram deste estudo transversal, 79 pessoas que responderam ao primeiro questionário de seguimento do projeto, entre março e agosto de 2018 (43 com e 36 sem o autorrelato de depressão). Uma equipe de quatro psiquiatras aplicou o DSM5, utilizando como referência a entrevista clínica estruturada para os transtornos mentais (SCID-5-CV), em consulta presencial dos participantes entre outubro e novembro de 2019. Foram calculados o percentual de concordância, sensibilidade, especificidade, valor preditivo positivo (VPP) e valor preditivo negativo (VPN) entre os diagnósticos autodeclarado de depressão e aquele confirmado pelo psiquiatra, além da aplicação do teste Kappa. Também, avaliou-se os percentuais de falsos positivos e negativos produzidos pelo diagnóstico autodeclarado em relação àquele diagnosticado pelo psiquiatra. Resultados: A maioria dos participantes era do sexo feminino (82,3%), adulto jovem (60,8% entre 20 e 39 anos), sem união estável (54,4%), com pós-graduação (75,9%) e trabalhadores em atividade (72,2%), não fumantes (69,6%), inativos/insuficientemente ativos (69,6%). Ainda, altas proporções dos participantes referiram consumo pesado episódico de bebidas alcoólicas (36,7%) e ingestão inadequada de carboidratos (50,6%) e de lipídios (78,5%). Não houve diferenças estatísticas para as variáveis de acordo com o autorrelato ou não de depressão. A concordância entre os diagnósticos de depressão foi de 81,0%, com sensibilidade de 80,6%, especificidade de 81,4%, VPP de 78,4%, VPN de 83,3% e valor Kappa de 0,62. Adicionalmente, foram observados 18,6% e 19,4%, respectivamente, de falsos positivos e de falsos negativos. Conclusão: O diagnóstico autodeclarado de depressão pelos participantes do projeto CUME apresenta boa acurácia, sendo válido para utilização em estudos sobre esse desfecho em saúde nesta população.

Introduction: Depression is an important public health problem, being globally considered to be the main cause of incapability. The prevalence of this disease, in 2015, was estimated in 4.4%, corresponding to 322 million cases all around the world. In Brazil, it is estimated that 5.8% of the population suffer from depression. Besides the incapability caused by the disease, it is associated with a greater occurrence of suicide and it is also a risk factor for the development of noncommunicable diseases. In this context, the Cohort of Universities of Minas Gerais (CUME) has, as one of its objectives, to evaluate the relationship between depression, lifestyle and chronic health outcomes in federal university graduates in the state of Minas Gerais. However, the depression diagnoses have been selfdeclared by the participants, making it necessary to be validated in order to increase the quality of the scientific evidences produced by that project. Objective: To validate the self-declared diagnoses of depression given by participants in the CUME project. Methods: In this cross-sectional study, were included 79 participants who answered to the project's follow-up questionnaire, between March and August of 2018 (43 with and 36 without self-report depression). A team of four psychiatrists applied the DSM5, using the structured clinical interview for mental disorders (SCID-5-CV) as a reference, during appointments in person with the participants between October and November of 2019. The percentage of agreement, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) have been calculated between the self-reported depression diagnoses and those confirmed by the psychiatrists, besides the Kappa test application. The percentage of false positives and false negatives produced by the self-reported diagnoses in relation to those given by the psychiatrists has also been calculated. Results: The majority of the participants were female (82.3%), young adult (60.8% between 20 and 39 years of age), with no common-law marriage (54%), with postgraduate studies (75.9%) and currently employed (72.2%), non-smokers (69.6%), sedentary/insufficiently physically active (69.6%). In addition, high proportions of participants reported binge drinking (36.7%) and inadequate ingestion of carbohydrates (50.6%) and lipids (78.5%). There were no statistical differences for variables according to self-report of depression or non-depression. The agreement between the diagnose of depression was 81.0% with sensitivity of 80.6%, specificity of 81.4%, PPV of 78,4%, NPV of 83,3% and Kappa value of 0.62. Additionally, 18.6% and 19.4% of false positive and false negative were observed, respectively. Conclusion: The depression diagnoses self-declared by the participants of the CUME project presents good accuracy, being valid for utilization in studies about this health outcome in this population.

Human renal adipose tissue from normal and tumor kidney: its influence on renal cell carcinoma.

Tumor cells can interact with neighboring adipose tissue. We evaluated components present in human adipose explants from normal (hRAN) and kidney cancer (hRAT) tissue, and we evaluated the effects of conditioned media (CMs) from hRAN and hRAT on proliferation, adhesion and migration of tumor and non-tumor human renal epithelial cell lines. In addition, we evaluated the expression of AdipoR1, ObR, CD44, vimentin, pERK and pPI3K on cell lines incubated with CMs. hRAN were obtained from healthy operated donors, and hRAT from patients who underwent a nephrectomy. hRAT showed increased levels of versican, leptin and ObR; and decreased levels of perilipin, adiponectin and AdipoR1, compared to hRAN. Cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRAT-CMs vs. hRAN- or control-CMs. Surprisingly, HK-2, 786-O and ACHN cells showed a significant decrease in cell migration after incubation with hRAN-CMs vs. control-CMs. No difference in proliferation of cell lines was found after 24 or 48 h of treatment with CMs. AdipoR1 in ACHN and Caki-1 cells decreased significantly after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. ObR and CD44 increased in tumor line cells, and vimentin increased in non-tumor cells, after incubation with hRAT-CMs vs. hRAN-CMs and control-CMs. We observed an increase in the expression of pERK and pPI3K in HK-2, 786-O and ACHN, incubated with hRAT-CMs. In conclusion, results showed that adipose microenvironment can regulate the behavior of tumor and non tumor human renal epithelial cells.

Systemically administered allogeneic mesenchymal stem cells do not aggravate the progression of precancerous lesions: a new biosafety insight.

BACKGROUND: Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal cells currently tested for multiple therapeutic purposes. Their potential to home into tumors, to secrete trophic/vasculogenic factors, and to suppress immune response raises questions regarding their biosafety. Our aim was to evaluate whether systemically administered allogeneic MSCs modify the natural progression of precancerous lesions and whether their putative effect depends on cancer stage and/or cell dose. METHODS: Oral squamous cell carcinoma (OSCC) was induced in Syrian golden hamsters by topical application of 7,12-dimethylbenz[a]anthracene in one buccal pouch. At hyperplasia, dysplasia, or papilloma stage, animals received intracardially the vehicle or 0.7 × 106, 7 × 106, or 21 × 106 allogeneic bone marrow-derived MSCs/kg. OSCC progression was assessed according to the presence of erythroplakia and leukoplakia, extent of inflammation and vascularization, and appearance, volume, and staging of tumors. Also, the homing of donor cells was studied. RESULTS: Precancerous lesions progressed from hyperplasia to dysplasia in 2 weeks, from dysplasia to papilloma in 3 weeks, and from papilloma to carcinoma in 4 weeks. This time course was unmodified by the systemic administration of MSCs at hyperplasia or dysplasia stages. When MSCs were administered at papilloma stage, lesions did not progress to carcinoma stage. Tumors developed in hamsters receiving 0.7 × 106 or 7 × 106 MSCs/kg at hyperplasia stage were significantly smaller than those found in control animals (25 ± 4 or 23 ± 4 mm3 versus 72 ± 19 mm3, p < 0.05). Similar results were obtained when 0.7 × 106, 7 × 106, or 21 × 106 MSCs/kg were administered at papilloma stage (44 ± 15, 28 ± 7, or 28 ± 5 mm3 versus 104 ± 26 mm3, p < 0.05). For dysplasia stage, only the lower concentration of MSCs reached statistical significance (21 ± 9 mm3 versus 94 ± 39 mm3, p < 0.05). Animals receiving 21 × 106 MSCs/kg at hyperplasia stage developed tumors larger than those found in animals that received the vehicle (147 ± 47 mm3 versus 72 ± 19 mm3, p < 0.05). Donor cells were rarely found in precancerous lesions. CONCLUSIONS: Systemically administered allogeneic MSCs do not aggravate the progression of precancerous lesions. Moreover, they preclude cancer progression and tumor growth.

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

Omental adipose tissue is a more suitable source of canine Mesenchymal stem cells.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are a promising therapeutic tool in veterinary medicine. Currently the subcutaneous adipose tissue is the leading source of MSCs in dogs. MSCs derived from distinct fat depots have shown dissimilarities in their accessibility and therapeutic potential. The aims of our work were to determine the suitability of omental adipose tissue as a source of MSCs, according to sampling success, cell yield and paracrine properties of isolated cells, and compared to subcutaneous adipose tissue. RESULTS: While sampling success of omental adipose tissue was 100% (14 collections from14 donors) for subcutaneous adipose tissue it was 71% (10 collections from 14 donors). MSCs could be isolated from both sources. Cell yield was significantly higher for omental than for subcutaneous adipose tissue (38 ± 1 vs. 30 ± 1 CFU-F/g tissue, p < 0.0001). No differences were observed between sources regarding cell proliferation potential (73 ± 1 vs. 74 ± 1 CDPL) and cell senescence (at passage 10, both cultures presented enlarged cells with cytoplasmic vacuoles and cellular debris). Omental- and subcutaneous-derived MSCs expressed at the same level bFGF, PDGF, HGF, VEGF, ANG1 and IL-10. Irrespective of the source, isolated MSCs induced proliferation, migration and vascularization of target cells, and inhibited the activation of T lymphocytes. CONCLUSION: Compared to subcutaneous adipose tissue, omental adipose tissue is a more suitable source of MSCs in dogs. Since it can be procured from donors with any body condition, its collection procedure is always feasible, its cell yield is high and the MSCs isolated from it have desirable differentiation and paracrine potentials.

Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score.

As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

Regenerative Potential of Mesenchymal Stromal Cells: Age-Related Changes.

Preclinical and clinical studies have shown that a therapeutic effect results from mesenchymal stromal cells (MSCs) transplant. No systematic information is currently available regarding whether donor age modifies MSC regenerative potential on cutaneous wound healing. Here, we evaluate whether donor age influences this potential. Two different doses of bone marrow MSCs (BM-MSCs) from young, adult, or old mouse donors or two doses of their acellular derivatives mesenchymal stromal cells (acd-MSCs) were intradermally injected around wounds in the midline of C57BL/6 mice. Every two days, wound healing was macroscopically assessed (wound closure) and microscopically assessed (reepithelialization, dermal-epidermal junction, skin appendage regeneration, granulation tissue, leukocyte infiltration, and density dermal collagen fibers) after 12 days from MSC transplant. Significant differences in the wound closure kinetic, quality, and healing of skin regenerated were observed in lesions which received BM-MSCs from different ages or their acd-MSCs compared to lesions which received vehicle. Nevertheless, our data shows that adult's BM-MSCs or their acd-MSCs were the most efficient for recovery of most parameters analyzed. Our data suggest that MSC efficacy was negatively affected by donor age, where the treatment with adult's BM-MSCs or their acd-MSCs in cutaneous wound promotes a better tissue repair/regeneration. This is due to their paracrine factors secretion.

Multipotent mesenchymal stromal cells: A promising strategy to manage alcoholic liver disease.

Chronic alcohol consumption is a major cause of liver disease. The term alcoholic liver disease (ALD) refers to a spectrum of mild to severe disorders including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. With limited therapeutic options, stem cell therapy offers significant potential for these patients. In this article, we review the pathophysiologic features of ALD and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs), based on their potential to differentiate into hepatocytes, their immunomodulatory properties, their potential to promote residual hepatocyte regeneration, and their capacity to inhibit hepatic stellate cells. The perfect match between ALD pathogenesis and MSC therapeutic mechanisms, together with encouraging, available preclinical data, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage ALD onset and progression.

Morbimortalidade de usuários de um plano privado de saúde de Belo Horizonte, Minas Gerais / Morbimortalidad de usuarios de un plan privado de salud de Belo Horizonte, Minas Gerais / Morbimortality of private health plan users in Belo Horizonte, Minas Gerais

Caracterizar os perfis de morbidade e de mortalidade de beneficiários de um plano privado de saúde deBelo Horizonte.Estudo descritivo-exploratório desenvolvido com 4.913 internações no período entre 2009 e 2012. A coleta de dadosenvolveu informações secundárias e as principais variáveis analisadas foram sexo, idade, causas de internação e óbito. Asprincipais causas de internação segundo faixas etárias foram: doenças respiratórias em crianças (37,5%), doençasdigestivas em adolescentes (19,9%), doenças geniturinárias em adultos (16,9%) e doenças cardiovasculares em idosos(19,4%). Entre os adolescentes, se destacaram, ainda, as causas externas no sexo masculino (18,2%). Dos beneficiáriosinternados, 2,2% faleceram e a principal causa foram as doenças cardiovasculares (24,1%). O perfil demorbidade dosbeneficiários do plano de saúde se caracterizou pelo predomínio de doenças respiratórias em crianças ecrônicas emadultos e idosos. Ademais, as causas externas se destacaram entre os adolescentes do sexo masculino.