Construction a novel osteoporosis model in immune-deficient mice with natural ageing.

Osteoporosis (OP) predominantly affects elderly individuals. Stem cells show potential for treating OP. However, animal models with normal immune function can eliminate implanted human cells. This study utilized naturally aging NOD/SCID mice, which exhibit immunodeficiency, to create a human osteoporosis model. This approach helps to minimize the premature immune clearance of transplanted allogeneic or xenogeneic cells in preclinical studies, allowing for a more accurate replication of the clinical pharmacological and pharmacokinetic processes involved in stem cell interventions for osteoporosis. NOD/SCID mice were fed until 12, 32, and 43 weeks of age, respectively, and then euthanized. We harvested lumbar vertebra for Micro-Computed Tomography (Micro-CT) scanning and pathological examination. Additionally, we performed biomechanical testing of lumbar vertebra to assess the severity of osteoporosis. We utilized real-time RT-PCR to assess gene expression changes associated with bone metabolism, aging, inflammation, oxidative stress, and the Tgf-ß1/Smad3 signaling pathway. In addition, the protein expression levels of P16, Tgf-ß1 and Smad3 were detected using Western Blotting (WB). In comparison to 12-week-old mice, the 32-week-old and 43-week-old mice displayed significantly sparser and fractured trabeculae in their lumbar vertebra, lower bone mineral density (BMD), and changes in bone microstructural parameters (∗∗P < 0.01, ∗∗∗P < 0.001). Additionally, compared to 12-week-old mice, the 32-week-old and 43-week-old mice exhibited decreased expression of osteogenic genes (Alp, Opg, Sp7, Col1a1), increased expression of osteoclastic gene (Rankl), the number of TRAP-positive osteoclasts significantly increased in 32-week-old and 43-week-old mice compared to 12-week-old mice. The expression of genes related to aging and inflammatory (P16, Il-1ß, Tnf-α) increases with advancing age (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). The expression of oxidative stress-related genes (Sod1, Sod2, Foxo3, Nrf2), as well as Tgf-ß1 and Smad3 decreased with age (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). As age increases, the levels of P16 protein increase, Tgf-ß1 and Smad3 proteins decrease. Our study successfully replicated osteoporosis models in NOD/SCID mice at both 32 and 43 weeks, with the latter exhibiting more severe osteoporosis. This condition seems to be driven by factors such as aging, inflammation, oxidative stress, and the Tgf-ß1/Smad3 signaling pathway.

Analysis of the zoonotic tick-borne encephalitis virus (TBEV) in raw milk and dairy products in mountain pastures of the Lombardy region, Italy.

Over the last few decades, tick-borne encephalitis (TBE) has become a growing public health problem in Europe. The tick-borne encephalitis virus (TBEV) is a zoonotic virus that affects the central nervous system (CNS). TBEV has been detected in 27 European countries, and the rise in TBE cases is mainly due to environmental and ecological factors, and factors that increase the risk of human exposure to infected ticks. The infection via the alimentary route is the second most common means of TBEV transmission to humans. Raw milk from infected goats, sheep, or cows has been identified as a source of human food-borne infections. This study aims to gather new information on the prevalence of tick-borne encephalitis virus (TBEV) in raw goat's and cow's milk and related raw products in the Lombard Alps (Italy). This is important due to the close proximity of Lombardy to the Triveneto region, where TBE is endemic, and southern Switzerland, where numerous TBEV-positive mammals have been found. Throughout 2023, a passive monitoring plan was implemented on samples delivered for TBEV analyses from the Alpine pastures. In total, 248 specimens including raw milk, raw milk cheese, and butter were tested. This is the first monitoring of food at risk of TBEV transmission in a non-endemic region with evidence of TBEV circulation. Despite testing a wide range of dairy products, no sample tested positive for RNA-TBEV by real-time RT-PCR. Preliminary results suggest that raw milk and raw dairy products do not pose a significant risk of TBEV transmission to humans in the territory of Lombardy.

Discriminating North American Swine Influenza Viruses with a Portable, One-Step, Triplex Real-Time RT-PCR Assay, and Portable Sequencing.

Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (ß), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential.

Detection and Quantification of Soil-Borne Wheat Mosaic Virus, Soil-Borne Cereal Mosaic Virus and Japanese Soil-Borne Wheat Mosaic Virus by ELISA and One-Step SYBR Green Real-Time Quantitative RT-PCR.

Furoviruses are bipartite viruses causing mosaic symptoms and stunting in cereals. Infection with these viruses can lead to severe crop losses. The virus species Furovirus tritici with soil-borne wheat mosaic virus (SBWMV), Furovirus cerealis with soil-borne cereal mosaic virus (SBCMV) and Furovirus japonicum with Japanese soil-borne wheat mosaic virus (JSBWMV) and French barley mosaic virus (FBMV) as members are biologically and genetically closely related. Here, we develop SYBR green-based real-time quantitative RT-PCR assays to detect and quantify the RNA1 and RNA2 of the three virus species. Using experimental data in combination with Tm-value prediction and analysis of primer and amplicon sequences, we determine the capacity of our method to discriminate between the different viruses and evaluate its genericity to detect different isolates within the same virus species. We demonstrate that our method is suitable for discriminating between the different virus species and allows for the detection of different virus isolates. However, JSBWMV RNA1 primers may amplify SBWMV samples, bearing a risk for false positive detection with this primer. We also test the efficiency of polyclonal antibodies to detect the different viruses by ELISA and suggest that ELISA may be applied as a first screening to identify the virus. The real-time qRT-PCR assays developed provide the possibility to screen for quantitative disease resistance against SBCMV, SBWMV and JSBWMV. Moreover, with our method, we hope to promote research to unravel yet unresolved questions with respect to furovirus-host interaction concerning host range and resistance as well as regarding the role of multipartite genomes.

Æ©S COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay.

Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2-4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15-30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed Æ©S COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen's Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam.

OBJECTIVE: This study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation. RESULTS: The rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

Comparison of four concentration methods of adenovirus, norovirus and rotavirus in tap water.

Human enteric viruses, as adenovirus (HAdV), norovirus (HuNoV) and rotavirus (RVA) are significant causes of gastroenteritis associated with consumption of contaminated water worldwide. Various methods have been described for their detection and monitoring in water. The aim of this study was to compare the performance of four conditions for concentrating HAdV, HuNoV and RVA from water matrices, in order to develop a single protocol that could simultaneously concentrate all target viruses from tap water. The tested conditions were based on the adsorption-elution using electronegative filters, in which we evaluated cation-coated filtration by MgCl2 with or without acid rinse by H2SO4 and two elution buffers, namely NaOH and tris-glycine-beef extract. Genomic material was extracted and amplified by real-time PCR and real-time RT-PCR using commercial kits. Based on the statistical analysis of amplification results (cycles of quantification), the condition involving cation-coated filtration by MgCl2 using electronegative filters with acid rinse by H2SO4 combined with NaOH elution allowed efficient recovery of both HAdV, HuNoV and RVA from tap water compared to the other conditions. These findings confirm the effectiveness of the approach used to monitor three major enteric viruses in tap water.

Real-Time RT-PCR Assays for Typing of Epizootic Hemorrhagic Disease Virus.

Real-time RT-PCR for the detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples is a fast and sensitive tool for the diagnosis and confirmation of disease. Several real-time RT-PCR methods have been reported over the last 10 years. In this chapter, we describe seven duplex real-time RT-PCR assays to amplify part of genome segment 2 of EHDV to enable serotype identification. The assay includes the detection of an endogenous control gene-beta-actin.

Real-Time RT-PCR for the Diagnosis of Epizootic Hemorrhagic Disease Virus.

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) has become an essential tool in rapid and reliable detection of animal diseases such as epizootic hemorrhagic disease (EHD). Here we provide a protocol for the detection of epizootic hemorrhagic disease virus (EHDV) genetic material in blood and tissue samples, using a real-time RT-PCR that targets a conserved region in segment 9 of the EHDV genome. This protocol can be used to detect up to approximately 90 samples in a single run and can be completed in less than 4 h.

Comparative analysis of reverse-transcription-polymerase chain reaction for Aichivirus detection.

Aichivirus-A (AiV-A), a member of the Kobuvirus genus of the family Picornaviridae, was first reported in stool samples of patients with non-bacterial gastroenteritis in Aichi Prefecture, Japan, in 1989. AiV has been reported from in various aquatic environments, such as surface water and sewage, can be transmitted via the fecal-oral route through contaminated water. As AiV is known to acute gastroenteritis worldwide, developing methods for AiV detection from contaminated environments and food is required. In the present study, we established an effective polymerase chain reaction (PCR) method to detect AiV. Various real-time reverse transcription (RT)-PCR and conventional PCR methods for AiV detection were compared, and the limit of detection was confirmed by comparing the sensitivity at varied primer concentrations and PCR conditions. The final detection limits were 102 copy/µL in conventional PCR, and 101 copy/µL in the real-time RT-PCR. The optimized method used in this study might aid in detecting AiV contamination.

Genetic Characterization of Palyam Serogroup Viruses Isolated in Japan from 1984 to 2018 and Development of a Real-Time RT-PCR Assay for Broad Detection of Palyam Serogroup Viruses and Specific Detection of Chuzan (Kasba) and D'Aguilar Viruses.

We performed whole genome sequencing (WGS) of 15 Palyam serogroup virus (PALV) strains isolated from cattle or Culicoides biting midges in Japan from 1984 to 2018. We found that the PALV strains consisted of Chuzan (Kasba) virus (CHUV), D'Aguilar virus (DAGV), Bunyip Creek virus, and another PALV, Marrakai virus (MARV). The Japanese MARV strains isolated in 1997 were closely related to Australian PALV strains isolated in 1968-1976 in genome segments 2 and 10, but they were most closely related to other Japanese PALV strains in the other genome segments. Our data suggest that the Japanese MARV strains were reassortant viruses between Asian and Australian PALVs. In addition to the WGS, we developed a real-time reverse-transcription polymerase chain reaction assay that can broadly detect PALV and specifically detect CHUV and DAGV, utilizing the data obtained by the WGS in this study. We detected the DAGV gene in bovine stillborn fetuses and congenitally abnormal calves in 2019 using the newly developed assay. To our knowledge, this is the first report of isolation of MARV outside of Australia and the first report of detection of PALV in bovine fetuses or calves with congenital abnormality outside of Africa.

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses.

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/µL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

RNA-Seq analysis of the fruiting bodies and mycelia of angel-wing mushroom Pleurocybella porrigens that cause acute encephalopathy.

OBJECTIVE: In 2004, after consuming angel-wing mushrooms, Pleurocybella porrigens, 59 incidents of food poisoning were reported in Japan. Consequently, 17 individuals died of acute encephalopathy. In 2023, we proved that a lectin, pleurocybelline, and pleurocybellaziridine from this mushroom caused damage to the brains of mice. Although we reported genomic and transcriptomic data of P. porrigens in 2013, the assembly quality of the transcriptomic data was inadequate for accurate functional annotation. Thus, we obtained detailed transcriptomic data on the fruiting bodies and mycelia of this mushroom using Illumina NovaSeq 6000. RESULTS: De novo assembly data indicated that the N50 lengths for the fruiting bodies and mycelia were improved compared with those previously reported. The differential expression analysis between the fruiting bodies and the mycelia revealed that 1,937 and 1,555 genes were significantly up-regulated in the fruiting bodies and the mycelia, respectively. The biological functions of P. porrigens transcripts, including PA biosynthetic pathways, were investigated using BLAST search, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The obtained results revealed L-valine, a predicted precursor of PA, is biosynthesized in the fruiting bodies and mycelia. Furthermore, real-time RT-PCR was performed to evaluate the accuracy of the results of differential expression analysis.

Development of a CRISPR/SHERLOCK-Based Method for Rapid and Sensitive Detection of Selected Pospiviroids.

Pospiviroids infect a wide range of plant species, and many pospiviroids can be transmitted to potato and tomato. Pospiviroids continue to be a major production constraint as well as of quarantine concern for the movement of germplasm, and are regulated in several countries/regions. The USDA APHIS issued a federal order requiring all imported tomato and pepper seeds be certified free of six pospiviroids of quarantine significance. The six pospiviroids of quarantine interest include CLVd, PCFVd, PSTVd, TASVd, TCDVd, TPMVd. Currently, those six viroids are detected by real-time RT-PCR. CRISPR/Cas-based genome editing has been increasingly used for virus detection in the past five years. We used a rapid Cas13-based Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) platform for pospiviroid detection, determined the limits of detection and specificity of CRISPR-Cas13a assays. This platform combines recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated (CRISPR-Cas) RNA-guided endoribonuclease that is rapid and does not require expensive equipment, and can be adapted for on-site detection.

Qualitative real-time RT-PCR assay for nOPV2 poliovirus detection.

Based on the success of the Sabin2-based vaccine, a next-generation nOPV2 poliovirus vaccine has been developed. For epidemic monitoring and conducting epidemiological investigations, it is necessary to have a diagnostic assay with the ability to differentiate this variant from others. Here we describe such a real-time RT-PCR assay. The region with the cre insertion in the 5'-UTR was chosen as the target, and the limit of detection was 103 copies/mL (2.5×103 copies/mL using Probit analysis) determined using armored RNA particles. Sensitivity and specificity were 86.28 - 100 % and 76.84 - 100 %, respectively (with 95 % CI). Thus, this method can be effectively used when it is necessary to make a differential diagnosis of poliovirus strains.

A novel and cost-effective real-time RT-PCR targeting 24 nucleotides deletion to differentiate PEDV wild-type and classical attenuated vaccine strains.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the swine industry, causing severe disease and resulting in substantial economic losses. Despite China's implementation of a large-scale vaccine immunization strategy in recent years, various strains of PEDV, including classical attenuated vaccine strains, continue to emerge in immunized pig herds. Here, we established a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains. This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, and we also explore the causes of this discriminatory target deficiency of this method through phylogenetic and recombination analysis. We found that these three classical attenuated vaccine strains exhibit closer phylogenetic relationships and higher sequence similarity with five cell-adapted strains. Recombination analysis revealed that although recombination is widespread in the PEDV genome, the 24-nucleotide deletion site remains stable without undergoing recombination and can be utilized as a target for identification. Further analysis revealed there are no enzyme cleavage sites near the 24-nucleotide site, suggesting that this deletion may have been lost during the process of culturing these viral strains in cells.The detection method we have established exhibits high specificity and sensitivity to PEDV, without cross-reactivity with other viruses causing diarrheal diseases. A total of 117 swine fecal samples were analyzed using this established one-step real-time reverse transcription PCR assay, indicating the presence of classical attenuated vaccine strains in pig herds in Gansu province, China. Additionally, the designed primer pairs and two probes can be placed in a single reaction tube to differentiate between these two types of strains, effectively reducing detection costs. These findings offer an efficient and cost-effective technological platform for clinical rapid identification testing of both wild-type and classical attenuated vaccine strains of PEDV, as well as for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.

Evaluating an extraction-free sample preparation method for multiplex detection of SARS-Cov-2, influenza A/B, and RSV with implementation on a microfluidic chip.

Following the relaxation of COVID-19 restrictions, other respiratory viruses such as influenza and respiratory syncytial virus (RSV), whose transmission were decreased due to COVID-19 precautions, are rising again. Because of similar clinical features and reported co-infections, multiplex detection of SARS-CoV-2, influenza A/B, and RSV is required to use specific treatments. This study assessed an extraction-free sample preparation (heat treatment at 95°C for 3 minutes) for multiplex detection using rRT-PCR. Despite an observed Ct-delay (∆Ct) averageing 1.26 compared to the standard method, an acceptable total sensitivity of 92 % and a negative predictive value (NPV) of 96 % were obtained. Moreover, Implementation on a microfluidic chip demonstrated efficiency, maintaining an excellent correlation (R2=0.983) with the standard method. Combining this extraction-free procedure with rRT-PCR on a microfluidic chip seems promising, because it simplifies the design and reduces the cost and complexity of the integrated assay for multiplex detection of SARS-CoV-2, influenza A/B, and RSV.

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

TGFß signaling pathway in salivary gland tumors.

OBJECTIVE: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß cascade. DESIGN: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. RESULTS: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFß signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. CONCLUSIONS: Our study demonstrated the differential expression of TGFß cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.