A dynamic air-liquid interface system for in vitro mimicking of the nasal mucosa.

The development of an in vitro 3D model for the growth of the nasal mucosa cells can improve the therapy and the study of pathological states for subjects with chronic airway conditions. We have previously characterized a system consisting of a scaffold with an internal channel and a perfusion bioreactor with two independent flows provided by an external and an internal circuit, respectively. In this paper, this system was designed as a model of the nasal cavity, in which cells, grown on the inner surface of the scaffold channel, would be in contact at the same time with both culture medium, supplied by the external circuit, and air, provided with the internal flow. To ensure adequate nutrient supply to the cells in the scaffold channel, the radial diffusion of the culture medium through the porous matrix was evaluated first in qualitative and, then, in quantitative terms, demonstrating the capability of the system to control the value and direction of this flux. As a preliminary study, the culture of epithelial cells in the scaffold channel is also discussed in static, maintaining the air-liquid interface condition for up to 3 weeks. Despite minor abnormalities, such as a gap between cell layers and some detachments from the scaffold, the scaffold ensured cell survival and growth during the experimental time.

Molecular Chaperones and Thyroid Cancer.

Thyroid cancers are the most common of the endocrine system malignancies and progress must be made in the areas of differential diagnosis and treatment to improve patient management. Advances in the understanding of carcinogenic mechanisms have occurred in various fronts, including studies of the chaperone system (CS). Components of the CS are found to be quantitatively increased or decreased, and some correlations have been established between the quantitative changes and tumor type, prognosis, and response to treatment. These correlations provide the basis for identifying distinctive patterns useful in differential diagnosis and for planning experiments aiming at elucidating the role of the CS in tumorigenesis. Here, we discuss studies of the CS components in various thyroid cancers (TC). The chaperones belonging to the families of the small heat-shock proteins Hsp70 and Hsp90 and the chaperonin of Group I, Hsp60, have been quantified mostly by immunohistochemistry and Western blot in tumor and normal control tissues and in extracellular vesicles. Distinctive differences were revealed between the various thyroid tumor types. The most frequent finding was an increase in the chaperones, which can be attributed to the augmented need for chaperones the tumor cells have because of their accelerated metabolism, growth, and division rate. Thus, chaperones help the tumor cell rather than protect the patient, exemplifying chaperonopathies by mistake or collaborationism. This highlights the need for research on chaperonotherapy, namely the development of means to eliminate/inhibit pathogenic chaperones.

Immunomorphological Pattern of Molecular Chaperones in Normal and Pathological Thyroid Tissues and Circulating Exosomes: Potential Use in Clinics.

The thyroid is a major component of the endocrine system and its pathology can cause serious diseases, e.g., papillary carcinoma (PC). However, the carcinogenic mechanisms are poorly understood and clinical useful biomarkers are scarce. Therefore, we determined if there are quantitative patterns of molecular chaperones in the tumor tissue and circulating exosomes that may be useful in diagnosis and provide clues on their participation in carcinogenesis. Hsp27, Hsp60, Hsp70, and Hsp90 were quantified by immunohistochemistry in PC, benign goiter (BG), and normal peritumoral tissue (PT). The same chaperones were assessed in plasma exosomes from PC and BG patients before and after ablative surgery, using Western blotting. Hsp27, Hsp60, and Hsp90 were increased in PC in comparison with PT and BG but no differences were found for Hsp70. Similarly, exosomal levels of Hsp27, Hsp60, and Hsp90 were higher in PC than in BG, and those in PC were higher before ablative surgery than after it. Hsp27, Hsp60, and Hsp90 show distinctive quantitative patterns in thyroid tissue and circulating exosomes in PC as compared with BG, suggesting some implication in the carcinogenesis of these chaperones and indicating their potential as biomarkers for clinical applications.

Nandrolone decanoate interferes with testosterone biosynthesis altering blood-testis barrier components.

The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography-mass spectrometry. Western blot analysis and quantitative real-time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood-testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein-1 (TJP1). ND stimulation deregulated metalloproteinase-9, metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2. Moreover, ND administration resulted in a mislocalization of mucin-1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP-2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone.

Mild Aerobic Exercise Training Hardly Affects the Diaphragm of mdx Mice.

In the mdx mice model of Duchenne Muscular Dystrophy (DMD), mild endurance exercise training positively affected limb skeletal muscles, whereas few and controversial data exist on the effects of training on the diaphragm. The diaphragm was examined in mdx (C57BL/10ScSn-Dmdmdx) and wild-type (WT, C57BL/10ScSc) mice under sedentary conditions (mdx-SD, WT-SD) and during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days (training: 5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonins Hsp60 and Hsp70 and the p65 subunit of nuclear factor-kB (NF-kB) were measured in diaphragm, gastrocnemius, and quadriceps in each experimental group at all time points. Although morphological analysis showed unchanged total area of necrosis/regeneration in the diaphragm after training, there was a trend for larger areas of regeneration than necrosis in the diaphragm of mdx-EX compared to mdx-SD mice. However, the levels of Cx39, a protein associated with active regeneration in damaged muscle, were similar in the diaphragm of mdx-EX and mdx-SD mice. Hsp60 significantly decreased at 45 days in the diaphragm, but not in limb muscles, in both trained and sedentary mdx compared to WT mice. In limb muscles, but not in the diaphragm, Hsp70 and NF-kB p65 levels were increased in mdx mice irrespective of training at 30 and 45 days. Therefore, the diaphragm of mdx mice showed little inflammatory and stress responses over time, and appeared hardly affected by mild endurance training. J. Cell. Physiol. 232: 2044-2052, 2017. © 2016 Wiley Periodicals, Inc.

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit.

BACKGROUND/AIM: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. MATERIALS AND METHODS: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). RESULTS: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. CONCLUSIONS: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells.

Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real-time PCR. The results obtained show that testosterone levels increase at a 3.9 µM concentration of nandrolone and return to the basal level a 15.6 µM dose of nandrolone. Nandrolone-induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a-hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down-regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects.

Heat shock protein 60 levels in tissue and circulating exosomes in human large bowel cancer before and after ablative surgery.

BACKGROUND: Heat shock protein 60 (Hsp60) is a chaperonin involved in tumorigenesis, but its participation in tumor development and progression is not well understood and its value as a tumor biomarker has not been fully elucidated. In the current study, the authors presented evidence supporting the theory that Hsp60 has potential as a biomarker as well as a therapeutic target in patients with large bowel cancer. METHODS: The authors studied a population of 97 subjects, including patients and controls. Immunomorphology, Western blot analysis, and quantitative real-time polymerase chain reaction were performed on tissue specimens. Exosomes were isolated from blood and characterized by electron microscopy, biochemical tests, and Western blot analysis. RESULTS: Hsp60 was found to be increased in cancerous tissue, in which it was localized in the tumor cell plasma membrane, and in the interstitium associated with cells of the immune system, in which it was associated with exosomes liberated by tumor cells and, as such, circulated in the blood. An interesting finding was that these parameters returned to normal shortly after tumor removal. CONCLUSIONS: The data from the current study suggested that Hsp60 is a good candidate for theranostics applied to patients with large bowel carcinoma and encourage similar research among patients with other tumors in which Hsp60 has been implicated.

Fibrosis markers and CRIM1 increase in chronic heart failure of increasing severity.

BACKGROUND: Fibrosis suppressors/activators in chronic heart failure (CHF) is a topic of investigation. AIM: To quantify serum levels of fibrosis regulators in CHF. METHODS: ELISA tests were used to quantify fibrosis regulators, procollagen type-(PIP)I, (PIP)III, collagen-I, III, BMP1,2,3,7, SDF1α, CXCR4, fibulin 1,2,3, BMPER, CRIM1 and BAMBI in 66 CHF (NYHA class I, n = 9; II, n = 34; III n = 23), and in 14 controls. RESULTS: In CHF, TGFßR2, PIPIII, SDF1α and CRIM1 were increased. PIPIII correlated with CRIM1. CONCLUSIONS: The BMPs inhibitor CRIM1 is increased and correlates with higher levels of serum PIPIII showing an imbalance in favor of pro-fibrotic mechanisms in CHF.

An Uncommon Case of Pediatric Nasopharyngeal Carcinoma with Bone Metastases and Enchondromas Evaluated Using 18F-FDG PET/CT.

Nowadays, the utility of positron emission tomography/computed tomography (PET/CT) is well established in nasopharyngeal carcinoma (NPC). The incidence of NPC in the West population, especially in children, is very low. We present the first Italian case of a pediatric patient with NPC followed up with 18F-fluorodeoxyglucose (18F-FDG) PET/CT scan in addition to the standard follow-up imaging methods, including CT and magnetic resonance imaging. The 18F-FDG PET/CT scan was helpful in discriminating between metastatic and benign osseous lesions, thereby helping clinicians to determine the most appropriate therapeutic regimen. These findings support the clinical utility of 18F-FDG PET/CT in the diagnostic work-up of pediatric patients with NPC.

From Dysbiosis to Neurodegenerative Diseases through Different Communication Pathways: An Overview.

The microbiome research field has rapidly evolved over the last few decades, becoming a major topic of scientific and public interest. The gut microbiota (GM) is the microbial population living in the gut. The GM has many functions, such as maintaining gut homeostasis and host health, providing defense against enteric pathogens, and involvement in immune system development. Several studies have shown that GM is implicated in dysbiosis and is presumed to contribute to neurodegeneration. This review focuses mainly on describing the connection between the intestinal microbiome alterations (dysbiosis) and the onset of neurodegenerative diseases to explore the mechanisms that link the GM to nervous system health, such as the gut-brain axis, as well as the mitochondrial, the adaptive humoral immunity, and the microvesicular pathways. The gut-brain communication depends on a continuous bidirectional flow of molecular signals exchanged through the neural and the systemic circulation. These pathways represent a possible new therapeutic target against neuroinflammation and neurodegeneration. Progress in this context is desperately needed, considering the severity of most neurodegenerative diseases and the current lack of effective treatments.

Digital Technologies Applied to Control the One-Step Process of Cannabis Olive Oil Preparations.

The reproducibility of an extemporaneous preparation is an essential condition for guaranteeing the quality, efficacy, and safety of the medicinal product. This study aimed to develop a controlled one-step process for cannabis olive oil preparations by applying digital technologies. For this purpose, the chemical profile of cannabinoid contents in oil extracts of Bedrocan, FM2, and Pedanios varieties obtained with the already in use method, proposed by the Italian Society of Compounding Pharmacists (SIFAP), was compared with two new methods, specifically the Tolotto Gear® extraction method (TGE) and the Tolotto Gear® extraction method preceded by a pre-extraction procedure (TGE-PE). HPLC analyses showed that the concentration of THC using cannabis flos with a high THC content (over 20% w/w) was always higher than 21 mg/mL for the Bedrocan variety and close to 20 mg/mL for the Pedanios variety when applying TGE, while with TGE-PE, the THC concentration was higher than 23 mg/mL for the Bedrocan variety. For the FM2 variety, the amounts of THC and CBD in the oil formulations obtained using TGE were higher than 7 mg/mL and 10 mg/mL, respectively, and for TGE-PE, the concentrations of THC and CBD were higher than 7 mg/mL and 12 mg/mL, respectively. GC-MS analyses were performed to define the terpene contents in the oil extracts. The samples of Bedrocan flos extracted with TGE-PE displayed a distinctive profile, highly rich in terpenes and devoid of oxidized volatile products. Thus, TGE and TGE-PE allowed performing a quantitative extraction of cannabinoids and increasing the total mono-di-tri terpenes and sesquiterpene concentrations. The methods were repeatable and applicable to any quantity of raw material, preserving the phytocomplex of the plant.

Immunomorphological Patterns of Chaperone System Components in Rare Thyroid Tumors with Promise as Biomarkers for Differential Diagnosis and Providing Clues on Molecular Mechanisms of Carcinogenesis.

Hurthle cell (HC), anaplastic (AC), and medullary (MC) carcinomas are low frequency thyroid tumors that pose several challenges for physicians and pathologists due to the scarcity of cases, information, and histopathological images, especially in the many areas around the world in which sophisticated molecular and genetic diagnostic facilities are unavailable. It is, therefore, cogent to provide tools for microscopists to achieve accurate diagnosis, such as histopathological images with reliable biomarkers, which can help them to reach a differential diagnosis. We are investigating whether components of the chaperone system (CS), such as the molecular chaperones, can be considered dependable biomarkers, whose levels and distribution inside and outside cells in the tumor tissue could present a distinctive histopathological pattern for each tumor type. Here, we report data on the chaperones Hsp27, Hsp60, and Hsp90. They presented quantitative levels and distribution patterns that were different for each tumor and differed from those of a benign thyroid pathology, goiter (BG). Therefore, the reported methodology can be beneficial when the microscopist must differentiate between HC, AC, MC, and BG.

Robotic-Assisted Neck Dissection: Our Experience.

Introduction Robotic neck dissection surgery allows less invasiveness to significantly improve the aesthetic impact even though it does not compromise the principles of radical cancer procedure. Objective The aim of our work is to describe our personal experience with robotic neck dissection surgery. Methods A retrospective study was conducted by analyzing 10 patients subjected to a robotic neck dissection surgery. In the period from August 2012 to December 2018, these patients have been treated exclusively with robotic lateral-cervical dissection. Five of them were subjected to robotic-assisted transaxillary neck dissection (RATAND) and the other 5 treated with robotic-assisted retroauricular neck dissection (RARAND), then the surgical results have been compared with 5 similar dissections performed by open neck dissection (OND). Results The average surgical time of RATAND was estimated in 166 minutes, the average surgical time of RARAND was estimated in 153 minutes and the average surgical time of OND was estimated in 48 minutes. Both robotic techniques are valid from the oncological and aesthetic point of view, but in terms of surgical time, they are much longer than the open technique. Conclusions In terms of the post-operative decree, in our opinion, the retroauricular technique is more rapid for the purposes of recovery.

Nicotine modulation of the lateral habenula/ventral tegmental area circuit dynamics: An electrophysiological study in rats.

Nicotine, the addictive component of tobacco, has bivalent rewarding and aversive properties. Recently, the lateral habenula (LHb), a structure that controls ventral tegmental area (VTA) dopamine (DA) function, has attracted attention as it is potentially involved in the aversive properties of drugs of abuse. Hitherto, the LHb-modulation of nicotine-induced VTA neuronal activity in vivo is unknown. Using standard single-extracellular recording in anesthetized rats, we observed that intravenous administration of nicotine hydrogen tartrate (25-800 µg/kg i.v.) caused a dose-dependent increase in the basal firing rate of the LHb neurons of nicotine-naïve rats. This effect underwent complete desensitization in chronic nicotine (6 mg/kg/day for 14 days)-treated animals. As previously reported, acute nicotine induced an increase in the VTA DA neuronal firing rate. Interestingly, only neurons located medially (mVTA) but not laterally (latVTA) within the VTA were responsive to acute nicotine. This pattern of activation was reversed by chronic nicotine exposure which produced the selective increase of latVTA neuronal activity. Acute lesion of the LHb, similarly to chronic nicotine treatment, reversed the pattern of DA cell activation induced by acute nicotine increasing latVTA but not mVTA neuronal activity. Our evidence indicates that LHb plays an important role in mediating the effects of acute and chronic nicotine within the VTA by activating distinct subregional responses of DA neurons. The LHb/VTA modulation might be part of the neural substrate of nicotine aversive properties. By silencing the LHb chronic nicotine could shift the balance of motivational states toward the reward.

OSAHS Growth Impairment and Resolution after Adenotonsillectomy in Children.

Introduction: One of the most important complications of OSAHS in children is growth delay. The aim of this study was to investigate changes in clinical body growth, and laboratory growth in children with OSAHS after adeno-tonsillar surgery. Materials and Methods: In our study, among 102 children suffering from sleep-disordered breathing, 70 met the inclusion criteria because they were affected by OSAHS and adenotonsillar hypertrophy. In total, 96 children affected by adeno-tonsillar hypertrophy (55 males and 41 females) underwent nocturnal cardiorespiratory monitoring with Embletta MPR, monitoring for post-operative 24 hours. Patients underwent blood sampling to evaluate preoperative GH and IGF-1 serum levels, "placement" in Cacciari's growth charts and adenotonsillectomy and saturation monitoring for post-operative 24 hours. According to auxological parameters, 82.86% of the patients were below the fiftieth percentile of BMI Cacciari's growth charts and IGF-1 preoperative serum levels were below the normal range. All patients underwent adenotonsillectomy. Results: All 70 patients recovered from OSAHS according to the results of nocturnal cardiorespiratory monitoring after six months. IGF-1 serum levels significantly increased after three months and one year after. All the auxological parameters showed a significant increase after surgery. We calculated the average annual growth in height of the patients before and after adenotonsillectomy (AT): the growth rate was impaired by OSAHS (5.4±1.3 cm/year), while in the following year post-surgery we found a significant growth speed acceleration (9.9±1.7 cm/year, P=0.001). Conclusions: In conclusion, growth delay in children can be caused by OSAHS, and when it is due to adenotonsillar hypertrophy, adenotonsillectomy is to be considered as the therapy of choice.

The chaperone system in glioblastoma multiforme and derived cell lines: diagnostic and mechanistic implications.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Novel treatments are needed to counteract the molecular mechanisms of GBM growth and drug resistance. The chaperone system (CS) members are typically cytoprotective but some, termed Hsp, can become pathogenic and participate in carcinogenesis, along with the vascular endothelial growth factor (VEGF), and we investigated them in GBM biopsies and derived cell lines. The objectives were to identify diagnostic-prognostic biomarkers and gather information for developing chaperonotherapy. METHODS: Cell lines from GBMs were established, characterized (morphology, growth characteristics, and specific markers), and stored. Chaperones and angiogenic factors [Hsp10, Hsp27, Hsp60, Hsp70, Hsp90, FLT-1 (VEGFR-1), FLK1 (KDR, VEGFR-2), and FLT-4 (VEGFR-3)] were observed in cells by immunofluorescence while the chaperones were measured in tumor tissue by immunohistochemistry. RESULTS: Four cell lines were derived from four different GBMs; the cells were spindle shaped or polygonal and grew at high rates as adherent monolayers or clusters without evidence of contact inhibition. The astrocyte-specific glial fibrillary acidic protein (GFAP); and the neuronal NSE, malignancy VIM, and proliferation PCNA, markers were determined. The cells expressed GFAP but no NSE, indicating that they were primary glioblastoma cell lines, with high levels of Hsp10, Hsp27, Hsp60, Hsp90, and Flk1; and low levels of Hsp70, Flt1, and Flt4. CONCLUSIONS: Four cell lines were established derived from four out of ten GBM tumors studied. The cell lines showed intense positivity for chaperones studied and factors connected to malignancy and the tumors showed increased levels of chaperones, making them potential diagnostic-prognostic biomarkers and targets for anti-cancer compounds.

Extracellular vesicles derived from gut microbiota in inflammatory bowel disease and colorectal cancer.

The human gut microbiome encompasses inter alia, the myriad bacterial species that create the optimal host-microorganism balance essential for normal metabolic and immune function. Various lines of evidence suggest that dysregulation of the microbiota-host interaction is linked to pathologies such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). Extracellular vesicles (EVs), found in virtually all body fluids and produced by both eukaryotic cells and bacteria are involved in cell-cell communication and crosstalk mechanisms, such as the immune response, barrier function and intestinal flora. This review highlights advancements in knowledge of the functional role that EVs may have in IBD and CRC, and discusses the possible use of EVs derived from intestinal microbiota in therapeutic strategies for treating these conditions.

Morphological Alterations and Stress Protein Variations in Lung Biopsies Obtained from Autopsies of COVID-19 Subjects.

Molecular chaperones, many of which are heat shock proteins, play a role in cell stress response and regulate the immune system in various ways, such as in inflammatory/autoimmune reactions. It would be interesting to study the involvement of these molecules in the damage done to COVID-19-infected lungs. In our study, we performed a histological analysis and an immunomorphological evaluation on lung samples from subjects who succumbed to COVID-19 and subjects who died from other causes. We also assessed Hsp60 and Hsp90 distribution in lung samples to determine their location and post-translational modifications. We found histological alterations that could be considered pathognomonic for COVID-19-related lung disease. Hsp60 and Hsp90 immunopositivity was significantly higher in the COVID-19 group compared to the controls, and immunolocalization was in the plasma membrane of the endothelial cells in COVID-19 subjects. The colocalization ratios for Hsp60/3-nitrotyrosine and Hsp60/acetylate-lisine were significantly increased in the COVID-19 group compared to the control group, similar to the colocalization ratio for Hsp90/acetylate-lisine. The histological and immunohistochemical findings led us to hypothesize that Hsp60 and Hsp90 might have a role in the onset of the thromboembolic phenomena that lead to death in a limited number of subjects affected by COVID-19. Further studies on a larger number of samples obtained from autopsies would allow to confirm these data as well as discover new biomarkers useful in the battle against this disease.

Hsp27 and Hsp60 in human submandibular salivary gland: Quantitative patterns in healthy and cancerous tissues with potential implications for differential diagnosis and carcinogenesis.

Tumors of the submandibular salivary gland (SMG) are uncommon but sufficiently frequent for the physician to consider them in routine examinations and for the pathologist to be prepared to differentiate them from other tissue abnormalities. However, scarcity of specimens makes training difficult, a situation compounded by the lack of accepted universal diagnostic guidelines. Furthermore, there is little information on the chaperone system (CS) of the gland, despite the increasing evidence of its participation in carcinogenesis as a biomarker for diagnosis and patient follow up, and in the mechanisms by which the tumor cells thrive. We are investigating this aspect of various tumors, and here we describe standardized methods for assessing the tissue levels of two chaperones, Hsp27 and Hsp60, in normal SMG and its tumors. We present illustrative results obtained with immunohistochemistry (IHC) and immunofluorescence-confocal microscopy (IF-CM), which we propose as a platform onto which a data base could be built by adding new information and which would provide material for developing guidelines for tumor identification and monitoring. The initial findings are encouraging in as much as the tumors surveyed showed quantitative patterns of Hsp27 and Hsp60 that distinguished tumoral from normal tissue and certain tumors from the others, and the results from IHC were confirmed by IF-CM.