Thyroid hormones inhibit TGF-ß signaling and attenuate fibrotic responses.

TGF-ß, the most potent profibrogenic factor, acts by activating SMAD (mothers against decapentaplegic) transcription factors, which bind to SMAD-binding elements in target genes. Here, we show that the thyroid hormone triiodothyronine (T3), through binding to its nuclear receptors (TRs), is able to antagonize transcriptional activation by TGF-ß/SMAD. This antagonism involves reduced phosphorylation of SMADs and a direct interaction of the receptors with SMAD3 and SMAD4 that is independent of T3-mediated transcriptional activity but requires residues in the receptor DNA binding domain. T3 reduces occupancy of SMAD-binding elements in response to TGF-ß, reducing histone acetylation and inhibiting transcription. In agreement with this transcriptional cross-talk, T3 is able to antagonize fibrotic processes in vivo. Liver fibrosis induced by carbon tetrachloride is attenuated by thyroid hormone administration to mice, whereas aged TR knockout mice spontaneously accumulate collagen. Furthermore, skin fibrosis induced by bleomycin administration is also reduced by the thyroid hormones. These findings define an important function of the thyroid hormone receptors and suggest TR ligands could have beneficial effects to block the progression of fibrotic diseases.

Autoregulatory loop of nuclear corepressor 1 expression controls invasion, tumor growth, and metastasis.

Nuclear corepressor 1 (NCoR) associates with nuclear receptors and other transcription factors leading to transcriptional repression. We show here that NCoR depletion enhances cancer cell invasion and increases tumor growth and metastatic potential in nude mice. These changes are related to repressed transcription of genes associated with increased metastasis and poor prognosis in patients. Strikingly, transient NCoR silencing leads to heterochromatinization and stable silencing of the NCoR gene, suggesting that NCoR loss can be propagated, contributing to tumor progression even in the absence of NCoR gene mutations. Down-regulation of the thyroid hormone receptor ß1 (TRß) appears to be associated with cancer onset and progression. We found that expression of TRß increases NCoR levels and that this induction is essential in mediating inhibition of tumor growth and metastasis by this receptor. Moreover, NCoR is down-regulated in human hepatocarcinomas and in the more aggressive breast cancer tumors, and its expression correlates positively with that of TRß. These data provide a molecular basis for the anticancer actions of this corepressor and identify NCoR as a potential molecular target for development of novel cancer therapies.

Liver Growth Factor (LGF) Upregulates Frataxin Protein Expression and Reduces Oxidative Stress in Friedreich's Ataxia Transgenic Mice.

Friedreich's ataxia (FA) is a severe disorder with autosomal recessive inheritance that is caused by the abnormal expansion of GAA repeat in intron 1 of FRDA gen. This alteration leads to a partial silencing of frataxin transcription, causing a multisystem disorder disease that includes neurological and non-neurological damage. Recent studies have proven the effectiveness of neurotrophic factors in a number of neurodegenerative diseases. Therefore, we intend to determine if liver growth factor (LGF), which has a demonstrated antioxidant and neuroprotective capability, could be a useful therapy for FA. To investigate the potential therapeutic activity of LGF we used transgenic mice of the FXNtm1MknTg (FXN)YG8Pook strain. In these mice, intraperitoneal administration of LGF (1.6 µg/mouse) exerted a neuroprotective effect on neurons of the lumbar spinal cord and improved cardiac hypertrophy. Both events could be the consequence of the increment in frataxin expression induced by LGF in spinal cord (1.34-fold) and heart (1.2-fold). LGF also upregulated by 2.6-fold mitochondrial chain complex IV expression in spinal cord, while in skeletal muscle it reduced the relation oxidized glutathione/reduced glutathione. Since LGF partially restores motor coordination, we propose LGF as a novel factor that may be useful in the treatment of FA.

Sterile inflammation in acetaminophen-induced liver injury is mediated by Cot/tpl2.

Cot/tpl2 (MAP3K8) activates MKK1/2-Erk1/2 following stimulation of the Toll-like/IL-1 receptor superfamily. Here, we investigated the role of Cot/tpl2 in sterile inflammation and drug-induced liver toxicity. Cot/tpl2 KO mice exhibited reduced hepatic injury after acetaminophen challenge, as evidenced by decreased serum levels of both alanine and aspartate aminotransferases, decreased hepatic necrosis, and increased survival relative to Wt mice. Serum levels of both alanine and aspartate aminotransferases were also lower after intraperitoneal injection of acetaminophen in mice expressing an inactive form of Cot/tpl2 compared with Wt mice, suggesting that Cot/tpl2 activity contributes to acetaminophen-induced liver injury. Furthermore, Cot/tpl2 deficiency reduced neutrophil and macrophage infiltration in the liver of mice treated with acetaminophen, as well as their hepatic and systemic levels of IL-1α. Intraperitoneal injection of damage-associated molecular patterns from necrotic hepatocytes also impaired the recruitment of leukocytes and decreased the levels of several cytokines in the peritoneal cavity in Cot/tpl2 KO mice compared with Wt counterparts. Moreover, similar activation profiles of intracellular pathways were observed in Wt macrophages stimulated with Wt or Cot/tpl2 KO damage-associated molecular patterns. However, upon stimulation with damage-associated molecular patterns, the activation of Erk1/2 and JNK was deficient in Cot/tpl2 KO macrophages compared with their Wt counterparts; an effect accompanied by weaker release of several cytokines, including IL-1α, an important component in the development of sterile inflammation. Taken together, these findings indicate that Cot/tpl2 contributes to acetaminophen-induced liver injury, providing some insight into the underlying molecular mechanisms.

NOD1 receptor is up-regulated in diabetic human and murine myocardium.

Type 2 diabetes has a complex pathology that involves a chronic inflammatory state. Emerging evidence suggests a link between the innate immune system receptor NOD1 (nucleotide-binding and oligomerization domain 1) and the pathogenesis of diabetes, in monocytes and hepatic and adipose tissues. The aim of the present study was to assess the role of NOD1 in the progression of diabetic cardiomyopathy. We have measured NOD1 protein in cardiac tissue from Type 2 diabetic (db) mice. Heart and isolated cardiomyocytes from db mice revealed a significant increase in NOD1, together with an up-regulation of nuclear factor κB (NF-κB) and increased apoptosis. Heart tissue also exhibited an enhanced expression of pro-inflammatory cytokines. Selective NOD1 activation with C12-γ-D-glutamyl-m-diaminopimelic acid (iEDAP) resulted in an increased NF-κB activation and apoptosis, demonstrating the involvement of NOD1 both in wild-type and db mice. Moreover, HL-1 cardiomyocytes exposed to elevated concentrations of glucose plus palmitate displayed an enhanced NF-κB activity and apoptotic profile, which was prevented by silencing of NOD1 expression. To address this issue in human pathology, NOD1 expression was evaluated in myocardium obtained from patients with Type 2 diabetes (T2DMH) and from normoglycaemic individuals without cardiovascular histories (NH). We have found that NOD1 was expressed in both NH and T2DMH; however, NOD1 expression was significantly pronounced in T2DMH. Furthermore, both the pro-inflammatory cytokine tumour necrosis factor α (TNF-α) and the apoptosis mediator caspase-3 were up-regulated in T2DMH samples. Taken together, our results define an active role for NOD1 in the heightened inflammatory environment associated with both experimental and human diabetic cardiac disease.

Beneficial effect of TLR4 blockade by a specific aptamer antagonist after acute myocardial infarction.

Experimental evidence indicates that the control of the inflammatory response after myocardial infarction is a key strategy to reduce cardiac injury. Cellular damage after blood flow restoration in the heart promotes sterile inflammation through the release of molecules that activate pattern recognition receptors, among which TLR4 is the most prominent. Transient regulation of TLR4 activity has been considered one of the potential therapeutic interventions with greater projection towards the clinic. In this regard, the characterization of an aptamer (4FT) that acts as a selective antagonist for human TLR4 has been investigated in isolated macrophages from different species and in a rat model of cardiac ischemia/reperfusion (I/R). The binding kinetics and biological responses of murine and human macrophages treated with 4FT show great affinity and significant inhibition of TLR4 signaling including the NF-κB pathway and the LPS-dependent increase in the plasma membrane currents (Kv currents). In the rat model of I/R, administration of 4FT following reoxygenation shows amelioration of cardiac injury function and markers, a process that is significantly enhanced when the second dose of 4FT is administered 24 h after reperfusion of the heart. Parameters such as cardiac injury biomarkers, infiltration of circulating inflammatory cells, and the expression of genes associated with the inflammatory onset are significantly reduced. In addition, the expression of anti-inflammatory genes, such as IL-10, and pro-resolution molecules, such as resolvin D1 are enhanced after 4FT administration. These results indicate that targeting TLR4 with 4FT offers new therapeutic opportunities to prevent cardiac dysfunction after infarction.

Histerocystoplasty: a novel surgical procedure in the rat.

BACKGROUND: Enterocystoplasties are associated to complications. To avoid them, different types of tissue templates have been used to augment the bladder and induce native bladder regeneration. MATERIALS AND METHODS: A novel surgical technique for bladder reconstruction using autologous uterine tissue was evaluated in a rat model. Forty-two female Wistar rats were randomly allocated into three groups: sham-operation hysterocystorrhaphy (n = 12), hysterocystoplasty (n = 18), and control (n = 12). Two weeks after surgery, ultrasound examination of the bladder was performed. At 2, 4, or 6 mo after surgery, the rats were anesthetized and blood and urine samples were taken. They were then euthanized and post-mortem and histologic examination were performed. Ultrasound examination, analytical parameters and weight control, as well as gross and histologic examination were performed in all the operated animals. The statistical analysis was performed using Kruskal-Wallis and the extension of Fisher's exact tests. Significance was set at 5% (P < 0.05). RESULTS: Serum chemistry, blood count and peripheral blood smears, electrolytes, and urinary parameters were all within the normal range for the rat. Histologic sections of the surgically augmented zone between the bladder and uterine horn demonstrated urothelial epithelization, providing adequate coverage of the transition area in 72.22% of the rats that underwent hysterocystoplasty. CONCLUSIONS: The hysterocystoplasty was technically viable in all the cases and proved to be an easy and safe surgical model for bladder reconstruction. All animals were healthy after surgery and all systemic parameters analyzed were within normal physiologic range for the rat.

Hypothyroidism confers tolerance to cerebral malaria.

The modulation of the host's metabolism to protect tissue from damage induces tolerance to infections increasing survival. Here, we examined the role of the thyroid hormones, key metabolic regulators, in the outcome of malaria. Hypothyroidism confers protection to experimental cerebral malaria by a disease tolerance mechanism. Hypothyroid mice display increased survival after infection with Plasmodium berghei ANKA, diminishing intracranial pressure and brain damage, without altering pathogen burden, blood-brain barrier disruption, or immune cell infiltration. This protection is reversed by treatment with a Sirtuin 1 inhibitor, while treatment of euthyroid mice with a Sirtuin 1 activator induces tolerance and reduces intracranial pressure and lethality. This indicates that thyroid hormones and Sirtuin 1 are previously unknown targets for cerebral malaria treatment, a major killer of children in endemic malaria areas.

Cot/tpl2 (MAP3K8) mediates myeloperoxidase activity and hypernociception following peripheral inflammation.

Cot/tpl2 (also known as MAP3K8) has emerged as a new and potentially interesting therapeutic anti-inflammatory target. Here, we report the first study of Cot/tpl2 involvement in acute peripheral inflammation in vivo. Six hours after an intraplantar injection of zymosan, Cot/tpl2(-/-) mice showed a 47% reduction in myeloperoxidase activity, concomitant with a 46% lower neutrophil recruitment and a 40% decreased luminol-mediated bioluminescence imaging in vivo. Accordingly, Cot/tpl2 deficiency provoked a 25-30% reduction in luminol-mediated bioluminescence and neutrophil recruitment together with a 65% lower macrophage recruitment 4 h following zymosan-induced peritonitis. Significantly impaired levels of G-CSF and GM-CSF and of other cytokines such as TNFα, IL-1ß, and IL-6, as well as some chemokines such as MCP-1, MIP-1ß, and keratinocyte-derived chemokine, were detected during the acute zymosan-induced intraplantar inflammatory response in Cot/tpl2(-/-) mice. Moreover, Cot/tpl2 deficiency dramatically decreased the production of the hypernociceptive ligand NGF at the inflammatory site during the course of inflammation. Most importantly, Cot/tpl2 deficiency significantly reduced zymosan-induced inflammatory hypernociception in mice, with a most pronounced effect of a 50% decrease compared with wild type (WT) at 24 h following intraplantar injection of zymosan. At this time, Cot/tpl2(-/-) mice showed significantly reduced NGF, TNFα, and prostaglandin E(2) levels compared with WT littermates. In conclusion, our study demonstrates an important role of Cot/tpl2 in the NGF, G-CSF, and GM-CSF production and myeloperoxidase activity in the acute inflammatory response process and its implication in inflammatory hypernociception.

Liver growth factor treatment restores cell-extracellular matrix balance in resistance arteries and improves left ventricular hypertrophy in SHR.

Liver growth factor (LGF) is an endogenous albumin-bilirubin complex with antihypertensive effects in spontaneously hypertensive rats (SHR). We assessed the actions of LGF treatment on SHR mesenteric resistance and intramyocardial arteries (MRA and IMA, respectively), heart, and vascular smooth muscle cells (VSMC). SHR and Wistar-Kyoto (WKY) rats treated with vehicle or LGF (4.5 µg LGF/rat, 4 ip injections over 12 days) were used. Intra-arterial blood pressure was measured in anesthetized rats. The heart was weighted and paraffin-embedded. Proliferation, ploidy, and fibronectin deposition were studied in carotid artery-derived VSMC by immunocytochemistry. In MRA, we assessed: 1) geometry and mechanics by pressure myography; 2) function by wire myography; 3) collagen by sirius red staining and polarized light microscopy, and 4) elastin, cell density, nitric oxide (NO), and superoxide anion by confocal microscopy. Heart sections were used to assess cell density and collagen content in IMA. Left ventricular hypertrophy (LVH) regression was assessed by echocardiography. LGF reduced blood pressure only in SHR. LGF in vitro or as treatment normalized the alterations in proliferation and fibronectin in SHR-derived VSMC with no effect on WKY cells. In MRA, LGF treatment normalized collagen, elastin, and VSMC content and passive mechanical properties. In addition, it improved NO availability through reduction of superoxide anion. In IMA, LGF treatment normalized perivascular collagen and VSMC density, improving the wall-to-lumen ratio. Paired experiments demonstrated a partial regression of SHR LVH by LGF treatment. The effective cardiovascular antifibrotic and regenerative actions of LGF support its potential in the treatment of hypertension and its complications.

Hematopoiesis in aged female mice devoid of thyroid hormone receptors.

Hypothyroidism is often associated with anemia and immunological disorders. Similar defects are found in patients and in mice with a mutated dominant-negative thyroid hormone receptor α (TRα) and in knockout mice devoid of this receptor, suggesting that this isoform is responsible for the effects of the thyroid hormones in hematopoiesis. However, the hematological phenotype of mice lacking also TRß has not yet been examined. We show here that TRα1/TRß-knockout female mice, lacking all known thyroid hormone receptors with capacity to bind thyroid hormones, do not have overt anemia and in contrast with hypothyroid mice do not present reduced Gata1 or Hif1 gene expression. Similar to that found in hypothyroidism or TRα deficiency during the juvenile period, the B-cell population is reduced in the spleen and bone marrow of ageing TRα1/TRß-knockout mice, suggesting that TRß does not play a major role in B-cell development. However, splenic hypotrophy is more marked in hypothyroid mice than in TRα1/TRß-knockout mice and the splenic population of T-lymphocytes is not significantly impaired in these mice in contrast with the reduction found in hypothyroidism. Our results show that the overall hematopoietic phenotype of the TRα1/TRß-knockout mice is milder than that found in the absence of hormone. Although other mechanism/s cannot be ruled out, our results suggest that the unoccupied TRs could have a negative effect on hematopoiesis, likely secondary to repression of hematopoietic gene expression.

Recessive mutations in muscle-specific isoforms of FXR1 cause congenital multi-minicore myopathy.

FXR1 is an alternatively spliced gene that encodes RNA binding proteins (FXR1P) involved in muscle development. In contrast to other tissues, cardiac and skeletal muscle express two FXR1P isoforms that incorporate an additional exon-15. We report that recessive mutations in this particular exon of FXR1 cause congenital multi-minicore myopathy in humans and mice. Additionally, we show that while Myf5-dependent depletion of all FXR1P isoforms is neonatal lethal, mice carrying mutations in exon-15 display non-lethal myopathies which vary in severity depending on the specific effect of each mutation on the protein.

Physiological androgen insensitivity of the fetal, neonatal, and early infantile testis is explained by the ontogeny of the androgen receptor expression in Sertoli cells.

CONTEXT: Although gonadotropins and testosterone are high in the fetal/early postnatal periods, Sertoli cells remain immature and spermatogenesis does not progress. We hypothesized that Sertoli cells do not respond to testosterone because they do not express the androgen receptor. OBJECTIVE: The objective of the study was to describe the precise ontogeny of androgen receptor expression in the human testis from fetal life through adulthood. DESIGN: This was an immunohistochemical study on testicular biopsies from fetal, neonatal, prepubertal, pubertal, and adult human testes. MAIN OUTCOME MEASURES: Quantification of androgen receptor expression in Sertoli cells was measured. Evaluation of androgen receptor expression in peritubular and interstitial cells as well as anti-Müllerian hormone and inhibin-alpha was also performed. RESULTS: Androgen receptor expression was first observed in the nuclei of few Sertoli cells at the age of 5 months. Labeling was weak in 2-15% of Sertoli cells until 4 yr of age and progressively increased thereafter. High levels of androgen receptor expression were observed in more than 90% from the age of 8 yr through adulthood. Androgen receptor was positive in peritubular cells and variable in interstitial cells. Anti-Müllerian hormone immunolabeling was strong in all Sertoli cells from fetal life throughout prepuberty and weakened progressively as spermatogenesis developed. Inhibin-alpha expression was detected in all Sertoli cells from fetal life through adulthood. CONCLUSIONS: A lack of androgen receptor expression could explain a physiological Sertoli cell androgen insensitivity during fetal and early postnatal life, which may serve to protect the testis from precocious Sertoli cell maturation, resulting in proliferation arrest and spermatogenic development.

Benzylamine and Thenylamine Derived Drugs Induce Apoptosis and Reduce Proliferation, Migration and Metastasis Formation in Melanoma Cells.

Melanomas are heterogeneous and aggressive tumors, and one of the worse in prognosis. Melanoma subtypes follow distinct pathways until terminal oncogenic transformation. Here, we have evaluated a series of molecules that exhibit potent cytotoxic effects over the murine and human melanoma cell lines B16F10 and MalMe-3M, respectively, both ex vivo and in animals carrying these melanoma cells. Ex vivo mechanistic studies on molecular targets involved in melanoma growth, migration and viability were evaluated in cultured cells treated with these drugs which exhibited potent proapoptotic and cytotoxic effects and reduced cell migration. These drugs altered the Wnt/ß-catenin pathway, which is important for the oncogenic phenotype of melanoma cells. In in vivo experiments, male C57BL/6 or nude mice were injected with melanoma cells that rapidly expanded in these animals and, in some cases were able to form metastasis in lungs. Treatment with anti-tumor drugs derived from benzylamine and 2-thiophenemethylamine (F10503LO1 and related compounds) significantly attenuated tumor growth, impaired cell migration, and reduced the metastatic activity. Several protocols of administration were applied, all of them leading to significant reduction in the tumor size and enhanced animal survival. Tumor cells carrying a luciferase transgene allowed a time-dependent study on the progression of the tumor. Molecular analysis of the pathways modified by F10503LO1 and related compounds defined the main relevant targets for tumor regression: the activation of pro-apoptotic and anti-proliferative routes. These data might provide the proof-of-principle and rationale for its further clinical evaluation.

Short-term treatment of spontaneously hypertensive rats with liver growth factor reduces carotid artery fibrosis, improves vascular function, and lowers blood pressure.

OBJECTIVE: Liver growth factor (LGF), a mitogen for liver cells, reduces fibrosis in a rat model of cirrhosis. The present study assesses the possible vascular antifibrotic and antihypertensive effects of LGF treatment on spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR and normotensive Wistar Kyoto rats (WKY) were treated with LGF (4.5 microg LGF/rat i.p. twice a week for 2 weeks). Haemodynamic parameters were measured in anaesthetized rats. Vascular structure and function were studied in carotid arteries using optical and confocal microscopy, radioimmunoassay for desmosine, and isometric tension recording. RESULTS: LGF reduced systolic and diastolic blood pressure only in SHR. When compared to those of untreated SHR, carotid arteries from LGF-treated SHR showed: 1) a 50% reduction in collagen area and an increase in vascular smooth muscle cell number in the media, 2) no difference in total elastin content, but an increase in size of fenestrae in the internal elastic lamina, and 3) enhanced relaxation to acetylcholine, sodium nitroprusside, and forskolin. These effects were specific for SHR, since no changes were observed in LGF-treated WKY. CONCLUSION: Short-term treatment with a low dose of LGF induced a large improvement in vascular structure and function and significantly reduced blood pressure in a rat model of essential hypertension. The present results could open future research to explore the vascular effects of this endogenous factor in order to determine its potential as an antifibrotic and antihypertensive agent in humans.

Mild and Short-Term Caloric Restriction Prevents Obesity-Induced Cardiomyopathy in Young Zucker Rats without Changing in Metabolites and Fatty Acids Cardiac Profile.

Caloric restriction (CR) ameliorates cardiac dysfunction associated with obesity. However, most of the studies have been performed under severe CR (30-65% caloric intake decrease) for several months or even years in aged animals. Here, we investigated whether mild (20% food intake reduction) and short-term (2-weeks) CR prevented the obese cardiomyopathy phenotype and improved the metabolic profile of young (14 weeks of age) genetically obese Zucker fa/fa rats. Heart weight (HW) and HW/tibia length ratio was significantly lower in fa/fa rats after 2 weeks of CR than in counterparts fed ad libitum. Invasive pressure measurements showed that systolic blood pressure, maximal rate of positive left ventricle (LV) pressure, LV systolic pressure and LV end-diastolic pressure were all significantly higher in obese fa/fa rats than in lean counterparts, which were prevented by CR. Magnetic resonance imaging revealed that the increase in LV end-systolic volume, stroke volume and LV wall thickness observed in fa/fa rats was significantly lower in animals on CR diet. Histological analysis also revealed that CR blocked the significant increase in cardiomyocyte diameter in obese fa/fa rats. High resolution magic angle spinning magnetic resonance spectroscopy analysis of the LV revealed a global decrease in metabolites such as taurine, creatine and phosphocreatine, glutamate, glutamine and glutathione, in obese fa/fa rats, whereas lactate concentration was increased. By contrast, fatty acid concentrations in LV tissue were significantly elevated in obese fa/fa rats. CR failed to restore the LV metabolomic profile of obese fa/fa rats. In conclusion, mild and short-term CR prevented an obesity-induced cardiomyopathy phenotype in young obese fa/fa rats independently of the cardiac metabolic profile.

Altered mitochondrial function, capacitative calcium entry and contractions in the aorta of hypertensive rats.

OBJECTIVE: It has been suggested that Ca entry through store-operated Ca channels (SOCs) is regulated by a dynamic interplay between the endoplasmic reticulum Ca stores and the mitochondria. These relationships drive the activation and inactivation of SOCs, yet it remains unclear whether this regulation of SOCs by mitochondria is altered in the aorta of spontaneously hypertensive rats (SHRs). METHODS: We performed a thorough study of the mitochondrial membrane potential, the ability of mitochondria to deal with cytosolic Ca, capacitative Ca entry (CCE), and stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (orai1) protein expression, as well as the contractile capacity of aortic rings, in normotensive Wistar Kyoto rats (WKYs) and SHRs. RESULTS: Changes were observed in aortic tissue and cultured vascular smooth muscle cells isolated from SHRs relative to WKYs, including more depolarized mitochondria, stronger CCE upon the addition of Ca, larger cytosolic Ca transients (cytosolic Ca concentration) or aortic ring contraction elicited by endoplasmic reticulum depletion and a significant increase in STIM1 protein expression but not of orai1. CONCLUSION: These results suggest that the impaired Ca buffering capacity of partially depolarized mitochondria dysregulates CCE, leading to overfilling of the endoplasmic reticulum Ca store through enhanced STIM1/orai1 interactions and an increase in aorta contractions in SHRs. Thus, understanding the implications of the alterations to STIM1/orai1, and their relationship to mitochondria, may aid drug development and therapeutic strategies to treat hypertension, as well as its long-term sequelae in poorly controlled patients.

Primary testicular lesions are associated with testicular germ cell tumors of adult men.

The present study aims to establish the nature and frequency of testicular lesions in the parenchyma adjacent to testicular germ cell tumors (TGCT) to improve understanding of the factors involved in the development of testicular cancer. Fifty-three cases of TGCT that were fixed in both neutral-buffered formalin and Bouin solution, allowing for the nuclear characterization of Sertoli cells (SCs), were included in this study. In each case, at least 3 sections of different areas of preserved parenchyma surrounding the TGCT were studied. We found Leydig cell hyperplasia, microlithiasis, angiopathy, adenomatous hyperplasia of the rete testis, SC nodules, SC dysgenesis and involution, SC-only tubules, tubular atrophy, adluminal compartment lesions, hypospermatogenesis associated with spermatocyte sloughing, spermatogonial maturation arrest, and hypertrophic and multinucleated spermatogonia. These lesions were found in regions both adjacent and far away from the tumoral mass, and abnormal seminiferous tubules were found intermingled with those showing complete spermatogenesis, suggesting that these lesions are primary and existed before the development of the tumor. Our study suggests that SCs might play a more important role in the development of testicular tumors than previously thought. Our data supports the hypothesis that there is an abnormal differentiation of SCs, caused either by genetic anomalies or by environmental agents during fetal life. This abnormal SC differentiation may cause not only primary spermatogenesis failure and spermatogenesis arrest at different levels, but may also contribute to the poor differentiation of gonocytes into spermatogonia. The abnormal gonocyte differentiation might favor the development of dysplastic germ cells that may later transform into intratubular germ cell neoplasia, unclassified type.

PCPH expression is an early event in the development of testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) include various malignancies with distinct pathologies that share a common precursor lesion (intratubular germ cell neoplasia, unclassified, ITGCNU, or carcinoma in situ, CIS). TGCTs, as a whole, represent a highly curable tumor paradigm, with high sensitivity to radiotherapy and, especially, to cisplatin-based chemotherapy. However, a percentage of cases display therapeutic resistance, and the molecular mechanisms underlying such resistant phenotype remain to be elucidated. We put forward the notion that expression of oncogenic forms of the PCPH gene, which are known to confer resistance to radiation and chemotherapeutic drugs, including cisplatin, may be expressed in TGCTs, and thus contribute to the development of therapeutic resistance. To begin testing this concept, we studied PCPH expression in human TGCT cell lines and in 54 solid tumors by RT-PCR, western immunoblot and immunohistochemistry. The results demonstrated that: i) PCPH is expressed in TGCT cell lines and tumors, including CIS; ii) its expression levels vary among different TGCT pathologies, being generally higher in well differentiated regions and lower in areas of predominant proliferation; iii) PCPH expression is substantially increased in tumors relative to matched normal testicular tissue; iv) tumor samples express PCPH polypeptides of low molecular mass, consistent with the known size of the PCPH oncoprotein, that are either absent from, or markedly reduced in, matched normal tissue. Collectively, these results positively identify PCPH as a good early molecular marker for testicular neoplasms, and strongly indicate that immunodetection of truncated PCPH polypeptides may be a useful diagnostic tool for TGCT.

Age-related epididymis-like intratesticular structures: benign lesions of Wolffian origin that can be misdiagnosed as testicular tumors.

This study aims to characterize the epididymis-like intratesticular structures (ELITSs), a rare lesion found in elderly men. ELITSs were identified in 6 patients from a review of 1442 autopsies and 271 surgical specimens of adult men. Bilateral lesions were seen in 5 cases. The lesion was located in the proximity of the mediastinal rete testis (6 testes) and at the testicular periphery (4 testes), and at both central and peripheral locations in 1 case. The lesion is characterized by a pseudostratified cylindrical epithelium, with a robust pankeratin and 8, 18, and 19 keratin expression, focal vimentin expression, and apical CD 10 expression, similar to what is proper of the normal human epididymidis. The epithelial layer of ELITSs was surrounded by a thin layer of smooth-muscle cells. The adjacent testicular parenchyma was atrophied and the rete testis showed some associated degenerative lesions related to arteriosclerosis. The ELITSs are distinct from atrophic seminiferous tubules with a Sertoli cell-only pattern and from the benign glandular teratomatous component of an involution of a malignant testicular germ cell tumor, the so-called burn-out germ cell tumor. Clinical and histopathological data suggest that this lesion represents a late Wolffian differentiation similar to the initial segment of the epididymal duct, which represents an unusual manifestation of the aging process.