Publications by authors named "Paolo Romagnoli"

34 Publications

Archetype JC polyomavirus DNA associated with extracellular vesicles circulates in human plasma samples.

J Clin Virol 2020 07 19;128:104435. Epub 2020 May 19.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. Electronic address:

Background: JC polyomavirus (JCPyV) establishes a stable and successful interaction with the host, causing progressive multifocal leukoencephalopathy (PML) in immunocompromised subjects. Recently, it has been reported that JCPyV, like other viruses, may exploit extracellular vesicles (EV) in cell cultures.

Objective: To investigate the presence of JCPyV-DNA in EV circulating in human plasma obtained from patients at risk for PML.

Study Design: JCPyV-DNA status was studied in EV obtained from 170 plasma samples collected from 120 HIV positive patients and 50 healthy donors. EV were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81, annexin II, cythocrome C protein and, finally, by immunoelectron microscopy (IEM). Presence and quantitation of JCPyV-DNA were assessed with Multiplex real-time TaqMan PCR assay.

Results: The JCPyV-DNA plasma prevalence in 120 HIV positive patients and 50 healthy donors was 28% and 4%, respectively. The investigation performed on well-characterized plasma EV reported JCPyV-DNA detection in 15 out of 36 (42%) of the viremic samples (14 were from HIV patients and 1 from healthy people) at a mean level of 23.5 copies/mL. The examination of EV selected samples reported the percentage of JCPyV-DNA in EV of 5.4% of the total viral load. Moreover, IEM reported the presence of JCPyV Vp1 antigen in plasma-derived EV.

Conclusion: The potential role of EV-associated JCPyV-DNA open new avenues and mechanistic insights into the molecular strategies adopted by this polyomavirus to persist in the host and spread to the central nervous system.
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http://dx.doi.org/10.1016/j.jcv.2020.104435DOI Listing
July 2020

The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence from an In Vitro Tumor Microenvironment Crosstalk Model.

Cancers (Basel) 2019 Dec 4;11(12). Epub 2019 Dec 4.

Endocrinology Unit, Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50139 Florence, Italy.

Metabolic interplay between the tumor microenvironment and cancer cells is a potential target for novel anti-cancer approaches. Among stromal components, adipocytes and adipose precursors have been shown to actively participate in tumor progression in several solid malignancies. In adrenocortical carcinoma (ACC), a rare endocrine neoplasia with a poor prognosis, cancer cells often infiltrate the fat mass surrounding the adrenal organ, enabling possible crosstalk with the adipose cells. Here, by using an in vitro co-culture system, we show that the interaction between adipose-derived stem cells (ASCs) and the adrenocortical cancer cell line H295R leads to metabolic and functional reprogramming of both cell types: cancer cells limit differentiation and increase proliferation of ASCs, which in turn support tumor growth and invasion. This effect associates with a shift from the paracrine cancer-promoting IGF2 axis towards an ASC-associated leptin axis, along with a shift in the SDF-1 axis towards CXCR7 expression in H295R cells. In conclusion, our findings suggest that adipose precursors, as pivotal components of the ACC microenvironment, promote cancer cell reprogramming and invasion, opening new perspectives for the development of more effective therapeutic approaches.
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http://dx.doi.org/10.3390/cancers11121931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966501PMC
December 2019

Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

FASEB J 2019 02 24;33(2):2263-2277. Epub 2018 Sep 24.

Endocrinology Unit, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.

The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.
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http://dx.doi.org/10.1096/fj.201801028RRDOI Listing
February 2019

Torquetenovirus detection in exosomes enriched vesicles circulating in human plasma samples.

Virol J 2018 09 20;15(1):145. Epub 2018 Sep 20.

Department of Experimental and Clinical Medicine, University of Florence, Viale Morgagni 48, I-50134, Florence, Italy.

Background: Torquetenovirus (TTV) belongs to Anelloviridae family, infects nearly all people indefinitely without causing overt disease establishing a fine and successful interaction with the host. Increasing evidence have shown some human viruses exploit extracellular vesicles thereby helping viral persistence in the host. Here, the presence of TTV in extracellular vesicles circulating in human plasma was investigated.

Methods: TTV DNA was quantified in plasma-derived exosomes from 122 samples collected from 97 diseased patients and 25 healthy donors. Exosomes enriched vesicles (EEVs) were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81 and annexin II protein and, finally, by electron microscopy (EM). Presence and quantitation of TTV DNA were assessed with an universal single step real-time TaqMan PCR assay.

Results: Preliminary investigation showed that the human plasma extracted extracellular vesicles exhibited a main size of 70 nm, had concentration of 2.5 × 10/ml, and scored positive for tetraspanin CD63, CD81 and annexin II, typical characteristic of the exosomes vesicles. EEVs extracted from pooled plasma with TTV DNA viremia of 9.7 × 10 copies/ml showed to contain 6.3 × 10 TTV copies/ml, corresponding to 0.65% of total viral load. Important, TTV yield changed significantly following freezing/thawing, detergents and DNAse treatment of plasma before EEVs extraction. EEVs purified by sucrose-density gradient centrifugation and analysis of gradient fraction positive for exosomes marker CD63 harbored 10 TTV copies/ml. Moreover, EM evidenced the presence of TTV-like particles in EEVs. Successive investigation of plasma EEVs from 122 subjects (37 HIV-positive, 20 HCV infected, 20 HBV infected, 20 kidney transplant recipients, and 25 healthy) reported TTV DNA detection in 42 (34%) of the viremic samples (37 were from diseased patients and 5 from healthy people) at a mean level of 4.8 × 10 copies/ml. The examination of EEVs selected samples reported the presence of TTV genogroup 1, 3, 4 and 5, with genogroup 3 highly observed.

Conclusions: Collectively, although these observations should be confirmed by further studies, circulation of TTV particles in EEVs opens new avenues and mechanistic insights on the molecular strategies adopted by anelloviruses to persist in the host.
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http://dx.doi.org/10.1186/s12985-018-1055-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6149034PMC
September 2018

The microenvironment induces collective migration in -silenced mouse pheochromocytoma spheroids.

Endocr Relat Cancer 2017 10;24(10):555-564

Department of Experimental and Clinical MedicineUniversity of Florence, Florence, Italy

Pheochromocytomas (Pheos) and paragangliomas (PGLs) are neuroendocrine tumors. Approximately 30-40% of Pheos/PGLs are due to germline mutations in one of the susceptibility genes, including those encoding the succinate dehydrogenase subunits A-D (). Up to 2/3 of patients affected by mutated Pheo/PGL develop metastatic disease with no successful cure at present. Here, for the first time, we evaluated the effects of silencing in a three dimension (3D) culture using spheroids of a mouse Pheo cell line silenced or not (wild type/wt/control) for the SDHB subunit. We investigated the role of the microenvironment on spheroid growth and migration/invasion by co-culturing -silenced or wt spheroids with primary cancer-activated fibroblasts (CAFs). When spheroids were co-cultured with fibroblasts, -silenced cells showed a significant increase in matrigel invasion as demonstrated by the computation of the migratory areas ( < 0.001). Moreover, cells detaching from the -silenced spheroids moved collectively, unlike the cells of wt spheroids that moved individually. Additionally, silenced spheroids developed long filamentous formations along which clusters of cells migrated far away from the spheroid, whereas these structures were not present in wt spheroids. We found that lactate, largely secreted by CAFs, plays a specific role in promoting migration only of -silenced cells. In this study, we demonstrated that silencing increases tumor cell migration/invasion and that microenvironment, as represented by CAFs, plays a pivotal role in enhancing collective migration/invasion in Pheo -silenced tumor cells, suggesting their role in increasing the tumor metastasizing potential.
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http://dx.doi.org/10.1530/ERC-17-0212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441822PMC
October 2017

Inorganic nanoparticles as potential regulators of immune response in dendritic cells.

Nanomedicine (Lond) 2017 Jul 21;12(14):1647-1660. Epub 2017 Jun 21.

Department of Experimental & Clinical Medicine, University of Florence, via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy.

Aim: The spontaneous adsorption of proteins on nanoparticles (NPs) in biological media is exploited to prepare complexes of NPs and proteins from cancer cells' lysates for application in cancer immunotherapy.

Materials & Methods: Gold (Au) and silica NPs were synthesized, incubated with cancer cells' lysates and characterized. Dendritic cells (DCs) were challenged with protein-coated NPs, their maturation, viability and morphology were evaluated and lymphocytes T proliferation was determined.

Results: Silica and Au NPs bound different pools of biomolecules from lysates, and are therefore promising selective carriers for antigens. When incubated with immature DCs, NPs were efficiently endocytosed without cytotoxicity. Finally, protein-coated AuNPs promoted DC maturation and DC-mediated lymphocyte proliferation, at variance with lysate alone and protein-coated silica NPs, that did not promote DCs maturation.

Conclusion: These results demonstrate that the spontaneous formation of protein coronas on NPs represents a possible approach to fast, easy, cost-effective DCs stimulation.
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http://dx.doi.org/10.2217/nnm-2017-0061DOI Listing
July 2017

Interleukin 22 early affects keratinocyte differentiation, but not proliferation, in a three-dimensional model of normal human skin.

Exp Cell Res 2016 07 17;345(2):247-54. Epub 2016 May 17.

Department of Surgery and Translational Medicine, Università degli Studi di Firenze, 50125 Florence, Italy.

Interleukin (IL)-22 is a pro-inflammatory cytokine driving the progression of the psoriatic lesion with other cytokines, as Tumor Necrosis Factor (TNF)-alpha and IL-17. Our study was aimed at evaluating the early effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) keratinocyte (KC) proliferation, ii) terminal differentiation biomarkers as keratin (K) 10 and 17 expression, iii) intercellular junctions. Transmission electron microscopy (TEM) analysis was performed. A model of human skin culture reproducing a psoriatic microenvironment was used. Plastic surgery explants were obtained from healthy young women (n=7) after informed consent. Fragments were divided before adding IL-22 or a combination of the three cytokines, and harvested 24 (T24), 48 (T48), and 72 (T72)h later. From T24, in IL-22 samples we detected a progressive decrease in K10 immunostaining in the spinous layer paralleled by K17 induction. By TEM, after IL-22 incubation, keratin aggregates were evident in the perinuclear area. Occludin immunostaining was not homogeneously distributed. Conversely, KC proliferation was not inhibited by IL-22 alone, but only by the combination of cytokines. Our results suggest that IL-22 affects keratinocyte terminal differentiation, whereas, in order to induce a proliferation impairment, a more complex psoriatic-like microenvironment is needed.
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http://dx.doi.org/10.1016/j.yexcr.2016.05.004DOI Listing
July 2016

Patients with Griscelli syndrome and normal pigmentation identify RAB27A mutations that selectively disrupt MUNC13-4 binding.

J Allergy Clin Immunol 2015 May 11;135(5):1310-8.e1. Epub 2014 Oct 11.

Pediatric Oncology Network, Istituto Toscano Tumori (I.T.T.), Florence, Italy. Electronic address:

Background: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare and often fatal disorder characterized by defective cellular cytotoxicity and hyperinflammation, and the only cure known to date is hematopoietic stem cell transplantation. Mutations in RAB27A, LYST, and AP3B1 give rise to FHL associated with oculocutaneous albinism, and patients with FHL are usually only screened for mutations in these genes when albinism is observed. A number of patients with FHL and normal pigmentation remain without a genetic diagnosis.

Objective: We asked whether patients with FHL with immunodeficiency but with normal pigmentation might sometimes have mutations that affected cellular cytotoxicity without affecting pigmentation.

Methods: We carried out mutation analysis of RAB27A, LYST, and AP3B1 in patients with FHL with pigment dilution, as well as a cohort with no clinical evidence of pigment dilution but no mutations in the other known FHL-related genes (PRF1, STXBP2, and UNC13D).

Results: We identify patients with Griscelli syndrome type 2 with biallelic mutations in RAB27A in the absence of albinism. All 6 patients carried mutations at amino acids R141, Y159, or S163 of Rab27a that disrupt the interaction of Rab27a with Munc13-4, without impairing the interaction between melanophilin and Rab27a.

Conclusion: These studies highlight the need for RAB27A sequencing in patients with FHL with normal pigmentation and identify a critical binding site for Munc13-4 on Rab27a, revealing the molecular basis of this interaction.
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http://dx.doi.org/10.1016/j.jaci.2014.08.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418747PMC
May 2015

Stimulatory interactions between human coronary smooth muscle cells and dendritic cells.

PLoS One 2014 16;9(6):e99652. Epub 2014 Jun 16.

Department of Health Sciences, Clinical Pharmacology and Oncology Unit, University of Florence, Florence, Italy.

Despite inflammatory and immune mechanisms participating to atherogenesis and dendritic cells (DCs) driving immune and non-immune tissue injury response, the interactions between DCs and vascular smooth muscle cells (VSMCs) possibly relevant to vascular pathology including atherogenesis are still unclear. To address this issue, immature DCs (iDCs) generated from CD14+ cells isolated from healthy donors were matured either with cytokines (mDCs), or co-cultured (ccDCs) with human coronary artery VSMCs (CASMCs) using transwell chambers. Co-culture induced DC immunophenotypical and functional maturation similar to cytokines, as demonstrated by flow cytometry and mixed lymphocyte reaction. In turn, factors from mDCs and ccDCs induced CASMC migration. MCP-1 and TNFα, secreted from DCs, and IL-6 and MCP-1, secreted from CASMCs, were primarily involved. mDCs adhesion to CASMCs was enhanced by CASMC pre-treatment with IFNγ and TNFα ICAM-1 and VCAM-1 were involved, since the expression of specific mRNAs for these molecules increased and adhesion was inhibited by neutralizing antibodies to the counter-receptors CD11c and CD18. Adhesion was also inhibited by CASMC pre-treatment with the HMG-CoA-reductase inhibitor atorvastatin and the PPARγ agonist rosiglitazone, which suggests a further mechanism for the anti-inflammatory action of these drugs. Adhesion of DCs to VSMCs was shown also in vivo in rat carotid 7 to 21 days after crush and incision injury. The findings indicate that DCs and VSMCs can interact with reciprocal stimulation, possibly leading to perpetuate inflammation and vascular wall remodelling, and that the interaction is enhanced by a cytokine-rich inflammatory environment and down-regulated by HMGCoA-reductase inhibitors and PPARγ agonists.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099652PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059651PMC
January 2015

Pectus excavatum and heritable disorders of the connective tissue.

Pediatr Rep 2013 Sep 24;5(3):e15. Epub 2013 Sep 24.

Department of Pediatric Surgery, Children's Hospital A. Meyer Florence.

Pectus excavatum, the most frequent congenital chest wall deformity, may be rarely observed as a sole deformity or as a sign of an underlying connective tissue disorder. To date, only few studies have described correlations between this deformity and heritable connective tissue disorders such as Marfan, Ehlers-Danlos, Poland, MASS (Mitral valve prolapse, not progressive Aortic enlargement, Skeletal and Skin alterations) phenotype among others. When concurring with connective tissue disorder, cardiopulmonary and vascular involvement may be associated to the thoracic defect. Ruling out the concomitance of pectus excavatum and connective tissue disorders, therefore, may have a direct implication both on surgical outcome and long term prognosis. In this review we focused on biological bases of connective tissue disorders which may be relevant to the pathogenesis of pectus excavatum, portraying surgical and clinical implication of their concurrence.
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http://dx.doi.org/10.4081/pr.2013.e15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812532PMC
September 2013

Rib cartilage characterization in patients affected by pectus excavatum.

Anat Rec (Hoboken) 2013 Dec 29;296(12):1813-20. Epub 2013 Oct 29.

Department of Pediatric Surgery, University of Florence and Children's University Hospital A. Meyer, Florence, Italy.

Pectus excavatum (PE) is the most frequent anterior chest deformity which may be frequently associated with connective tissue disorders. We performed microscopic analyses to better understand cartilage behavior and obtain clues on its pathogenesis. In 37 PE patients, none with Marfan syndrome, we analyzed costal cartilage by light microscopy, immunohistochemistry and transmission electron microscopy. Control tissue specimens were harvested from four patients without any connective tissue disease. In both control and PE patients, chondrocytes were on the average <15 µm in diameter and occupied <10% of tissue volume; in most cases the extracellular matrix was stained by alcian blue, instead of safranin; no difference between PE and control samples was significant. All samples showed an uneven collagen type II immunolabeling both within the cells and pericellular matrix, and occasionally of the territorial matrix. In all cases numerous cells underwent apoptosis accompanied by matrix condensation as shown by electron microscopy. Our results suggest that matrix composition and the cell number and size of costal cartilage are dependent on the subject and not on the disease; the microscopic organization of cartilage is correlated with the stabilization of the defective shape rather than with the onset of the deformity.
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http://dx.doi.org/10.1002/ar.22824DOI Listing
December 2013

Rosiglitazone promotes the differentiation of Langerhans cells and inhibits that of other dendritic cell types from CD133 positive hematopoietic precursors.

Histol Histopathol 2014 03 24;29(3):323-32. Epub 2013 Jul 24.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Dendritic cells and their precursors express PPAR-gamma, whose stimulation has inhibitory effects on the maturation and function of dendritic cells in vivo. Dendritic cells can differentiate in vitro from CD133+ progenitors; the influence of PPAR-gamma stimulation on this process is unknown. We have addressed the effect of PPAR-gamma agonist rosiglitazone, at a concentration as used in clinics, on the differentiation of dendritic cells from human CD133+ progenitors. Cells were harvested from cord blood by density gradient and immunomagnetic separation, and cultured for 18 days with fetal calf serum, cytokines and 1 μmol/L rosiglitazone. Analyses included flow cytometry, electron microscopy and mixed lymphocyte reaction. As expected, control cells generated without rosiglitazone were dendritic, expressed MHC-II, CD80, CD83 and CD86 and stimulated mixed reaction potently. A minority of cells expressed the Langerhans cell marker CD207/langerin, but none contained Birbeck granules. With rosiglitazone much fewer cells were generated; they were all dendritic, expressed differentiation and maturation-related antigens in higher percentage and were better stimulators of lymphocytes than those generated without the drug. The vast majority of cells expressed CD207/langerin and many contained Birbeck granules, i.e. were full-fledged Langerhans cells. We conclude that stimulation of PPAR-gamma, while negatively affecting the number of generated cells, promotes the maturation of human cord blood CD133 positive precursors into efficient, immunostimulating dendritic cells with a Langerhans cell phenotype.
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http://dx.doi.org/10.14670/HH-29.323DOI Listing
March 2014

The ultrastructure of the muscle coat of human gastro-oesophageal junction, with special reference to "interstitial cells of Cajal".

Front Neurosci 2013 4;7:49. Epub 2013 Apr 4.

Department of Experimental and Clinical Medicine (Formerly: Department of Anatomy, Histology and Forensic Medicine, and in 1977 Institute of Histology and general Embryology), University of Florence Florence, Italy.

The muscle coat of the human lower oesophageal sphincter and stomach was studied 5 cm above and 4 cm below the gastro-oesophageal junction. Four subjects were operated on for motility disorders of the esophagus, two for a hypertensive lower oesophageal sphincter and two for an epiphrenic diverticulum; six subjects were operated on for oesophageal or gastric carcinomas. Specimens were fixed in phosphate-buffered OsO4, embedded in Epon, contrasted with uranyl acetate and lead citrate and observed under a Siemens Elmiskop Ia electron microscope. Both the oesophageal and gastric muscle cells, which showed features typical of this cell type, were innervated by multiple varicosities that were rich in synaptic vesicles; these varicosities were generally rarely encountered at distances less than 1000 Å from muscle cells. Only a very few, close neuromuscular junctions were detected. Special cells, which correspond to the "interstitial cells of Cajal" as reported by other authors, were discerned at the periphery of muscle cell bundles. These cells were characterized by an elongated cell body with many thin branches and an oval, sometimes indented nucleus. Some pinocytotic vesicles were located at the cell periphery. These cells were surrounded by a discontinuous basal lamina and were seen in close contact with each other and with muscle cells; the close contact areas were often very wide. The cytoplasm contained variable amounts of mitochondria, a well-developed smooth endoplasmic reticulum and a Golgi complex. As a characteristic feature, bundles of thin filaments were located at the cell periphery and were attached to electron-dense areas of the cell membrane. Morphologically, these filaments resembled myofilaments; they were present in variable amounts and were sometimes very numerous. The observation that the cytoplasmic organelles and filaments varied in number, is probably related to the different functional properties of these cells. Interstitial cells were richly innervated by varicose nerve fibers that were densely packed with synaptic vesicles; many close junctions to nerve endings were also detected. These morphological data lead us to assume that the interstitial cells demonstrated by the electron microscope do not correspond to the cells initially identified by Cajal and cannot even be considered connective tissue cells. We propose that they are specialized smooth muscle cells that are involved in generating spontaneous, myogenic electrical activity in the gastrointestinal tract.
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http://dx.doi.org/10.3389/fnins.2013.00049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616261PMC
April 2013

Advantage of affinity histochemistry combined with histology to investigate death causes: indications from sample cases.

J Forensic Sci 2011 Nov 9;56(6):1620-5. Epub 2011 Aug 9.

Department of Anatomy, Histology and Forensic Medicine, Section E Allara, Viale Pieraccini 6, 50139 University of Florence, Italy.

Mast cell histochemistry has been proposed in addition to classic histological methods to estimate the course of traumatic events before and after death. We have addressed the utility of this approach on nine victims of different types of trauma. Sections of wounded skin were stained with hematoxylin and eosin and with fluorescent avidin to tag mast cells. Mast cell numbers were evaluated by both direct and digitalized counts. Intact skin was used as control. The results on mast cells implemented the findings upon hematoxylin and eosin stain and helped to put the wounds and death in chronological sequence. Digitalized morphometry allowed to reduce intra- and inter-observer variation. We conclude that combined histological and histochemical analyses can be of practical use in forensic pathology, that a preliminary setting of the reference values is needed for each laboratory, and that image analysis can be of help for the quantification of the results.
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http://dx.doi.org/10.1111/j.1556-4029.2011.01866.xDOI Listing
November 2011

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors.

Blood 2011 Apr 8;117(15):3983-95. Epub 2011 Feb 8.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

CD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34(+) progenitors. Transforming growth factor-β1 (TGF-β1) and anti-TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti-TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133(+) progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.
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http://dx.doi.org/10.1182/blood-2010-08-299735DOI Listing
April 2011

Contacts between mast cells and dendritic cells in the human skin.

Ital J Anat Embryol 2010 ;115(1-2):25-30

Department of Anatomy, Histology and Forensic Medicine, Section "E. Allara", University of Florence, Italy.

Langerhans cells are a dendritic cell type characteristic of the epidermis. Since mast cells secrete molecules potentially influent on dendritic cell differentiation, we have addressed the degree of proximity between these two cell types in biopsies of skin diseases characterized by massive influx of dendritic cell precursors. By fluorescence microscopy, avidin labeled mast cells were found in contact with CD1a+ dendritic cells. By electron microscopy, contacts between mast cells and blunty dendritic cells were found in areas corresponding to those where CD1a+ cells were localized by immunohistochemistry. We propose that mast cells can induce the differentiation of precursors into Langerhans cells through both the release of short range acting soluble factors and contact-mediating plasma membrane molecules.
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January 2011

Inhibition of immune synapse by altered dendritic cell actin distribution: a new pathway of mesenchymal stem cell immune regulation.

J Immunol 2010 Nov 1;185(9):5102-10. Epub 2010 Oct 1.

Department of Neurological Sciences, University of Florence, Florence, Italy.

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306-318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell-cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC-DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.
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http://dx.doi.org/10.4049/jimmunol.1001332DOI Listing
November 2010

Drugs acting on mast cells functions: a cell biological perspective.

Inflamm Allergy Drug Targets 2010 Sep;9(4):214-28

Department of Anatomy, Histology and Forensic Medicine, Univeristy of Florence, Florence, Italy.

Mast cells are bone marrow derived cells capable of secreting many active molecules: mediators stored in specific granules, such as histamine and heparin; small molecules produced immediately upon stimulation, such as lipid derivatives and nitric oxide; and many constitutively secreted, pleiotropic cytokines. Thanks to these secretion products and perhaps direct cell-cell interactions, mast cells play roles in inflammation and tissue repair, angiogenesis and fibrosis. Mast cells themselves respond to many mediators of their own, giving rise to autocrine loops. Successful anti-allergic therapies have typically targeted the receptors for mast cell secretory products, particularly those for histamine. Among agents directly affecting mast cells, disodium chromoglycate and glucocorticoids are known since some time, while new pharmacological approaches may stem from the recognition of an interference with mast cell growth and differentiation by cyclosporine A, monoclonal antibodies, interferons, and JAK3 inhibitors. The action of agents that affect mast cell differentiation and function is considered here from a cell and tissue biological perspective as a premise to the application of these agents to the clinics, therefore special attention has been paid to references pertaining to humans.
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http://dx.doi.org/10.2174/187152810793358813DOI Listing
September 2010

Alcohol delamination in the treatment of recurrent corneal erosion: an electron microscopic study.

Br J Ophthalmol 2010 Jul;94(7):933-9

Department of Ophthalmology, University of Florence, Italy.

Aim: To investigate by electron microscopy the plane of separation of the epithelial sheet from its substratum in the procedure of alcohol delamination (ALD) in patients with recurrent corneal erosion syndrome.

Methods: Ten cases of recurrent corneal erosions (RCE) secondary to trauma and seven cases related to map-dot-fingerprint dystrophy (MDFP) were treated with ALD. The epithelial sheets obtained from these patients were examined by transmission electron microscopy. Similarly sheets obtained from 20 patients undergoing photorefractive keratectomy (10 by mechanical removal and 10 by ALD) were also examined as control group. Five further corneal buttons obtained at keratoplasty were treated with ALD and the epithelial sheet and corresponding stroma were both examined.

Results: In all specimens, whether removed mechanically or by ALD, the intercellular surfaces did not show any disruption and desmosomes were preserved. In patients with traumatic RCE and in corneal buttons obtained at keratoplasty, tissue separation occurred along the lamina lucida, whereas in patients with MDFP the whole basal lamina was removed along with the epithelium. Focal areas of basal cell degeneration and epithelial detachment from the basal lamina were also noted.

Conclusions: ALD enables efficient removal of the epithelium with an almost complete preservation of the lamina densa in traumatic RCE. In RCE due to MDFP the epithelium separates from the stroma below the basal lamina and may reflect the pathology of the condition.
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http://dx.doi.org/10.1136/bjo.2009.174409DOI Listing
July 2010

Safety and efficacy of rituximab in patients with hepatitis C virus-related mixed cryoglobulinemia and severe liver disease.

Blood 2010 Jul 22;116(3):335-42. Epub 2010 Mar 22.

Center for Systemic Manifestations of Hepatitis Viruses (MaSVE), Department of Internal Medicine, University of Florence, Florence, Italy.

The effectiveness of rituximab in hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC) has been shown. However, the risk of an increase in viral replication limits its use in cirrhosis, a condition frequently observed in patients with MC. In this prospective study, 19 HCV-positive patients with MC and advanced liver disease, who were excluded from antiviral therapy, were treated with rituximab and followed for 6 months. MC symptoms included purpura, arthralgias, weakness, sensory-motor polyneuropathy, nephropathy, and leg ulcers. Liver cirrhosis was observed in 15 of 19 patients, with ascitic decompensation in 6 cases. A consistent improvement in MC syndrome was evident at the end-of-treatment (EOT) and end-of-follow-up (EOF-U). Variable modifications in both mean viral titers and alanine aminotransferase values were observed at admission, EOT, third month of follow-up, and EOF-U (2.62 x 10(6), 4.28 x 10(6), 4.82 x 10(6), and 2.02 x 10(6) IU/mL and 63.6, 49.1, 56.6, and 51.4 IU/L, respectively). Improvement in liver protidosynthetic activity and ascites degree was observed at EOT and EOF-U, especially in more advanced cases. This study shows the effectiveness and safety of rituximab in MC syndrome with advanced liver disease. Moreover, the depletion of CD20(+) B cells was also followed by cirrhosis syndrome improvement despite the possibility of transient increases of viremia titers.
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http://dx.doi.org/10.1182/blood-2009-11-253948DOI Listing
July 2010

CO(2) laser therapy in Tis and T1 glottic cancer: indications and results.

Head Neck 2010 Mar;32(3):392-8

Department of Oto-Neuro-Ophthalmological Surgical Sciences, Division of Otorhinolaryngology-Head and Neck Surgery, University of Florence, Florence, Italy.

Background: Laser cordectomy for glottic cancer is still hampered by recurrence, which is more frequent upon anterior commissure (AC) involvement. Analysis of results may be a step to improve the efficacy of this therapy for early glottic cancer.

Methods: In all, 81 patients who underwent surgery with CO(2) laser for Tis and T1, AC0 to AC2 glottic carcinoma were followed up to 55 months.

Results: The incidence of recurrence increased significantly with T and AC classifications. The disease-free interval decreased with increasing T and AC classifications and with increasing severity of histology, but only the AC classification appeared significant. Recurrences occurred in 5 of 35 patients upon type I and type II cordectomy, in 16 of 24 patients upon type V cordectomy, and never upon type III and IV cordectomy.

Conclusions: Type I to type IV cordectomy, when indicated, can achieve radical treatment of most T1 glottic cancer. Type V cordectomy requires that any suspicion of cartilage invasion, even microscopic, be excluded.
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http://dx.doi.org/10.1002/hed.21200DOI Listing
March 2010

Rosiglitazone reduces the inflammatory response in a model of vascular injury in rats.

Shock 2009 Dec;32(6):638-44

Department of Experimental Medicine, Excellence Centre for Cardiovascular Diseases, Second University of Naples, Naples, Italy.

Thiazolidinediones are ligands that bind to and activate the nuclear peroxisome proliferator-activated receptor gamma. They are widely used as insulin sensitizers for the treatment of type 2 diabetes. Several studies have implicated the peroxisome proliferator-activated receptor gamma agonists rosiglitazone and pioglitazone in inflammatory events. To assess the anti-inflammatory properties of rosiglitazone, we investigated its effects on the molecular and cellular inflammatory response induced by a carotid injury in the rat. Male Wistar rats were randomized into a rosiglitazone-treated group (10 mg kg(-1) day(-1)) and a control group (0.9% w/v NaCl). The drug or vehicle was administered by gavage for 7 days before carotid injury and for up to 21 days after injury. The inflammatory markers p38 mitogen-activated protein kinase, cyclooxygenase 2, nuclear factor-kappaB, and heat shock protein 47 and the influx and activity of cells in response to injury were measured. Rosiglitazone treatment significantly reduced the expression of the inflammatory markers compared with control group. p38 mitogen-activated protein kinase and nuclear factor-kappaB started to decrease a few hours after injury, whereas cyclooxygenase 2 and heat shock protein 47 expression decreased 7 and 14 days, respectively, after injury. Rosiglitazone also reduced neointima formation and inflammatory cell infiltration. In conclusion, rosiglitazone negatively regulated the inflammatory events involved in tissue repair at molecular and cellular levels. These results suggest that rosiglitazone plays a protective role in inflammatory vascular diseases.
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http://dx.doi.org/10.1097/SHK.0b013e3181a5a377DOI Listing
December 2009

Smooth muscle cells, dendritic cells and mast cells are sources of TNFalpha and nitric oxide in human carotid artery atherosclerosis.

Thromb Res 2008 17;122(5):657-67. Epub 2008 Jun 17.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

Introduction: In atherogenesis, dendritic cells, beside presenting antigens, may be sources of tumour necrosis factor (TNF)alpha and nitric oxide (NO), together with mast cells and smooth muscle cells.

Material And Methods: We have looked at the expression of TNFalpha and inducible NO synthase (iNOs) by these cells by affinity cytochemistry in autoptical specimens from normal carotid arteries and not ruptured, hemorrhagic or calcified atheromata.

Results: Round to dendritic, major histocompatibility complex class II molecules (MHC-II+) cells and avidin-labeled mast cells were rare in normal arteries and significantly more numerous in atheromata. Many MHC-II+ cells expressed S-100 antigen; while a few were positive for phalloidin; appreciable fractions of these cells were immunoreactive for TNFalpha and iNOs, both in control specimens and atheromata. The fraction of mast cells labeled for iNOs was significantly lower in atheromata than in controls. Phalloidin positive cells were the most abundant cell type in the normal intima and atheromata; the fractions of these cells labeled for TNFalpha and iNOs were significantly higher in atheromata than in controls. Very few of these cells were also labeled for MHC-II. Computerized image analysis confirmed that the amounts of iNOs and TNFalpha were higher in atheromata than in controls. The increase in TNFalpha in atheromata was also confirmed by western blot.

Conclusions: Dendritic cells and mast cells can participate to the generation of TNFalpha and NO in the normal arterial wall and in atheromata, but myointimal cells are candidates as major sources of these molecules.
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http://dx.doi.org/10.1016/j.thromres.2008.04.013DOI Listing
December 2008

A role for transforming growth factor-beta1 in maintaining the differentiated state of Langerhans cells in human epidermis.

Ital J Anat Embryol 2006 Jul-Sep;111(3):133-49

Department of Anatomy, Histology and Forensic Medicine of the University of Florence, Florence, Italy.

Since during the generation of dendritic cells transforming growth factor (TGF)-beta1 is required to generate specifically Langerhans cells, we have addressed whether TGF-beta1 also affects the number and immunophenotype of Langerhans cells within the epidermis. Isolated human epidermal sheets were cultured as follows: serum free; with serum; serum free with TGF-beta1; with serum plus anti-TGF-betal; with serum plus an irrelevant antibody of the same isotype as anti-TGF-beta1. The expression of Langerhans cell antigens was analyzed by immunofluorescence and the preservation of epidermal structure and the expression of E-cadherin by electron microscopy. The number, surface area and perimeter length of Langerhans cells were measured and the results subjected to analysis of variance. Independent of serum, the architecture of the isolated epidermis was well preserved and E-cadherin was expressed for at least 48 h. In cultures without serum, Langerhans cells appeared well preserved when stained for MHC-II antigens. On the contrary, their number, surface area and perimeter length were significantly decreased upon labeling for CD1a and langerin, indicating altered expression and distribution of these differentiation specific antigens. These alterations were not accompanied by the expression of antigens of mature dendritic cells and were almost entirely prevented by serum. TGF-beta1, 1.0 ng/mL, had similar effects as serum on CD1a and langerin expression and distribution within cells, whereas anti-TGF-beta1 antibodies neutralized the effect of serum. The results indicate that Langerhans cells depend on soluble factors for the maintenance of the differentiated state within epidermis and that TGF-beta1 is a major one of such factors.
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April 2007

Inflammatory events in a vascular remodeling model induced by surgical injury to the rat carotid artery.

Br J Pharmacol 2006 Jan;147(2):175-82

Department of Experimental Medicine, Excellence Centre on Cardiovascular Diseases, Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy.

1.--The aim of our study was to gain insight into the molecular and cellular mechanisms of the inflammatory response to arterial injury in a rat experimental model. 2.--Rats (five for each experimental time) were subjected to brief clamping and longitudinal incision of a carotid artery and monitored for 30 days. Subsequently, Nuclear Factor-kappaB (NF-kappaB) expression was measured by electrophoretic mobility shift assay. Heat shock protein (HSP) 27, HSP47 and HSP70 were evaluated by Western blot. Morphological changes of the vessel wall were investigated by light and electron microscopy. 3.--In injured rat carotid artery NF-kappaB activity started immediately upon injury, and peaked between 2 and 3 weeks later. Western blot showed a significant increase of HSP47 and HSP70 7 days after injury. At 2 weeks postinjury, HSP27 expression peaked. Light microscopy showed a neointima formation, discontinuity of the media layer and a rich infiltrate. Among infiltrating cells electron microscopy identified dendritic-like cells in contact with lymphocytes. 4.--Our model of surgical injury induces a significant inflammatory process characterized by enhanced NF-kappaB activity and HSPs hyperexpression. Dendritic-like cells were for the first time identified as a novel component of tissue repair consequent to acute arterial injury.
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http://dx.doi.org/10.1038/sj.bjp.0706472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1615853PMC
January 2006

Differential diagnosis between uterine carcinosarcoma versus carcinoma with sarcomatous metaplasia: an immunohistochemical and ultrastructural case study.

Ultrastruct Pathol 2005 Mar-Apr;29(2):149-55

Department of Human Pathology and Oncology, University of Florence, School of Medicine, Florence, Italy.

Uterine carcinosarcomas are biphasic neoplasms with carcinomatous and sarcomatous elements. However, several elements suggest that carcinosarcomas may be more closely related to carcinoma of the endometrium and that they arise from an unique stem cell. Recently, the authors observed an uterine tumor that at histologic examination showed an apparently double population of cells: malignant epithelial element admixed with mesenchymal spindle-shaped cells. The immunohistochemical stainings instead showed cytokeratin positivity and negativity for stromal markers. Electron microscopy showed the neoplastic tissue to be made of a single population of poorly differentiated epithelial cells, thus confirming the immunohistochemical findings and leading to the diagnosis of uterine metaplastic carcinoma.
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http://dx.doi.org/10.1080/019131290924072DOI Listing
August 2005

Skin cancer and immunosuppression.

Crit Rev Oncol Hematol 2005 Oct;56(1):127-36

Department of Dermatological Sciences, University of Florence Medical School, Via degli Alfani, 37-50121 Florence, Italy.

All immunosuppressive treatments, either pharmacological or physical, have the potential to impair the skin immune system network of cells and cytokines, thus leading to an increased incidence of skin cancer. Since skin cancer in transplant recipients may show uncommon clinical features and have an unusually aggressive course, transplant patients should be strictly followed up by experienced dermatologists in order to diagnose and treat properly any skin cancer in an early phase. Importantly, due to the fact that sun exposure increases immunosuppression in the skin, patients should be clearly informed about the additional risk of sun exposure and the preventive measures to be taken.
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http://dx.doi.org/10.1016/j.critrevonc.2004.11.011DOI Listing
October 2005

Cerebellar liponeurocytoma: morphological, immunohistochemical, and ultrastructural study of a relapsed case.

Neuropathology 2005 Mar;25(1):77-83

Departments of Human Pathology and Oncology, University of Florence, Florence, Italy.

Cerebellar liponeurocytoma is a rare and newly identified neoplasm found in adults which is reputed to be benign. Its salient morphological characteristics are advanced neuronal/neurocytic differentiation, the presence of lipomatous areas, low mitotic rate, and the absence of necrosis, pleomorphism and vascular hyperplasia. Reported is a case of relapsing liponeurocytoma which occurred 3 and a half years after the radical excision of the primary lesion. Histopathological aggressive features (mitoses and a high proliferation index as evaluated by MIB-1) were shown in the primary lesion and recurrence of the tumor. We suggest that liponeurocytoma is an uncertain malignant potential lesion when mitoses are present and the MIB-1 positive cells are more than 10%.
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http://dx.doi.org/10.1111/j.1440-1789.2004.00574.xDOI Listing
March 2005

Embryological study of the glottic site and clinical implications.

Oral Oncol 2004 Nov;40(10):1017-25

Departments of Oto-Neuro-Ophthalmology, Section of Otolaryngology, University of Florence, I-50134 Florence, Italy.

The development of the glottic site, in particular of its ventral area, was studied to better understand the spreading pathways of T1, T2 cancer. Serial sections of larynges from human embryos, fetuses and adults were observed. A dorsal, a ventral and an intermediate compartment were found on the basis of their maturation schedule. A commissure muscle which develops in the anterior one third of the glottic site and wraps the connection system of vocal ligaments was recognized. The inferior paraglottic space, the compartment structures and the localization of superficial and deep blood vessels and of glands in the ventral compartment and the components of Broyles ligament were studied during ontogenesis. The compartments identified here have clinical and oncological relevance. Their detailed knowledge offers a prerequisite for planning and performing compartment conservative surgery in T1, T2 cancer, based on their spreading pathways.
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http://dx.doi.org/10.1016/j.oraloncology.2004.05.004DOI Listing
November 2004

Interferon-alpha affects the tumour necrosis factor-alpha content of mast cells in human nasal mucosa. A pilot study in allergic patients.

Ital J Anat Embryol 2004 Apr-Jun;109(2):115-22

Department of Anatomy, Histology, and Forensic Medicine, Sect. E. Allara, University of Florence, Italy.

Human nasal mucosal mast cells contain and secrete tumour necrosis factor (TNF)-alpha, which in turn can stimulate histamine secretion by these cells. Interferon (IFN)-alpha can inhibit TNF-alpha secretion by mast cells in vitro. We have addressed the interrelationships between IFN-alpha and the content in TNF-alpha and number of mast cells in vivo, in the human nasal mucosa. Biopsies were taken from two healthy control patients, two allergic patients and two more allergic patients treated topically with IFN-alpha for two weeks; biopsies from the last two patients were taken both before and after stimulation with the specific allergen. Mast cells were counted upon tagging with rhodaminated avidin and by indirect immunofluorescence for TNF-alpha. Data were subjected to analysis of variance. Mast cell numbers were significantly lower in all allergic patients than in controls (P<0.001). Upon IFN-alpha treatment, TNF-alpha positive mast cells were less than in allergic, untreated patients and the opposite was true for TNF-alpha negative mast cells (p<0.05). Allergen challenge caused selective, significant decrease only in the number of TNF-alpha negative mast cells (p<0.05). The results suggest that upon topical IFN-alpha treatment: (1) mast cells stores of TNF-alpha in the nasal mucosa of allergic patients are decreased; and (2) only TNF-alpha negative cells degranulate in response to allergen challenge. Therefore, one may expect that such a treatment reduces the TNF-alpha burden to the mucosa in these patients.
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December 2004