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Vascular calcification (VC) represents a major cardiovascular risk factor in chronic kidney disease patients. High phosphate (Pi) levels are strongly associated with VC in this population. Therefore, Pi binders are commonly used to control high Pi levels. The aim of this work was to study the mechanism of action of lanthanum chloride (LaCl3) on the progression of Pi-induced VC through its direct effect on vascular smooth muscle cells (VSMCs) in vitro. High Pi induced VSCM Ca deposition. We evaluated the action of LaCl3, compared to gadolinium chloride (GdCl3), and found different effects on the modulation of VSMC lineage markers, such as α-actin and SM22α. In fact, only LaCl3 preserved the expression of both VSMC lineage markers compared to high Pi-treated cells. Interestingly, both LaCl3 and GdCl3 reduced the high Pi-induced elevations of bone morphogenic protein 2 mRNA expression, with no reduction of the high core binding factor-alpha 1 mRNA levels observed in calcified VSMCs. Furthermore, we also found that only LaCl3 completely prevented the matrix GLA protein mRNA levels and osteonectin protein expression elevations induced by high Pi compared to GdCl3. Finally, LaCl3, in contrast to GdCl3, prevented the high Pi-induced downregulation of Axl, a membrane tyrosine kinase receptor involved in apoptosis. Thus, our results suggest that LaCl3 prevents VC by preserving VSMC lineage markers and by decreasing high Pi-induced osteoblastic differentiation.

Aims: Pulmonary adenofibromas are rare benign fibroepithelial tumours of the lung with unknown histogenesis and an indolent clinical behaviour. Their stroma resembles that of solitary fibrous tumours, whereas the glands are composed of respiratory epithelium organized in a phyllodes-like architecture. Differentiation of pulmonary adenofibromas from other more aggressive intrathoracic tumours is clinically relevant. However, their biology is unknown. Here, we sought to characterize pulmonary adenofibromas at a clinicopathological level and to define whether they could be underpinned by a highly recurrent somatic genetic alteration akin to tumours with similar morphology. Methods and results: Seven pulmonary adenofibromas were subjected to immunohistochemical analysis for thyroid transcription factor 1 (TTF1), napsin A, cytokeratin 7, E-cadherin, CD99, CD34, CD31, STAT6, oestrogen receptor (ER), progesterone receptor, androgen receptor, bcl-2, and vimentin, as well as electron microscopy and capillary sequencing on microdissected samples to evaluate the presence of NAB2–STAT6 fusion genes and MED12 exon 2 mutations in their discrete components. A control group comprising pulmonary solitary fibrous tumours, pulmonary hamartomas and breast fibroadenomas was also analysed. We confirmed that the stromal elements of pulmonary adenofibromas pertain to the fibroblastic lineage, and show ER overexpression in 71% of cases, whereas the epithelium consists of TTF1-positive, E-cadherin positive bronchiolar elements. A highly recurrent NAB2–STAT6 fusion variant (exon 4–exon 2) was detected in the stroma but not in the epithelium. No MED12 mutations were identified. Conclusions: Here, we demonstrate that pulmonary adenofibromas are neoplastic lesions harbouring the molecular hallmark of solitary fibrous tumours

Inflammatory bowel disease (IBD) represents a socially and clinically relevant disorder, characterized by intestinal chronic inflammation. Cystamine (CysN) is a multipotent molecule with healthy effects and, moreover, it is an inhibitor of transglutaminases (TGs), including the TG type 2 (TG2), an enzyme with pleiotropic functions, involved in different pathways of inflammation and central in the pathogenesis of some human disorders as the IBD. Our aim was to evaluate the effect of CysN in an IBD rat model. A total of 30 rats were divided into 4 groups: controls without treatment (CTR; n=7); receiving the 2,4,6-trinitrobenzene sulfonic acid enema (TNBS group; n=8); treated with TNBS enema plus oral CysN (TNBS-CysN group; n=8); treated with CysN (CysN group; n=7). After killing, bowel inflammation was evaluated applying specific scores. TG activity, TG2 and isopeptide bond immunohistochemical expression, and tumor necrosis factor-α (TNF-α) were evaluated in the colonic tissue, such as interleukin-6 (IL-6) serological levels (ELISA). TG2 was also evaluated on the luminal side of the colon by immunoautoradiography. Colonic samples from IBD patients were compared with animal results. TNBS-CysN group developed a less severe colitis compared with the TNBS group (macroscopic score 0.43±0.78 vs 3.28±0.95, microscopic score 6.62±12.01 vs 19.25±6.04, P

http://hdl.handle.net/20.500.11768/96503 The combined variant of small-cell lung carcinoma (SCLC) refers to the variable admixture of small cell and non-small cell carcinoma, whereas the association with sarcoma or sarcoma-like elements is exceedingly rare. A 76-year-old Caucasian man underwent right upper lobectomy with regional lymphadenectomy because of a symptomatic 7 cm-sized tumor mass. Formalin fixed-paraffin embedded material was used to highlight several differentiation cell lineages by means of immunohistochemistry, electron microscopy, and mutational assay. The tumor was discovered as being IIB stage (pT2b pN1(1/51) pM0) and featured biphasic appearance with close intermingling of SCLC (40%) and collagen-rich spindle cell sarcoma (60%). Epithelial (cytokeratins, TTF-1), neural (neurofilaments, GFAP), endocrine (chromogranin, synaptophysin, CD56), and skeletal muscle (desmin, sarcomeric actin, myogenin) markers were variably co-expressed by SCLC elements, whereas mesenchymal (vimentin), smooth muscle (actin, myosin, H-caldesmon, calponin), fibroblastic (CD10), and, more focally, skeletal muscle (desmin, sarcomeric actin and myogenin) markers were highlighted in the spindle cell sarcoma elements. TP53 codon V274F mutation in exon 8 was shared by either cell component. After undergoing adjuvant chemotherapy, the patient is currently alive and well at the 40-month follow-up. To the best of our knowledge, this is the first report of combined SCLC with quadripartite differentiation of epithelial, neuroendocrine, skeletal muscle, and myofibroblastic type, somewhere at the level of the same individual tumor cells. This tumor had probably derived for clonal evolution of a p53-mutated common ancestor lesion.

Circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) are currently being investigated in a variety of diseases as markers of vascular turnover or damage and, also in the case of CEPs, vasculogenesis. CEPs appear to have a "catalytic" role in different steps of cancer progression and recurrence after therapy, and there are preclinical and clinical data suggesting that CEC enumeration might be useful to select and stratify patients who are candidates for anti-angiogenic treatments. In some types of cancer, CECs and CEPs might be one of the possible hidden identities of cancer stem cells. The definition of CEC and CEP phenotype and the standardization of CEC and CEP enumeration strategies are highly desirable goals in order to exploit these cells as reliable biomarkers in oncology clinical trials.

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. Results: Sorted DNA/Syto16+CD45−CD31+CD146+ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P < 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

Intestinal lymphangiectasia is a rare condition, which is characterized by the dilation of small bowel lymphatics and presents with signs and symptoms of protein-losing enteropathy. Some patients have complained of occlusive symptoms attributable to the mechanical obstruction caused by the considerable mucosal edema associated with the lymphatic dilation. On the basis of the hypothesis that alterations in the neuromuscular structures controlling clearance function or gut tone may play a role in ileal dilation, we examined the resected ileum of a 48-year-old male patient with segmental lymphangiectasia histologically, immunohistochemically (for S100 protein, PGP 9.5, Bcl-2, neuron-specific enolase, neurofilaments, synaptophysin, and CD117/C-kit), and by means of electron microscopy. Histology showed pseudocystic dilation of the mucosal, submucosal, and muscular lymphatics with fragmentation of the circular and longitudinal muscle layers. Hardly any neural expression of synaptophysin was observed, but the neural structures were otherwise morphologically normal and reacted normally to the other neural markers. This case shows that neuromuscular alterations can be found in the dilated ileum of patients with segmental lymphangiectasia.

An increasing amount of patients affected by advanced chronic kidney disease suffer from vascular calcification (VC) that associates with cardiovascular morbidity and mortality. In this study, we created a new experimental in vitro model, trying to better elucidate high phosphate (Pi)-induced VC pathogenic mechanisms. Rat aortic vascular smooth muscle cells (VSMCs) were challenged for 7-10 days with high Pi with a repeated and short suspensions of high Pi treatment (intermittent suspension, IS) that was able to induce a significant inhibition of high Pi calcification, maximal at 5 h. Interestingly, the delay in calcification is a consequence of either the absence of free Pi or calcium-phosphate crystals being comparable to the total effect obtained during the 5 h-IS. The protective effect of IS was mediated by the reduction of apoptosis as demonstrated by the action of 20 μmol/L Z-VAD-FMK and by the preservation of the pro-survival receptor Axl expression. Furthermore, autophagy, during IS, was potentiated by increasing the autophagic flux, evaluated by LC3IIB western, while treating VSMCs with 1 mmol/L valproic acid did not affect VC. Finally, IS prevented VSMC osteoblastic differentiation by preserving smooth muscle lineage markers expression. Our data support the hypothesis that to delay significantly VC is necessary and sufficient the IS of high Pi challenge. The IS was able to prevent significantly apoptosis, to induce a potentiation in autophagy, and to prevent osteoblastic differentiation by preserving SM lineage markers.

Background: In an attempt to clarify the role of gliadin toxicity in the pathogenesis of gluten intolerance (celiac disease), previous in vitro studies have been based on two‐dimensional human cell...

Summary Background Quantitative fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low/unmeasurable plasma fibrinogen antigen levels. Their genetic basis is invariably represented by mutations within the fibrinogen genes (FGA, FGB and FGG coding for the Aα, Bβ and γ chains). Currently, only four mutations (p.Gly284Arg, p.Arg375Trp, delGVYYQ 346-350, p.Thr314Pro), all affecting the fibrinogen γ chain, have been reported to cause fibrinogen storage disease (FSD), a disorder characterized by protein aggregation, endoplasmic reticulum retention and hypofibrinogenemia. Objectives To investigate the genetic basis of FSD in two hypofibrinogenemic patients. Methods The mutational screening of the fibrinogen genes was performed by direct DNA sequencing. The impact of identified mutations on fibrinogen structure was investigated by in-silico molecular modeling. Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. Results Here, we describe two hypofibrinogenemic children with persistent abnormal liver function parameters. Direct sequencing of the coding portion of fibrinogen genes disclosed two novel FGG missense variants (p.Asp316Asn, fibrinogen Pisa; p.Gly366Ser, fibrinogen Beograd), both present in the heterozygous state and affecting residues located in the fibrinogen C-terminal γ-module. Liver sections derived from biopsies of the two patients were examined by immunocytochemical analyses, revealing hepatocyte cytoplasmic inclusions immunoreactive to anti-fibrinogen antibodies. Conclusions Our work strongly confirms the clustering of mutations causing FSD in the fibrinogen γ chain between residues 284 and 375. Based on an in-depth structural analysis of all FSD-causing mutations and on their resemblance to mutations leading to serpinopathies, we also comment on a possible mechanism explaining fibrinogen polymerization within hepatocytes.

ABSTRACT: Aim: The aim of our study was to investigate whether biofilm production by Candida albicans clinical isolates could be a hallmark of virulence in vivo. Materials & methods: Twenty clinical isolates of C. albicans were examined via histological studies on larvae infected with various fungal doses (from 103 to 105 CFU/larva) of biofilm producer and nonproducer strains. Results: The poor prognostic role of infection due to a biofilm-producing isolate was confirmed by the Wald test (hazard ratio: 2.63; 95% CI: 2.03–3.41). Histological examinations at 24 h showed a strong innate immune response, with evidence of melanization for both infection groups. However, at 48 h, we found huge differences in filamentation and tissue invasion capability between biofilm nonproducing and producing isolates, the latter being highly organized into biofilm and invading the larval intestinal tract. Invasion corroborated survival data. Conclusion: The histological results demonstrate that the production of biofilm could enhance the invasiveness of C. albicans.

The invertebrate model Galleria mellonella is a widely used factitious host to study the microbial pathogenesis in vivo. However, a specific procedure for the recovery and the processing of the infected tissues, important for a better understanding of the host-pathogen interactions, has not been reported to our knowledge. In the present study we describe a new procedure of fixation and processing of larval tissue that allows studying the larval topographic anatomy and assessing the morphological changes due to the fungal infection. Lepidopteran larvae were infected with Candida albicans strains displaying various biofilm-forming abilities. The whole larvae were then examined for tissue changes by histological techniques. We show that comparing cutting planes, serial transversal sections of paraffin-embedded larva result in better accuracy and information recovering. Using this technique, it was possible to preserve the integrity of G. mellonella internal structures allowing the detailed analysis of morphological differences in different experimental groups (i.e., healthy vs infected larvae). We were also able to study strain-related differences in the pathogenesis of C. albicans by observing the immune response elicited and the invasiveness of two isolates within the larval tissues. In general, by processing the whole larva and optimizing routinely histochemical stainings, it is possible to visualize and analyse infected tissues. Various degrees of pathogenicity (strain- or inoculum-related), and the infection time course can be described in details. Moreover, the host immune response events can be followed throughout the infectious process leading to a comprehensive picture of the studied phenomenon.

Autophagy is an inducible catabolic process that responds to environment and is essential for cell survival during stress, starvation and hypoxia. Its function in the human placenta it is not yet understood. We collected 14 placentas: 7 at vaginal delivery and 7 at elective caesarean section after uneventful term pregnancies. The presence of autophagy was assessed in different placental areas by immunoblotting, immunohistochemistry and electron microscopy. We found that autophagy is significantly higher in placentas obtained from cesarean section than in those from vaginal delivery. Moreover there is a significant inverse relationship between autophagy and umbilical arterial glucose concentration.

Cancer blood vessels consist of two interacting types of cells: inner lining endothelial cells (ECs) and surrounding perivascular cells (pericytes, vascular smooth muscle cells or mural cells). PDGFRbeta(CD140b)+ progenitor perivascular cells (PPC) can differentiate into pericytes and regulate vessel stability and vascular survival in tumors. Similarly to what we have done with circulating ECs and progenitors, we developed a flow cytometry procedure for the enumeration of circulating PPCs and the study of their viability in murine models of cancer and in cancer patients. DNA+CD45−CD31−CD140b+ cells were enumerated by six-colour flow cytometry, their morphology was studied by electron microscopy, PPC specificity confirmed by reverse trascription-PCR (RT-PCR) expression of CD140b mRNA, and viability assessed by Syto16 and 7AAD. In preclinical marrow transplantation studies, 9 ± 4% of circulating PPCs were derived from the marrow donor. PPCs were increased in cancer-bearing mice and in patients affected by some types of cancer. At variance with the kinetic of circulating endothelial progenitors, high-dose cyclophosphamide reduced the number of viable PPCs. The administration of sunitinib, a drug known to inhibit PDGFR, was associated in murine models and in cancer patients with an increase of apoptotic/necrotic circulating PPC, suggesting a direct targeting of these cells. PPC enumeration might be studied as a tool for the definition of the optimal biologic dose of anti-PDGFR drugs and investigated clinically as a possible predictive/prognostic tool in patients receiving anti-PDGFR drugs.

Tight junctions play a pivotal role in maintaining the integrity of the intestinal barrier. Their alteration is involved in the pathogenesis of celiac disease. Our aim was to investigate the gliadin effect on the tight junction proteins in an in vitro three-dimensional cell culture model through imaging analyses. Lovo multicellular spheroids were treated with enzymatically digested (PT) gliadin 500 μg/mL and its effect on actin, occludin and zonula occludens-1, was evaluated by means of confocal laser microscopy, transmission electron microscopy and image capture analysis. Compared to untreated spheroids, PT-gliadin-treated ones showed enlargement of the paracellular spaces (9.0±6.9 vs. 6.2±1.7 nm, p

Background Bone Marrow (BM) progenitor cells can target the site of myocardial injury, contributing to tissue repair by neovascolarization and/or by a possible direct paracrine effect on the inflammatory cascade. Angiotensin Converting Enzyme inhibitors (ACE-I) are effective in reducing mortality and preventing left ventricular (LV) function deterioration after myocardial infarction. Methods We investigated the short term effects of BM mononuclear cells (BMMNCs) therapy on the pro-inflammatory cytokines (pro-CKs) and on LV remodelling and compared these effects over a standard ACE-I therapy in a rat model of myocardial cryodamage. Forty two adult inbread Fisher-F344 rats were randomized into three groups: untreated (UT; n = 12), pharmacological therapy (ACE-I; n = 14, receiving quinapril), and cellular therapy (BMMNCs; n = 16, receiving BMMNCs infusion). Rats underwent to a standard echocardiogram in the acute setting and 14 days after the damage, before the sacrifice. Pro-CKs analysis (interleukin (IL)1β, IL-6, tumor necrosis factor (TNF)α was performed (multiplex proteome arrays) on blood samples obtained by direct aorta puncture before the sacrifice; a control group of 6 rats was considered as reference. Results Concerning the extension of the infarcted area as well as the LV dimensions, no differences were observed among the animal groups; treated rats had lower left atrial diameters and higher indexes of LV function. Pro-Cks were increased in infarcted-UT rats if compared with controls, and significantly reduced by BMMNCs and ACE-I ; TNFα inversely correlated with LV fractional shortening. Conclusion After myocardial infarction, both BMMNCs and ACE-I reduce the pattern of pro-Ck response, probably contributing to prevent the deterioration of LV function observed in UT rats.

Congenital dyserythropoietic anemia (CDA) type II is the most frequent type of congenital dyserythropoietic anemia; it is transmitted in an autosomal recessive fashion and is characterized by ineffective erythropoiesis, peripheral hemolysis, bi-multinuclearity in the erythroblasts, and hypoglycosylation of red blood cell (RBC) membrane proteins such as band 3. The disease is generally caused by biallelic mutations in the SEC23B gene. However, there are a small portion of patients with clinical and hematologic features of CDA II that are negative for mutations in SEC23B, suggesting that alternative etiologies for such disturbed erythropoiesis exist. We identified two siblings of Italian origin who had dyserythropoiesis with a chronic macrocytic anemia. Their parents were healthy with normal hematologic parameters. No history of consanguinity for at least three generations was noted. The affected siblings had anisopoikylocytosis on peripheral blood smear with stomatocytes (8-9%), spherocytes (4-5%), rare ovalocytes, and dacryocytes. RBCs osmotic fragility was increased but the red cells had normal eosin-5-maleimide (EMA)-binding. Serum ferritin and transferrin saturation were increased in only one sibling. Bone marrow morphology revealed erythroid hyperplasia (myeloid: erythroid ratio = 0.6) with binuclearity and megaloblastic changes, as well as occasional cytoplasmic bridging between cells at different stage of maturation; electron microscopy of bone marrow erythroblasts showed multiple membranes that ran parallel to the plasma membrane or that were grouped in stacked segments, possibly attributable to residual endoplasmic reticulum (ER) cisternae. SDS-PAGE analysis of RBC ghosts from both siblings demonstrated hypoglycosylation of band 3 and GLUT1, as well as residual residual Protein Disulphide Isomerase (PDI) positive ER remnants, as observed in classical CDA II cases. However, in contrast to CDAII, the Ham's test performed with 15 normal serum samples was negative, and no mutations were detected in the SEC23B gene. To uncover the underlying etiologies, whole-exome sequencing was conducted on all available family members. After filtering for common variants, only a single gene had biallelic mutations in the affected siblings, which were transmitted from the unaffected heterozygous parents. The identified mutations resided in the PARP4 gene, which encodes a poly-ADP ribose polymerase enzyme, and were predicted to be deleterious. We demonstrate that knockdown of PARP4 using shRNA in primary human erythroid progenitors results in impaired erythroid differentiation and increased apoptosis. In addition, morpholino-mediated knockdown of the PARP4 orthologue in the zebrafish resulted in dyserythropoiesis and anemia in developing embryos. Sequencing of PARP4 in additional rare cases of CDA II without an identified molecular basis will help to uncover the frequency and spectrum of PARP4 mutations leading to dyserythropoiesis. The finding of a new gene implicated in a similar type of CDA with features such as redundant ER membranes offers the potential for more mechanistic dissection of the role of both SEC23B and PARP4 in erythroid development and suggests that new insight can be gained into the underlying pathophysiology of both normal and disordered erythropoiesis through the study of such rare cases. Disclosures No relevant conflicts of interest to declare.

Background Carcinosarcoma of the colon is a rare histopathological entity with uncertain histogenesis, that shows both epithelial and mesenchymal malignant differentiation. Carcinosarcoma rarely affects the gastrointestinal tract and only few cases are reported in the colon. Herein we describe a carcinosarcoma of the ascending colon, with morphological, ultrastructural and molecular analysis. Case presentation An 81-year-old man was hospitalised for asthenia, weight loss and iron-deficiency anaemia. The patient underwent colonoscopy and adenocarcinoma was diagnosed by endoscopic biopsy. A right hemicolectomy was performed and, during surgical operation, liver metastases were detected. Histological examination of the surgical specimen revealed areas of both carcinomatous and sarcomatous differentiation, completely separated by fibrous septae. The sarcomatous component exhibited areas of smooth muscle and osteoblastic differentiation, with focal osteoid material deposition. Molecular analysis conducted separately on the epithelial and mesenchymal components revealed the same p53 gene mutation (R282W in exon 8) and identical polymorphisms in p53 exon 4, in EGFR exons 20 and 21, and in c-kit exon 17. Microsatellite markers analysis revealed a common loss of heterozygosis on 18q. Overall, the data are consistent with a common origin of the two tumor components. The patient was treated with 8 cycles of oral capecitabine (1250 mg/m2 twice a day for 14 days repeated every 28 days) and two years after surgery is alive with liver metastases. Conclusion Carcinosarcoma of the colon is a rare tumour with both epithelial and sarcomatous components. Molecular analysis of the current case suggests the histogenesis from a common cell progenitor.

AIM: To evaluate the effects of gliadin on the oxidative environment in the “ in vivo-like ”model of a three-dimensional cell culture system. METHODS: LoVo cell line (intestinal adenocarcinoma) multicellular spheroids were treated with digested gliadin (with albumin used as a control). Spheroid volumes, cell viability and morphology, lactate dehydrogenase (LDH) release, content of reduced glutathione (GSH) and activity of GSH-related enzymes were examined. The data were statistically analyzed using the Student’s t-test (P

Summary. Background: Type I fibrinogen deficiencies (hypofibrinogenemia and afibrinogenemia) are rare congenital disorders characterized by low or unmeasurable plasma fibrinogen antigen levels. Their genetic bases are represented by mutations within the three fibrinogen genes. Among the 11 reported missense mutations, a few have been characterized by expression studies and found to have an impaired fibrinogen assembly and/or secretion. Histopathological analyses were previously reported in two hypofibrinogenemic cases with discernible hepatic disease, revealing that both underlying mutations (γ-Gly284Arg and γ-Arg375Trp) were associated with hepatic fibrinogen endoplasmic reticulum storage disease (ERSD). Objective: The objective of this study was to investigate the liver histology in an afibrinogenemic patient, homozygous for the Bβ-Leu353Arg mutation, and to study the intracellular processing of the mutant protein. Patients and methods: Liver histology was evaluated by light microscopy, electron microscopy and immunocytochemistry. Intracellular processing of mutant fibrinogen was analyzed by pulse–chase labeling and immunoprecipitation experiments. Messenger RNA levels were determined by real-time reverse transcription–polymerase chain reaction (RT-PCR). Results: The histopathological characterization of the liver showed no signs of fibrinogen accumulation, a difference from the previously reported findings in two hypofibrinogenemic kindreds with ERSD. To evaluate whether the Bβ-Leu353Arg mutation and the ERSD-associated γ-Gly284Arg mutation affected intracellular fibrinogen trafficking differently, both mutant proteins were expressed in COS-1 cells. Bβ-Leu353Arg led to a more severe secretion defect, but no differences that could explain phenotype–genotype correlation were found in the intracellular processing. Endoglycosidase-H analysis demonstrated a secretion block before translocation to the Golgi medial stacks. Real-time RT-PCR studies showed normal levels of the Bβ mRNA in the patient's liver. Conclusions: The results confirm that Bβ-Leu353Arg is associated with impaired fibrinogen secretion, but not with hepatic ERSD.

The two regulatory subunits (R1 and R2) of protein kinase A (PKA) are differentially expressed in cancer cell lines and exert diverse roles in growth control. Recently, mutations of the PKA regulatory subunit 1A gene (PRKAR1A) have been identified in patients with Carney complex. The aim of this study was to evaluate the expression of the PKA regulatory subunits R1A, R2A, and R2B in a series of 30 pituitary adenomas and the effects of subunit activation on cell proliferation. In these tumors, neither mutation of PRKAR1A nor loss of heterozygosity was identified. By real-time PCR, mRNA of the three subunits was detected in all of the tumors, R1A being the most represented in the majority of samples. By contrast, immunohistochemistry documented low or absent R1A levels in all tumors, whereas R2A and R2B were highly expressed, thus resulting in an unbalanced R1/R2 ratio. The low levels of R1A were, at least in part, due to proteasome-mediated degradation. The effect of the R1/R2 ratio on proliferation was assessed in GH3 cells, which showed a similar unbalanced pattern of R subunits expression, and in growth hormone-secreting adenomas. The R2-selective cAMP analog 8-Cl cAMP and R1A RNA silencing, stimulated cell proliferation and increased Cyclin D1 expression, respectively, in human and rat adenomatous somatotrophs. These data show that a low R1/R2 ratio promoted proliferation of transformed somatotrophs and are consistent with the Carney complex model in which R1A inactivating mutations further unbalance this ratio in favor of R2 subunits. These results suggest that low expression of R1A protein may favor cAMP-dependent proliferation of transformed somatotrophs.

We studied the presence of surfactant protein A (Sp-A) immunoreactivity and messenger RNA in 62 normal and abnormal breast samples. Sections were immunostained with polyclonal anti-Sp-A antibody. The association between Sp-A immunoreactivity and histologic grade of 32 invasive ductal carcinomas was assessed by 3 pathologists who scored the intensity of Sp-A immunoreactivity times the percentage of tumor immunostained; individual scores were averaged, and the final scores were correlated with tumor grade, proliferative index, and expression of estrogen and progesterone receptors. Strong Sp-A immunoreactivity was present at the luminal surface of ductal epithelial cells in normal breast samples and in benign lesions; carcinomas displayed variable immunoreactivity, inversely proportional to the degree of differentiation. Sp-A messenger RNA was detected by reverse transcriptase-polymerase chain reaction in 3 of 3 normal breast samples and 9 of 9 carcinomas. The significance of Sp-A expression in breast epithelium requires further study; possibly it has a role in native host defense or epithelial differentiation.

A case of cirrhotic liver harbouring three atypical macroregenerative nodules and an hepatocellular carcinoma was immunocytochemically investigated for the expression of VLA-beta 1, VLA-alpha 2 and VLA-alpha 5 integrins and for different extracellular matrix (ECM) components (collagen I, collagen IV, laminin, fibronectin and tenascin). In addition, the proliferative activity within the nodules was evaluated, using the MIB 1 monoclonal antibody (MAb). The cirrhotic liver disclosed a continuous staining pattern of the ECM proteins investigated, as well as a "sinusoidal" immunostaining of VLA-beta 1, VLA-alpha 2 and VLA-alpha 5. The macroregenerative nodules showed a discontinued immunoreactivity for ECM proteins while maintaining a VLA-beta 1 sinusoidal immunostaining, coupled with intercellular immunostaining. VLA-alpha 2 and VLA-alpha 5 expression was lacking. The growth fraction was low in both the above pathological conditions. The hepatocellular carcinoma was devoid of any ECM immunostaining. VLA-beta 1 immunoreactivity exhibited a honeycomb pattern of staining, whereas VLA alpha subunits were absent. MIB1 expression was high, being present in 30% of neoplastic nuclei. A possible relationship between atypical macroregenerative nodules and hepatocellular carcinoma is discussed.

Russell body gastritis is an unusual form of chronic gastritis characterized by the permeation of lamina propria by numerous plasma cells with eosinophilic cytoplasmic inclusions. Very few cases have been reported in the literature; the majority of which have shown Helicobacter Pylori (H. pylori) infection, thus suggesting a correlation between plasma cell presence and antigenic stimulation by H. pylori. We present a case of Russell body gastritis in a 78-year-old woman who was undergoing esophagogastroduodenoscopy for epigastric pain. Gastric biopsy of the gastroesophageal junction showed the presence of cells with periodic acid-Schiff-positive hyaline pink bodies. Giemsa staining for H. pylori infection was negative, as well as immunohistochemical detection. The cells with eosinophilic inclusions stained positive for CD138, CD79a, and κ and lambda light chains, which confirmed plasma cell origin. In particular, κ and lambda light chains showed a polyclonal origin and the patient was negative for immunological dyscrasia. The histological observations were confirmed by ultrastructural examination. The cases reported in the literature associated with H. pylori infection have shown regression of plasma cells after eradication of H. pylori. Nothing is known about the progression of H. pylori-negative cases. The unusual morphological appearance of this type of chronic gastritis should not be misinterpreted during routine examination, and it should be distinguished from other common forms of chronic gastritis. It is mandatory to exclude neoplastic diseases such as gastric carcinoma, lymphoma and plasmocytoma by immunohistochemistry and electron microscopy, which can help with differential diagnosis. The long-term effects of plasma cells hyperactivation are still unknown, because cases of gastric tumor that originated in patients affected by Russell body gastritis have not been described in the literature. We are of the opinion that these patients should be scheduled for endoscopic surveillance.

The spermatozeugmata (sperm bundles lacking a distinct wall) from the spermathecae of Tubifex tubifex are composed of two different zones: an internal axial cylinder containing conventional spermatozoa and an external cortex composed of modified spermatozoa, tightly packed together. The conventional spermatozoa conform to the classical clitellate scheme: very long and thin with a complex acrosome, a filiform nucleus, small mitochondria, and a flagellum with Y links and β glycogen granules as accessory structures. The modified spermatozoa show "empty" acrosomes, degenerating nuclei, and tails which contain γ glycogen granules. The tails are helically wound around the spermatozeugma and are connected to each other by junctional complexes. The tips of the cortical tails are free and move with a metachronal wave. The presence of two sperm types in tubificids is discussed and a protective function for the modified cortical spermatozoa is proposed.

The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.

A corneal button excised from a 2-month-old infant with congenital posterior polymorphous dystrophy of the cornea, a rare disease affecting Descemet's membrane and endothelium, was examined by electron microscopy. We observed irregularly arranged, sometimes multilayered cells with marked epithelial features, lining the posterior surface of the cornea in place of the endothelium, and Descemet's membrane with focal alterations sometimes involving all of its layers. We interpreted these abnormal cells as epithelial-like cells. As these findings were in a very young patient, which is unusual, we concluded that the onset of the disease may take place in the early period of intrauterine life, corresponding to the beginning of Descemet's membrane production.

The ultrastructural localization of intracellular immunoglobulins on ultrathin sections of glutaraldehyde-fixed, postosmicated, and Epon-embedded human lymph nodes has been achieved using such highly sensitive immunocytochemical techniques as immunogold staining and avidin-biotin-peroxidase complex. These immunoelectron microscopic techniques allow the identification of intracellular immunoglobulins without affecting the ultrastructural morphology of the tissue, since they do not require any pretreatment of the sections with proteolytic enzymes or deresinating agents. Therefore, immunoglobulins can be precisely localized in the cell organelles; structures whose morphology is well preserved. The availability of a reliable postembedding staining procedure for the ultrastructural localization of immunoglobulins is of definite value for investigations on human lymphoid tissue, both normal and pathological.

The current view that coexpression of intermediate filaments (IFs) must be considered a bizarre and unpredictable phenomenon, which seriously jeopardizes the use of their localization in diagnostic applications, is critically reviewed in light of the evidence so far acquired by investigations in vivo and in vitro. A less dogmatic approach, which considers IF expression the result of a series of interactions between cells and their microenvironment instead of a function of their histogenesis, not only justifies the complex variety of coexpressions observed in normal and neoplastic tissues but also confirms the usefulness of IF expression in diagnostic applications and offers new opportunities for investigations, with special regard to immunoelectron microscopy.

(2009). Ki67 cytoplasmic expression: Observations in normal tissue from heart atrial appendages of healthy rats. Cell Cycle: Vol. 8, No. 13, pp. 2125-2125.

beta 2-Microglobulin (beta 2m) is considered to be the amyloidogenic precursor in dialysis-related amyloidosis, although the implication of other relevant cofactors in the pathogenesis of this disease has also been hypothesized. It is conceivable that substances found in amyloid deposits might represent something more than simple codeposition, possibly playing a pathogenic role in amyloidogenesis. Along these lines, a detailed analysis of the protein composition of amyloid fibrils purified from synovial material surgically obtained from nine patients on long-term dialysis was carried out. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several other protein components, in addition to beta 2m, were found. These were characterized by NH2 amino-terminal sequencing and immunoblotting. In fibrils obtained by water extraction, which fulfill the electron microscopy criteria of highly pure amyloid material, polyclonal kappa and lambda light chains were detected with a concentration of 15 micrograms/mL in the water extraction material; the beta 2m concentration was 200 micrograms/mL. Light microscopy immunohistochemistry was performed on samples from five patients. Amyloid deposits reacted with anti-beta 2m, and anti-light (kappa, lambda), chain antibodies. The immunoreaction of amyloid filaments to anti-beta 2m, anti-lambda, and anti-kappa light chain antibodies was also tested by electron microscopy by use of the immunogold staining procedure. Amyloid filaments were labeled by the three antibodies and showed a different intensity of immunostaining apparently related to their different aggregation pattern. These observations demonstrate that polyclonal immunoglobulin light chains (kappa and lambda) are not contaminants but, together with beta 2m, represent a major constituent of amyloid deposits in dialysis-related osteoarticular amyloidosis, thus indicating their possible role in amyloidogenesis.

Background We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. Methods Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients. Results Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. Conclusions The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.