Hepatic fibrinogen storage disease: identification of two novel mutations (p.Asp316Asn, fibrinogen Pisa and p.Gly366Ser, fibrinogen Beograd) impacting on the fibrinogen γ-module.

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, Bß 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.

A histological procedure to study fungal infection in the wax moth Galleria mellonella.

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.

Organization, achievement and results of a pelvis surgery center.

INTRODUCTION: Pelvic fractures are not frequent, yet severe injuries, often associated to other lesions. Well defined diagnostic and therapeutic procedures are lacking, and their economical assessment is inadequate. The goal of this study is to propose the organization of a multidisciplinary center that can develop diagnosis, treatment, and follow up protocols. MATERIALS AND METHODS: 25 patients were treated from August 2008 to July 2010, 5 women and 20 men, average age 34.5 years. Twenty patients had acetabular fractures (8 posterior wall fractures, 2 anterior column fractures and 10 mixed fractures, Judet and Letournel). Five patients suffered from diastasis symphisis pubis (three patients with a CAP type I, and 2 with a CAP type II, Young-Burgess). RESULTS: Average delay between trauma and operation was 15.6 days. Average hospital stay after surgery was 45 days. Five had excellent results, 15 were good, and 4 presented poor results. One patient deceased. Four patients underwent hip arthroplasty 1 year after surgery. DISCUSSION: It was essential to identify the collaborating units. The center aims at a uniform and rapid treatment for patients with lesions which are treated differently depending on the department of hospitalization and on the surgeon's experience. The target is to avoid treatment delays, costs and complications. The DRG evaluation grants the highest value to pelvis surgery. This should be followed by dedicated structures that can become reference centers. CONCLUSION: The results can be improved, but considering this is not well known context both clinically and economically, they can be seen positively.

Primary extra-cranial meningioma of head and neck: clinical, histopathological and immunohistochemical study of three cases.

Extracranial meningiomas of the head and neck region are rare neoplasms, the majority being a secondary location of a primary intracranial tumour. We herewith report three rare cases of extracranial meningiomas, located in the temporal muscle, parotid gland and nasal cavity, together with complete pathological, immunohistochemical and ultrastructural studies. Prognosis of this tumour is generally excellent. Surgical excision is the treatment of choice, with no need for further treatment; nevertheless, differential diagnosis must consider other more common tumours of the head and neck and be based on histopathologic examination and relative techniques, including examination of frozen sections. This procedure is particularly useful assessing surgical treatment and should be performed whenever possible to exclude the malignant nature of the lesion and avoid over-treatment. All three patients underwent surgery and are alive and disease-free.

Autophagy in term normal human placentas.

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.

A critical damping approach for assessing the role of marrow fat on the mechanical strength of trabecular bone.

Several clinical findings revealed that post-menopausal osteoporosis and age-related osteopenia are accompanied by trabecular bone marrow fat (BMF) increase. To help understand this phenomenon, a vibrating string model is proposed, based on the hypothesis that, when bone marrow properties change, the trabecular bone structure remodels itself to preserve its critical damping state. It is found that an inverse relationship holds between trabecular average length and marrow damping coefficient. Such a result leads us to hypothesize the following bone-weakening mechanism. Since fat-rich bone marrow is a worse damper, a BMF increment causes an increase of trabecular average length, which is accomplished by the absorption of horizontal trabeculae (structurally less important than vertical trabeculae). The resulting bone patterns are in excellent agreement with clinical observations of osteoporotic bone. A definitive confirmation of the proposed mechanism will support a therapeutical approach to widespread osteopenic diseases aimed at avoiding, or limiting, BMF increase.

Direct visualization of neo-vessel formation following peripheral injection of bone marrow derived CD34+ cells in experimental myocardial damage.

The definitive fate of peripherally injected PKH26 labelled bone marrow mononuclear cells expressing the CD34+ antigen following experimental myocardial cryodamage in rats (n=10) has been examined by direct visualization on photoconverted light and electron microscopy images. One week after the injection in each rat of about 150,000 CD34+ cells early stage PKH26+ vascular structures were localized in the infarcted areas, suggesting that a potential benefit of this therapeutic approach consists in the regeneration of the vasculature.

Assessment of selective homing and contribution to vessel formation of cryopreserved peripherally injected bone marrow mononuclear cells following experimental myocardial damage.

In view of a potential clinical use we aimed this study to assess the selective homing to the injured myocardium and the definitive fate of peripherally injected labeled and previously cryopreserved Bone Marrow Mononuclear cells (BMMNCs). The myocardial damage (cryoinjury) was produced in 59 rats (45 treated, 14 controls). From 51 donor rats 4.4 x 10(9) BMMNCs were isolated and cryopreserved (slow-cooling protocols); the number of CD34+ and the viability of pooled cells was assessed by flow-cytometry analysis before and after cryopreservation and simulated delivery through a 23G needle. Seven days after injury, BMMNCs were thawed, labeled with PKH26 dye and peripherally injected (20 x 10(6) cells in 500 microl) in recipient rats. Two weeks after experimental injury, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. Except a small amount in the spleen, PKH26+ cells were found only in the infarcted myocardium of the treated animals. Typical vascular structures CD34+ were found in the infarcted areas of all animals; treated rats showed a significantly higher number of these structures if compared with untreated. Morphological ultra-structural examination of infarcted areas confirmed in treated rats the presence of early-stage PKH26+ vascular structures derived from injected BMMNCs. The estimated mean CD34+ cells loss due to the cryopreservation procedure and to the system of delivery was 0.24% and 0.1%, respectively, confirming the feasibility of the procedure. This study supports the possible therapeutic use of cryopreserved peripherally injecetd BMMNCs as a source of CD34+ independent vascular structures following myocardial damage.

Medullary and papillary carcinoma of the thyroid gland occurring as a collision tumour: report of three cases with molecular analysis and review of the literature.

We report the simultaneous occurrence of medullary thyroid carcinoma (MTC) and papillary thyroid carcinoma (PTC), presenting as spatially distinct and well-defined tumour components, in three cases. In the first patient, histology, immunohistochemistry and electron microscopy demonstrated an MTC in the one nodule and PTC in two additional lesions. Non-neoplastic thyroid parenchyma separated the three nodules. Metastasis from PTC was diagnosed in a regional lymph node. Genetic analysis of both tumour components showed a distinctive mutational pattern: in the MTC a Cys634Arg substitution in exon 11 of the RET gene and in the two PTC foci a Val600Glu substitution in exon 15 of the BRAF gene. The other two patients are members of a large multigenerational family affected with familial MTC due to a germline mutation of the RET gene (Ala891Ser). Both patients harboured, besides medullary cancer and C-cell hyperplasia, distinct foci of papillary thyroid cancer, which was positive for Val600Glu BRAF mutation. Review of the literature disclosed 18 similar lesions reported and allowed the identification of different patterns of clinical presentation and biological behaviour. So far, the pathogenesis of these peculiar cases of thyroid malignancy has been completely unknown, but an underlying common genetic drive has been hypothesised. This is the first report in which two mutations, in the RET and BRAF genes, have been identified in three cases of MTC/PTC collision tumour, thus documenting the different genetic origin of these two coexisting carcinomas.

Liver histology of an afibrinogenemic patient with the Bbeta-L353R mutation showing no evidence of hepatic endoplasmic reticulum storage disease (ERSD); comparative study in COS-1 cells of the intracellular processing of the Bbeta-L353R fibrinogen vs. the ERSD-associated gamma-G284R mutant.

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 (gamma-Gly284Arg and gamma-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 Bbeta-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 Bbeta-Leu353Arg mutation and the ERSD-associated gamma-Gly284Arg mutation affected intracellular fibrinogen trafficking differently, both mutant proteins were expressed in COS-1 cells. Bbeta-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 Bbeta mRNA in the patient's liver. CONCLUSIONS: The results confirm that Bbeta-Leu353Arg is associated with impaired fibrinogen secretion, but not with hepatic ERSD.

DMBT1 expression is down-regulated in breast cancer.

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.

Bread wheat gliadin cytotoxicity: a new three-dimensional cell model.

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 cultures. However, the specific morphological and biochemical properties of in vivo tissue are better maintained in three-dimensional cell cultures (multicellular spheroids, MCS). The aim of this study was to develop a three-dimensional in vitro model to investigate the effects of gliadin on epithelial cells and broaden our understanding of the early tissue damage occurring in celiac disease. METHODS: The three-dimensionally growing Lovo cell line was exposed to increasing concentrations of peptic-tryptic-digested bread wheat gliadin (from 125 to 1000 microg/mL) for 7 days in order to evaluate cell viability (colony-forming assay), and at the standard concentration of 500 microg/mL for 7 days in order to evaluate MCS diameters, volumes and cell morphology using light and electron microscopy. RESULTS: In comparison with the controls, the cell viability of the gliadin-treated MCS was significantly reduced (20-80%), but there was no difference in size. Various degrees of cell damage (autophagic vacuoles and intra-cytoplasmic lipid-like droplets) were detected by both light and electron microscopy. CONCLUSION: This is the first study investigating the effects of gliadin on MCS. Lovo MCS seem to be responsive to gliadin exposure, thus confirming previous results obtained using two-dimensional cell cultures. The data suggest that three-dimensional cell cultures may be useful in broadening our understanding of some of the early effects of gliadin peptides on epithelial cells.

Clinical histopathology and ultrastructural analysis of myocardium following microwave energy ablation.

OBJECTIVE: Due to weaknesses of conventional modes for treating atrial fibrillation (AF), surgical energy ablation methods and tools to cure AF have been under rapid development. One of these methods, microwave energy, is beginning to be applied clinically. The purpose of this study was to examine histology and ultrastructure of lesions produced by microwave energy in the myocardium. METHODS: Fifteen consecutive patients underwent surgical microwave energy ablation (Microwave Ablation System with FLEX 4 probe, AFx Inc., Fremont, CA) concomitant to a valve procedure. Epicardial ablation was carried out on the beating normothermic heart prior to performing the valve procedure. Two tissue specimens (1cm(2)) were obtained from each patient; one from the lesion site (right appendage) and the other from an adjacent, non-ablated site, which was used as control. Tissue samples were fixed and stained as appropriate for histological and ultrastructural analysis. RESULTS: All ablated samples revealed observable microscopic alteration, including loss of nuclei, foci of coagulative necrosis or induced irregular bands of contraction. Ultrastructurally, ablated cells demonstrated architectural disarray, loss of contractile filaments, mitochondrial swelling and focal interruption of plasma membrane. CONCLUSIONS: Histologic appearance of lesions created by epicardial microwave energy ablation was consistent over tissue samples, although acute findings demonstrated differences from cryoablation. In most of the cases, lesions were transmural, as was demonstrated by loss of cellular viability throughout the depth of tissue specimens.

Unusual clear cell variant of epithelioid mesothelioma.

Clear cell mesothelioma is an extremely rare neoplasm of the pleura, which can easily be mistaken for a metastasis of clear cell carcinoma to the pleura. We report here the histochemical, immunohistochemical, and ultrastructural aspects of a new case of clear cell pleural mesothelioma in a 52-year-old man with no known asbestos exposure. He was admitted to the hospital for recurrent pleural effusion, which was negative for neoplastic cells at the cytologic examination. A partial decortication of the right pleura was performed. The morphologic, immunohistochemical, and ultrastructural features reported for this case are consistent with the diagnosis of clear cell mesothelioma. The differential diagnosis and immunohistochemical features in comparison with other clear cell neoplasms are discussed.

Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver.

Tissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver.

Surfactant protein A expression in human normal and neoplastic breast epithelium.

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.

Primary intrathoracic meningioma: histopathological, immunohistochemical and ultrastructural study of two cases.

Meningiomas are common, usually benign slow-growing neoplasms of the central nervous system thought to arise from meningocytes capping arachnoid villi. Primary ectopic meningiomas are exceedingly rare extracranial and extraspinal tumors of controversial origin; they are usually limited to the head and neck region or to the paravertebral soft tissues. Only one mediastinal ectopic meningioma and few pulmonary ectopic meningiomas have been described in the literature until now. Because of their rarity and their intriguing pathogenesis, we report here a second case of primary mediastinal meningioma and an additional case of primary pulmonary meningioma. Their possible origin and differential diagnosis are discussed.

Usefulness of histological examination for the diagnosis of onychomycosis.

BACKGROUND: [corrected] Direct microscopy and culture tests currently used in the diagnosis of nail mycosis can yield false-negative results, and confirmation of the pathogenic agent, especially in non-dermatophyte infections, is often a lengthy process. OBJECTIVE: The aim of this study was to investigate the usefulness of the histological examination of nail clipping samples in supplementing the standard microscopic and culture techniques for the diagnosis of onychomycosis. PATIENTS AND METHODS: One hundred and seventy-two subjects affected by nail alterations suggestive of onychomycosis were evaluated. Nail specimens were studied with 3 different techniques: direct microscopic examination of a 40% KOH clarified preparation, fungal culture and histological examination. Patients positive for fungal infection were re-evaluated with the same techniques after treatment with oral terbinafine, fluconazole or itraconazole and topical application of bifonazole or ciclopirox for 2 months. RESULTS: Direct microscopy was positive in 102 (59.3%) nail specimens. The culture test was positive in 90 cases (52.9%), showing a dermatophyte in 45, a yeast in 23 and a mould in 22 samples. The histological examination was positive in 94 (54.6%) samples. In 4 cases, it was the only investigation confirming the clinical diagnosis of nail mycosis. In most of the cases, the morphological aspect of the hyphae and/or spores suggested also to which group of pathogens (dermatophytes, yeasts or moulds) the mycetes observed in the histological sections could be ascribed. The concurrent presence of a dermatophyte and a mould was evidenced in a few specimens. The control histological examination at the end of the treatment showed negative results or residual non-vital hyphae and/or spores. CONCLUSIONS: Results of the present study indicate that the histological examination of nail clipping specimens is a relatively inexpensive, rapid and easily performed procedure. It is useful to confirm or refute the results of routine microscopy and culture tests. Moreover, nail histopathological observation may help in ascribing a pathogenic role of non-dermatophyte isolates and evaluating the effectiveness of antifungal treatment.