Analysis of myeloperoxidase activity in wound fluids as a marker of infection.

BACKGROUND: Neutrophilic polymorphonuclear leukocytes play a crucial role in the host defence against bacterial and fungal infections. They participate in the inflammatory response through the liberation of peptides and enzymes like myeloperoxidase (MPO). Therefore, MPO has a potential as a marker enzyme for the diagnosis of wound infection. METHODS: Substrate specificities and reaction pathways of MPO were investigated for new MPO substrates: crystal violet, leuco crystal violet, fast blue RR (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt) and various systematically substituted model substrates based on 2,7-dihydroxy-1-(4-hydroxyphenylazo)naphtalene-3,6-disulphonic acid. In addition, fast blue RR was covalently bound to siloxanes allowing immobilization of the substrate, while cellobiosedehydrogenase was integrated for generation of hydrogen peroxide required by MPO. RESULTS: Elevated concentrations of MPO were found in infected wounds compared with non-infected wounds (92.2 ± 45.0 versus 1.9 ± 1.8 U/mL). Various soluble and immobilized substrates were oxidized by MPO in wound samples and the influence of substrate structure and reaction pathways were elucidated for selected compounds. CONCLUSIONS: Incubation of different MPO substrates with infected wound fluid samples resulted in a clear colour change in the case of elevated MPO concentrations, thus allowing early diagnosis of wound infection.

Enhancer of Zeste 2 (EZH2) is up-regulated in malignant gliomas and in glioma stem-like cells.

AIMS: Proteins of the Polycomb repressive complex 2 (PRC2) are epigenetic gene silencers and are involved in tumour development. Their oncogenic function might be associated with their role in stem cell maintenance. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is a key member of PRC2 function: we have investigated its expression and function in gliomas. METHODS: EZH2 expression was studied in grade II-IV gliomas and in glioma stem-like cells (GSC) by quantitative PCR and immunohistochemistry. Effects of EZH2 down-regulation were analysed by treating GSC with the histone deacetylase (HDAC) inhibitor suberoylanide hydroxamic acid (SAHA) and by shRNA. RESULTS: DNA microarray analysis showed that EZH2 is highly expressed in murine and human GSC. Real-time PCR on gliomas of different grade (n = 66) indicated that EZH2 is more expressed in glioblastoma multiforme (GBM) than in low-grade gliomas (P = 0.0013). This was confirmed by immunohistochemistry on an independent set of 106 gliomas. Treatment with SAHA caused significant up-regulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was associated with a significant decrease of glioma proliferation. CONCLUSION: The data suggest that EZH2 plays a role in glioma progression and encourage the therapeutic targeting of these malignancies by HDAC inhibitors.

PNET/ESFT of the cranial vault: a case report.

A case of peripheral PNET (PNET/ESFT) of the cranial vault is described. A 56-year-old woman showed a mass with a large cyst in the right temporal region, adherent to the meninges, which caused a left hemiparesis with headache and confusion. The mass was totally removed. The histological examination showed a dense proliferation of small elements, organized in lobules separated by reticulin septa. Many circumscribed necroses, vessels with a thick handcuff of reticulin, a diffuse mucous degeneration and abundant mitoses were present. The cells were positive for Vimentin and CD99. RT-PCR revealed the EWS/FLI1 fusion transcript of the t(11,22) (q24;q12) translocation. The patient presented is the oldest one of the rare cases of dura-based meningioma-mimicking pPNETs till now described. In line with the possible origin from peripheral nerves or roots of cauda equina of non-intracranial tumors, those of the vault may derive from peripheral sensory nerves of the dura. The differential diagnosis must be made with cPNETs which show a worse prognosis and both can benefit from a different chemotherapy.

The history of neuropathology in Italy.

The history of Italian Neuropathology begins in the XIX century with Lombroso with his studies of criminal and prostitutes, inspired by the positivism of the era, and on the brain of epileptic patients. It reached its peak at the beginning of XX century with Camillo Golgi, Nobel laureate for his impregnation of neurons and the theory of the diffuse neuronal net. Neuropathology was then cultivated in Asylums and Universities where the main subject of interest were dementias and degenerative diseases, followed by vascular and inflammatory diseases. Some Laboratories arose in the country, especially in neurological institutes and some people later began to emigrate, especially to France and Germany and then to USA in order to improve their Neuropathology. Starting in the late fifties of the 20th century there was a progressive enrichment of Neuropathology with histochemistry, electron microscopy, immunohistochemistry and then molecular biology and the number of Laboratories increased consistently. As in other developed countries, Neuropathology with the enlargement of its scientific fields, began to split in sub-disciplines. It remained as a wide spectrum of knowledge, but neuropathologists were obliged to specialize in specific areas of the discipline. The continuous change of the set up of the university studies in the country in the last 20 years did not favor Neuropathology from which, moreover, some new independent disciplines originated.

Parenchymal and vascular lesions in ageing equine brains: histological and immunohistochemical studies.

Many age-related changes are described in the nervous system of different species, but detailed studies of brain lesions in ageing horses are lacking. The aim of the present study was to systematically characterize lesions in the brains of 60 horses aged from 7 to 23 years. No gross changes were present in any brain. Microscopically, spongiform changes, lipofuscin storage, corpora amylacea, gliosis and satellitosis were common, together with axonal and neuronal swellings. The most important findings were the presence of pseudocalcium-calcium (pCa-Ca) deposits and arterial wall degeneration. Scanning electron microscopical examination of two cases with vascular mineralization revealed marked deposition of an amorphous substance in the vessel walls that was probably formed by a polyanionic protein matrix and a mineral component. Immunohistochemically, numerous axonal spheroids were positively labelled for ubiquitin. No PrPsc was detected in sections with neuronal vacuolation. Neuronal swelling, corpora amylacea, hippocampal Tau-positive neurons and methenamine-positive diffuse (preamyloid) plaques were also detected. Congo red staining failed to detect amyloid deposition. The characterization of age-related lesions in the brains of these horses will allow these changes to be discriminated from pathological processes in future studies. Some lesions described here, including some vascular changes, the presence of diffuse plaques and tau accumulation in hippocampal neurons, have not been described previously in the horse.

Krabbe's disease in two West Highland White terriers.

Two 3-month-old male West Highland White terriers were referred for progressive neurological disease. Histological examination of the central nervous system of the animals euthanized at the owner' request, revealed diffuse, bilateral and symmetrical white matter lesion consisting of varying degrees of demyelination and axonal degeneration. Accumulation of round to ovoid large mononuclear cells was especially observed along the blood vessels in the white matter. These cells were characterized by central or eccentric nuclei and highly eosinophilic, granular and PAS-positive cytoplasm. Stored material was stained with toluidine blue both at pH 4 and pH 11 and exhibited a strong PAC and no PALK activities. Staining for lectins revealed a positivity using Ricinus communis agglutinin-I, Ricinus communis agglutin-II, Triticum vulgaris and Concavalin A. Histochemical evaluation of intracellular material was performed on the kidney and on the liver, too. Ultrastructural investigations allowed to observe the cytoplasmic contents of globoid cells that is an admixture of degraded myelin membranes and different kinds of tubular aggregates. To verify if the two dogs bore the mutation at position 473, a method involving PCR amplification of genomic DNA followed by restriction-digestion was used. The diagnosis of Krabbe's disease was performed based on the clinical evaluation, morphological, histochemical and ultrastructural features.

Radiotherapy by particle beams (hadrontherapy) of intracranial tumours: a survey on pathology.

A review of the principal contributions of radio-therapy of brain tumours by beam particles is carried out. Neutrons, protons and light ions are considered along with their pros and cons in relation to types and locations of brain tumours. A particular emphasis is given to the pathologic studies of their effects directly o n tumours and on the normal nervous tissue, considering mainly the relevant action mechanisms of the radiation types and the requirements of the clinical therapeutic strategies. For comparison the main features of the pathologic effects of radiotherapy by photons are described. From the review it emerges that the new modality of radiation by protons and light ions, because of their peculiar physical characteristics, may represent a new way of destroying the tumour and sparing normal nervous tissue, especially when deeply located and irregularly shaped tumours are concerned. More neuropathological studies are needed in order to better understand the potentiality of the new treatment of modalities.

Progressive multifocal leukoencephalopathy: diffusion-weighted imaging and pathological correlations.

We examined MRI of two patients with progressive multifocal leukoencephalopathy (PML), including diffusion-weighted imaging (DWI), with calculation of apparent diffusion coefficients (ADC). The pathology findings of one patient were compared with those of MRI. The lesions had different ADC and DWI appearances, depending on the stage of the disease. Newer lesions and the advancing edge of large lesions had normal-to-low ADC and gave high signal on DWI. Older lesions and the centre of large lesions had increased ADC and gave low signal. High signal on DWI and low ADC mark the regions of active infection and cell swelling, distinguishing them from areas of reparative gliosis.

Histological and immunohistochemical study of a neuroblastoma in a dog.

A 13-year-old, male German Shepherd dog was euthanasized for a frontal temporal mass revealed by the MRI. The histological examination showed a proliferation composed of small round undifferentiated cells arranged in sheets or nests and sometimes in pseudorosettes interrupted by hypocellular zones of fibrovascular stroma. Immunohistochemical studies revealed the expression of neuroblastic epitopes. The presented neoplasm has many histological and immunohistochemical features in common with the group of olfactory neuroblastomas reported in man, so it could be classified as primitive neuroectodermal tumor with neuronal differentiation.

Malignant fibrous histiocytoma associated with a meningothelial meningioma.

A 72-year-old woman was operated for a left parietal tumor of the meninges. The symptomatology began 22 years earlier with a right hemiparesis. The histological examination of the tumor showed a proliferation of meningothelial cells with whorl formation, associated with a pleomorphic proliferation of a malignant fibrous histiocytoma. The meningothelial meningioma showed a typical aspect, with nuclear inclusions and absence of mitotic activity. The second tumor component showed a storiform architecture with giant multinucleated cells, necrosis and many typical and atypical mitoses. There was also an inflammatory component with infiltrates of lymphocytes. This tumor component at the border with the meningioma appeared to arise from the septa of the meningioma with many prongs merging into one large and pleomorphic histiocytomatous tumor. The dual histological aspect and the case are discussed in the light of what is already known in the literature. The woman has been irradiated after surgery and she is doing well 6 months after operation.

Heterogeneity of apoptotic pathways and c.Jun/JNK expression in malignant gliomas.

In brain tumors, the prognostic value of apoptosis is still debated, even though recent observations are rather negative. In 50 astrocytic gliomas, apoptotic nuclei were recognized by TUNEL technique and morphology and apoptotic index (AI), mitotic index (MI) and the MI/AI ratio were calculated in proliferative areas and in areas containing perinecrotic palisadings and hypercellular centres. In proliferative areas, a positive linear correlation between MI and AI and a MI/AI ratio > I were found. The latter was < I in perinecrotic palisadings and hypercellular centres. This suggests the possibility that apoptosis is triggered respectively by cell proliferation and hypoxia. A small number of apoptotic nuclei was positive for c-Jun and JNKI, suggesting the involvement of this pathway in apoptosis as well as that of sphingomyelinase/ceramide. No relationship was found between AI and labeling indices of Bcl-2, Bcl-x, Bax, p53, APO.I/Fas. The rationale for a prognostic value of apoptosis in gliomas is thus poor.

Cyclin D1 expression in normal oligodendroglia and microglia cells: its use in the differential diagnosis of oligodendrogliomas.

Cyclin D1 regulates G1-S progression. In many carcinomas it is overexpressed and it might even correlate with prognosis. However, the amplification of CCND1 contributes to the loss of cell cycle control only in a small fraction of malignant gliomas. Cyclin D1 can be immunohistochemically demonstrated by DCS-6 mAb. In astrocytic gliomas the fraction of tumor cells with positive nuclei is almost null in well differentiated tumors and increases with the increase of proliferation rate that occurs in anaplasia. The correct evaluation of this fraction is hindered by the positive staining of normal oligodendrocytes and microglia cells. The cyclin D1-positive staining of normal oligodendrocytes and microglia cells has been studied in a series of 20 oligodendrogliomas, five diffuse astrocytomas and five oligoastrocytomas and in 10 samples of normal cortex and white matter, using cyclin D1 DCS-6 mAb, Feulgen reaction and CR3.43 mAb for microglia cells. As well as microglial nuclei, the nuclei of normal oligodendrocytes of the cortex and white matter, including peri-neuronal satellites and pericapillary cells, were immunostained by DCS-6 mAb. In infiltrative areas of oligodendrogliomas, normal, cyclin D1-positive oligodendrocytes and cyclin D1-negative tumor cells coexisted. In anaplastic oligodendrogliomas, cycling tumor oligodendrocytes may regain the capacity to express cyclin D1, which is thus positive in some tumor cells. The occurrence of positive oligodendrocytes in the peripheral parts of tumors can be useful in distinguishing astrocytomas from oligoastrocytomas.

Expression of angiopoietin-1 in human glioblastomas regulates tumor-induced angiogenesis: in vivo and in vitro studies.

To define a role for the angiopoietin/Tie2 system in astrocytoma angiogenesis, we examined the expression of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) in these tumors by immunohistochemistry and in situ hybridization. Furthermore, we studied in vitro the effects elicited by glioblastoma cell-secreted Ang1 or by recombinant Ang1 on functions of endothelial cells (ECs). Our observations of astrocytomas show that a stage-specific induction of angiopoietins occurs and is correlated with angiogenic phases of different intensity. Ang1 expression was found in a few astrocytes scattered in the tumor at all stages of astrocytoma progression. In blood vessels, Ang1 mRNA increased progressively in high-grade glioblastomas, in which the number of vessels was higher than in low-grade tumors. Ang2 was detected in tumor cells and in ECs in high-grade astrocytomas, whereas its expression was negligible in low-grade tumors. Coculture of glioblastoma cell lines producing Ang1 with endothelium demonstrated a key role of this ligand in the control of EC network organization. We found that recombinant Ang1 in vitro induces EC spreading and reorganization of the cell monolayer into cordlike structures. These results suggest that Ang1 directly acts on ECs by modulating cell-cell and cell-matrix associations and promoting the differentiation phase of angiogenesis.

Distribution of activated caspase-3 in relation with apoptosis in human malignant gliomas.

Activated caspase-3has been immunohistochemically studied in 30glioblastomas. Its distribution has been compared with that of apoptotic nuclei demonstrated by terminal dUTP nick-end labeling (TUNEL) and morphology. The best procedure for the demonstration of caspase-3 requires formalin fixation, followed by Carnoy fixation, with microwave irradiation. The number of positive cells is lower than that of apoptotic nuclei shown by TUNEL technique, especially in perinecrotic pseudo-palisadings, and there are also qualitative variations. Positive staining occurs in nuclei, cytoplasms or in both cell compartments. The interpretation of Caspase-3 positive staining is based on its crucial position in the final pathway to apoptosis and on the mechanisms by which it cleaves cytoplasmic and nuclear proteins among which inhibitory/caspase-activated DNase system is included.

CDKN2A/p16 in ependymomas.

Sixteen cases of ependymoma were studied for CDKN2A/p16 inactivation by immunohistochemistry using a p16 monoclonal antibody, by homozygous deletion (HD) assay and 5'CpG promoter methylation assay (methylation-specific PCR). Three out of 16 cases were p16 immuno-negative: two corresponded to grade II ependymomas and one to grade III. The latter ependymoma, characterized by a high Ki-67/MIB-1 LI, was the only one of the whole series to show CDKN2A HD. No promoter methylation was found in the two immuno-negative cases without CDKN2A HD. Alternative mechanisms, such as point mutations or alterations in p16 post-translational regulation, may be responsible for p16 inactivation. Since in our series just one out of eight anaplastic cases showed negative immunostaining and CDKN2A HD, p16/CDKN2A inactivation may not play an important role in the malignant transformation of ependymomas. Amplification of CCNDI and CDK4, p27/Kipl degradation and TP53 mutations were previously studied by other authors and were demonstrated not to correlate with anaplasia. Up to date, molecular genetic studies have not been useful in recognizing the anaplastic variant in ependymomas.

Neuroendocrine tumors in the brain.

Somatostatin and other neuropeptides are expressed in tumors originating from neuronal precursors and paraganglia, namely medulloblastoma, central Primitive Neuro-Ectodermal Tumors (cPNETs), neurocytoma, gangliocytoma. olfactory neuroblastoma, paraganglioma. In medulloblastoma, the most common malignant tumor in childhood, there is an extensive expression of somatostatin in addition to somatostatin receptors (SSTR) type 2. Although density of SSTR-2 and intensity of expression of somatostatin genes have no prognostic significance in medulloblastoma. their presence may bring along important information on oncogenesis and relate medulloblastoma to cPNETs. Radio-labeled octreotide scintigraphy may be useful in the follow-up of these patients. allowing differentiation between scar and tumoral tissue. Moreover, on the basis of octreotide-induced inhibition of cell proliferation in medulloblastoma, a trial with octreotide in patients with recurrent or high-risk tumor is warranted. Meningiomas and low-grade astrocytic gliomas, even if not displaying a clear neuroendocrine phenotype, have high levels of SSTR-2. In meningiomas, SSTRs-scintigraphy is not part of the routine pre-operative assessment; moreover, a therapeutic trial with somatostatin-analogues in patients with recurrent or inoperable meningiomas should be carried-out with great caution, because somatostatin and octreotide slightly increase cell proliferation in cultured meningiomatous cells. Low-grade gliomas (WHO grade 2), and a smaller fraction of anaplastic astrocytomas, express SSTR-2, while glioblastomas usually do not. Unfortunately, radiolabeled-octreotide scintigraphy is not useful in the differential diagnosis of gliomas, because the results are altered by the disruption of the blood brain barrier (BBB); in addition, radionuclide-labeled somatostatin analogues are not useful in the therapy of low-grade gliomas, because the intact BBB prevents them from reaching the target SSTR-2. Recently, a pilot study in gliomas, has proposed the use of a radio-labeled somatostostatin analogue with a loco-regional approach in order to overcome the intact BBB.