Glucocorticoid-induced tumor necrosis factor receptor-related (GITR)-Fc fusion protein inhibits GITR triggering and protects from the inflammatory response after spinal cord injury

Glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein is a costimulatory molecule that plays a role in inflammation so that GITR-Fc fusion protein can exert an anti-inflammatory effect. To investigate the mechanism by which GITR-Fc exerts its effects, we first used GITR knock-out (GITR(-/-)) mice to verify whether GITR ligand (GITRL)/GITR system played a pro-inflammatory role in the spinal cord injury (SCI) model. It is noteworthy that less pronounced disease was induced in GITR(-/-) compared with GITR(+/+) mice. We then evaluated the effect of GITR-Fc fusion protein against SCI-induced injuries in GITR(-/-) and wild-type (GITR(+/+)) mice. Administration of GITR-Fc ameliorated SCI-induced inflammation in GITR(+/+) mice as evaluated through: 1) histological damage and apoptosis, 2) modulation of apoptosis-related transduction factors (Bax and Bcl-2), 3) expression of inflammatory markers [nitrotyrosine, inducible nitric-oxide synthase, interleukin (IL)-2, IL-12, and tumor necrosis factor-alpha], and 4) T-lymphocyte infiltration. GITR-Fc was effective in GITR(+/+) but not in GITR(-/-), suggesting that in this experimental model, its anti-inflammatory action was due to inhibition of GITR triggering and not to GITRL activation. In conclusion, GITR plays a role in SCI, and administration of GITR-Fc results in amelioration of SCI severity, prompting further studies on the potential anti-inflammatory properties of GITR-Fc.     

Matrix Gla protein is involved in elastic fiber calcification in the dermis of pseudoxanthoma elasticum patients

Mature MGP (Matrix gamma-carboxyglutamic acid protein) is known to inhibit soft connective tissues calcification. We investigated its possible involvement in pseudoxanthoma elasticum (PXE), a genetic disorder whose clinical manifestations are due to mineralization of elastic fibers. PXE patients have lower serum concentration of total MGP compared to controls (P<0.001). Antibodies specific for the noncarboxylated (Glu-MGP) and for the gamma-carboxylated (Gla-MGP) forms of MGP were assayed on ultrathin sections of dermis from controls and PXE patients. Normal elastic fibers in controls and patients were slightly positive for both forms of MGP, whereas Gla-MGP was more abundant within control's than within patient's elastic fibers (P<0.001). In patients' calcified elastic fibers, Glu-MGP intensively colocalized with mineral precipitates, whereas Gla-MGP precisely localized at the mineralization front. Data suggest that MGP is present within elastic fibers and is associated with calcification of dermal elastic fibers in PXE. To investigate whether local cells produce MGP, dermal fibroblasts were cultured in vitro and MGP was assayed at mRNA and protein levels. In spite of very similar MGP mRNA expression, cells from PXE patients produced 30% less of Gla-MGP compared to controls. Data were confirmed by immunocytochemistry on ultrathin sections. Normal fibroblasts in vitro were positive for both forms of MGP. PXE fibroblasts were positive for Glu-MGP and only barely positive for Gla-MGP (P<0.001). In conclusion, MGP is involved in elastic fiber calcification in PXE. The lower ratio of Gla-MGP over Glu-MGP in pathological fibroblasts compared to controls suggests these cells may play an important role in the ectopic calcification in PXE.     

Promyelocytic leukemia protein (PML) and Daxx participate in a novel nuclear pathway for apoptosis

The promyelocytic leukemia protein (PML) gene of acute promyelocytic leukemia (APL) encodes a cell growth and tumor suppressor essential for multiple apoptotic signals. Daxx was identified as a molecule important for the cytoplasmic transduction of the Fas proapoptotic stimulus. Here, we show that upon mitogenic activation of mature splenic lymphocytes, Daxx is dramatically upregulated and accumulates in the PML nuclear body (NB) where PML and Daxx physically interact. In the absence of PML, Daxx acquires a dispersed nuclear pattern, and activation-induced cell death of splenocytes is profoundly impaired. PML inactivation results in the complete abrogation of the Daxx proapoptotic ability. In APL cells, Daxx is delocalized from the NB. Upon retinoic acid treatment, which induces disease remission in APL, Daxx relocalizes to the PML NBs. These results indicate that PML and Daxx cooperate in a novel NB-dependent pathway for apoptosis and shed new light in the role of PML in tumor suppression.     

Beneficial effects of the phytoestrogen genistein on hippocampal impairments of spontaneously hypertensive rats (SHR)

Hippocampal neuropathology is a recognized feature of the spontaneously hypertensive rat (SHR). The hippocampal alterations associate with cognitive impairment. We have shown that hippocampal abnormalities are reversed by 17β-estradiol, a steroid binding to intracellular receptors (estrogen receptor α and β subtypes) or the membrane-located G-protein coupled estradiol receptor. Genistein (GEN) is a neuroprotective phytoestrogen which binds to estrogen receptor β and G-protein coupled estradiol receptor. Here, we investigated whether GEN neuroprotection extends to SHR. For this purpose, we treated 5-month-old SHR for 2 weeks with 10 mg kg⁻¹ daily s.c injections of GEN. We analyzed the expression of doublecortin+ neuronal progenitors, glial fibrillary acidic protein+ astrocytes and ionized calcium-binding adapter molecule 1+ microglia in the CA1 region and dentate gyrus of the hippocampus using immunocytochemistry, whereas a quantitative real-time polymerase chain reaction was used to measure the expression of pro- and anti-inflammatory factors tumor necrosis factor α, cyclooxygenase-2 and transforming growth factor β. We also evaluated hippocampal dependent memory using the novel object recognition test. The results showed a decreased number of doublecortin+ neural progenitors in the dentate gyrus of SHR that was reversed with GEN. The number of glial fibrillary acidic protein+ astrocytes in the dentate gyrus and CA1 was increased in SHR but significantly decreased by GEN treatment. Additionally, GEN shifted microglial morphology from the predominantly activated phenotype present in SHR, to the more surveillance phenotype found in normotensive rats. Furthermore, treatment with GEN decreased the mRNA of the pro-inflammatory factors tumor necrosis factor α and cyclooxygenase-2 and increased the mRNA of the anti-inflammatory factor transforming growth factor β. Discrimination index in the novel object recognition test was decreased in SHR and treatment with GEN increased this parameter. Our results indicate important neuroprotective effects of GEN at the neurochemical and behavioral level in SHR. Our data open an interesting possibility for proposing this phytoestrogen as an alternative therapy in hypertensive encephalopathy.     

Comparative evaluation of nm23 and p16 expression as biomarkers of high-risk human papillomavirus infection and cervical intraepithelial neoplasia 2(+) lesions of the uterine cervix

Benevolo M, Terrenato I, Mottolese M, Marandino F, Muti P, Carosi M, Rollo F, Ronchetti L, Mariani L, Vocaturo G & Vocaturo A
(2010) Histopathology 57 , 580–586
Comparative evaluation of nm23 and p16 expression as biomarkers of high‐risk human papillomavirus infection and cervical intraepithelial neoplasia 2⁺ lesions of the uterine cervix Aims: To investigate the clinical role of nm23 expression in identifying both high‐risk human papillomavirus (HR‐HPV) and high‐grade cervical lesions or carcinomas [cervical intraepithelial neoplasia 2⁺ (CIN2⁺)], and to compare it with p16 overexpression, as this latter biomarker has already been reported widely in HR‐HPV infected cervical lesions. Methods and results: Immunohistochemical evaluation of nm23 and p16 in 143 cervical biopsy specimens including negative, low‐ and high‐grade lesions and squamous carcinomas (SC). HR‐HPV testing by Digene hybrid capture 2 (HC2) and polymerase chain reaction (PCR) on the cervico‐vaginal samples of the same patients. In detecting CIN2⁺, p16 was significantly more sensitive and specific than nm23 (96.3% versus 81.8% and 66% versus 36.4%, respectively, both P < 0.0001). Concerning HR‐HPV detection by HC2, p16 showed a significantly higher specificity than nm23 (82% versus 47%, P <0.0001), although the sensitivities were comparable (71% versus 76%). We found a significantly direct correlation between nm23 and HC2 findings. However, nm23 expression did not correlate with HPV16/18 infection. In contrast, we observed a significant association between p16 overexpression and HPV16/18 genotypes. Conclusions: We confirm the diagnostic value of p16 overexpression. Moreover, despite in vitro data regarding the interaction with the HPV‐E7 protein, nm23 does not appear to be a more useful biomarker than p16 in identifying CIN2⁺ or HR‐HPV infection.     

Elastofibroma: An in Vivo Model of Abnormal Neoelastogenesis

Thirteen cases of elastofibroma have been studied by conventional light and electron microscopy, as well as by histochemistry and immunohisto-chemistry. By light microscopy elastinophilic material appeared as huge fibers crossing collagen bundles. Immunohistochemistry demonstrated a strong positivity for elastin in numerous and circumscribed areas of the extracellular matrix. By electron microscopy, collagen consisted of 40-50-nm wide fibrils, and elastin was made of large aggregates of moderately electron-dense material surrounding a very thin, apparently normal, elastin core. At high magnification these aggregates consisted of short tubules, often in regular arrays, surrounded by microfibrils and microfilaments. These data, associated with selective digestions on thin sections with elastase, purified collagenase, hyaluronidase, and chondroitinase ABC, revealed that elastic fibers in elastofibroma seem to be made of true elastin surrounded by an enormous amount of hydrophilic material, in which some elastin, chondroitin sulfates, and collagenase type-VII sensitive material are aggregated forming a rather ordered array of short tubules.     

Randomized controlled study of an oral jaw-positioning appliance for the treatment of obstructive sleep apnea in children with malocclusion.

To evaluate the clinical usefulness and tolerability of an oral jaw-positioning appliance in the treatment of obstructive sleep apnea syndrome in children, we studied 32 patients (mean age, 7.1 +/- 2.6 yr; 20 males) with symptoms of obstructive sleep apnea, malocclusion, and a baseline apnea index > 1 event/h. A group of 19 subjects was randomly assigned to a 6-mo trial of an oral appliance; the remainder acted as control subjects. At baseline and after the trial all patients underwent physical examination, a standard polysomnography, and orthodontic assessment. A modified version of the Brouillette questionnaire related to obstructive sleep apnea symptoms was administered to parents before and after the trial and a clinical score was calculated. Of the 32 subjects enrolled, 4 treated subjects and 5 control subjects were lost to follow-up. Polysomnography after the trial showed that treated subjects all had significantly lower apnea index (p < 0.001) and hypopnea index values (p < 0.001) than before the trial, whereas in untreated control subjects these values remained almost unchanged. Clinical assessment before and after treatment showed that in 7 of the 14 subjects (50%) the oral appliance had reduced (a fall of at least 2 points in the respiratory score) and in 7 had resolved the main respiratory symptoms, whereas untreated patients continued to have symptoms. In conclusion, treatment of obstructive sleep apnea syndrome with an oral appliance in children with malocclusion is effective and well tolerated.     

Markers of eosinophilic inflammation in allergic diseases

The paper reviews personal studies aimed at identifying clinical markers of eosinophil inflammation in allergic diseases. Preliminary data on the use of flow cytometry as a method to detect eosinophil activation through phenotypic activation markers are reported. The concept of eosinophil releasability is introduced on the basis of in vim eosinophil activation and mediator release by different stimuli. Data showing an increase of serum levels of ECP mainly in subjects with sensitivity to perennial allergens and related to histamine bronchial reactivity are discussed in view of their practical impact in diagnosing and monitoring of allergic diseases and asthma.