Donkey's Milk in the Management of Children with Cow's Milk protein allergy: nutritional and hygienic aspects.

BACKGROUND: The therapeutic strategy for children with cow's milk allergy (CMA) consists in the elimination of cow's milk (CM) from their diet. Donkey's milk (DM) has been reported to be an adequate alternative, mainly to his nutritional similarities with human milk (HM) and excellent palatability. The aim of present prospective study was to evaluate the nutritional impact of DM on the diet of children with CMA in term of children growth. METHODS: Before the nutritional trial on children and during the study the health and hygiene risks and nutritional and nutraceuticals parameters of DM were monitored. Children with CMA were identified by the execution of in vivo and in vitro tests for CM and subsequent assessment of tolerability of DM with oral food challenge (OFC). Finally, we prescribed DM to a selected group of patients for a period of 6 months during which we monitored the growth of children. A total of 81 children, 70 with IgE mediated cow's milk protein allergy (IgE-CMPA) and 11 with Food Protein Induced Enterocolitis Syndrome to CM (CM-FPIES), were enrolled. RESULTS: Seventy-eight out of 81 patients underwent the OFC with DM and only one patient with IgE-CMPA (1.5 %) reacted. Twenty-two out of 81 patients took part of the nutritional trial. All the 22 patients took and tolerated the DM, moreover DM did not change the normal growth rate of infants. CONCLUSIONS: In conclusion, DM resulted safe in term of health and hygiene risks and nutritionally adequate: no negative impact on the normal growth rate of children was assessed. Therefore, it may be a suitable alternative for the management of IgE mediated CMA and FPIES, also in the first 6 months of life, if adequately supplemented.

Heme Oxygenase-1 and Brain Oxysterols Metabolism Are Linked to Egr-1 Expression in Aged Mice Cortex, but Not in Hippocampus.

Throughout life, stress stimuli act upon the brain leading to morphological and functional changes in advanced age, when it is likely to develop neurodegenerative disorders. There is an increasing need to unveil the molecular mechanisms underlying aging, in a world where populations are getting older. Egr-1 (early growth response 1), a transcriptional factor involved in cell survival, proliferation and differentiation - with a role also in memory, cognition and synaptic plasticity, can be implicated in the molecular mechanism of the aging process. Moreover, Heme Oxygenase-1a (HO), a 32 kDa heat-shock protein that converts heme to iron, carbon monoxide and biliverdin, is a key enzyme with neuroprotective properties. Several in vitro and in vivo studies reported that HO-1 could regulate the metabolism of oxysterols, oxidation products of cholesterol that include markers of oxidative stress. Recently, a link between Egr-1 and HO-1 has been demonstrated in mouse lung cells exposed to cigarette smoke. In view of these data, we wanted to investigate whether Egr-1 can be implicated also in the oxysterol metabolism during brain aging. Our results show that Egr-1 expression is differently expressed in the cortex and hippocampus of old mice, as well as the oxysterol profile between these two brain areas. In particular, we show that the cortex experiences in an age-dependent fashion increasing levels of the Egr-1 protein, and that these correlate with the level of HO-1 expression and oxysterol abundance. Such a situation was not observed in the hippocampus. These results are further strenghtened by our observations made with Egr-1 KO mice, confirming our hypothesis concerning the influence of Egr-1 on oxysterol production and accumulation via regulation of the expression of HO-1 in the cortex, but not the hippocampus, of old mice. It is important to notice that most of the oxysterols involved in this process are those usually stimulated by oxidative stress, which would then represent the triggering factor for this mechanism.

Effects of pasteurization and storage conditions on donkey milk nutritional and hygienic characteristics.

Until now there are only few data on the effects of thermal treatments on the nutritional and hygienic characteristics of donkey milk. This Research Communication aims to provide information on the effects of pasteurization (at +65 °C for 30 min) and prolonged storage at refrigeration and freezing temperatures (21 d at + 3 °C ± 2 °C and up to 90 d at -20 °C ± 5 °C) on some nutritional and hygienic characteristics of Amiata donkey milk. The milk was monitored by chemical and microbiological analysis. Pasteurization ensured compliance with EC Regulation No 1441/2007, as Enterobacteriaceae were never found in the milk, or during storage at refrigeration and freezing temperatures. Colony count at 30 °C in pasteurized milk never went beyond 1 log CFU/ml. The heat treatment and the storage did not result in any variations in the main constituents of the milk. Only a decrease in lactose and few variations in some fatty acids at 90 d of freezing were observed. In conclusion, pasteurization was able to achieve and maintain a high hygienic-sanitary quality over time; storage at refrigeration or freezing temperatures did not alter the nutritional quality of fat and the gross composition of the product. These findings are useful to improve knowledge on the milk shelf life in order to guarantee safety and nutritional quality for infants who need small quantities of daily milk.

BK channels blockage inhibits hypoxia-induced migration and chemoresistance to cisplatin in human glioblastoma cells.

Glioblastoma (GBM) cells express large-conductance, calcium-activated potassium (BK) channels, whose activity is important for several critical aspects of the tumor, such as migration/invasion and cell death. GBMs are also characterized by a heavy hypoxic microenvironment that exacerbates tumor aggressiveness. Since hypoxia modulates the activity of BK channels in many tissues, we hypothesized that a hypoxia-induced modulation of these channels may contribute to the hypoxia-induced GBM aggressiveness. In U87-MG cells, hypoxia induced a functional upregulation of BK channel activity, without interfering with their plasma membrane expression. Wound healing and transwell migration assays showed that hypoxia increased the migratory ability of U87-MG cells, an effect that could be prevented by BK channel inhibition. Toxicological experiments showed that hypoxia was able to induce chemoresistance to cisplatin in U87-MG cells and that the inhibition of BK channels prevented the hypoxia-induced chemoresistance. Clonogenic assays showed that BK channels are also used to increase the clonogenic ability of U87-MG GBM cells in presence, but not in absence, of cisplatin. BK channels were also found to be essential for the hypoxia-induced de-differentiation of GBM cells. Finally, using immunohistochemical analysis, we highlighted the presence of BK channels in hypoxic areas of human GBM tissues, suggesting that our findings may have physiopathological relevance in vivo. In conclusion, our data show that BK channels promote several aspects of the aggressive potential of GBM cells induced by hypoxia, such as migration and chemoresistance to cisplatin, suggesting it as a potential therapeutic target in the treatment of GBM.

Amiata Donkey Milk Chain: Animal Health Evaluation and Milk Quality.

This study presents an investigation of Amiata donkey health and quality of milk for human consumption. Thirty-one lactating dairy jennies were examined. The following samples were collected: faecal samples from the rectum of animals for parasitological examination; cervical swabs for the detection of bacteria causing reproductive disorders; and blood samples for serological diagnosis of main zoonotic (Brucella spp., Leptospira spp.) and donkey abortion agents (Brucella spp., Leptospira spp., Salmonella abortus equi, Equine viral arterithis virus, Equine herpesvirus type 1). In addition, individual milk samples were collected and analysed for mastitis-causing pathogens and milk quality. Regarding animal health, we detected a high prevalence of strongyle parasites in donkeys. It is very important to tackle parasitic diseases correctly. Selective control programmes are preferable in order to reduce anthelmintic drug use. For dairy donkeys, withdrawal periods from anthelmintic drugs need to be carefully managed, in accordance with EU and national regulations. The isolation of Staphylococcus aureus in milk highlights the importance of preventing contamination during milking, by adopting appropriate hygiene and safety practices at a farm level. Lysozyme activity was high compared to cow's milk, contributing to the inhibitory activity against certain bacteria. Donkey milk was characterised by high lactose content, low caseins, low fat, higher levels of unsaturated fatty acids compared to ruminant milks. Unsaturated fatty acids and omega 3 fatty acids in particular have become known for their beneficial health effect, which is favourable for human diet. These characteristics make it suitable for infants and children affected by food intolerance/allergies to bovine milk proteins and multiple food allergies as well as for adults with dyslipidemias. It is also recommended to prevent cardiovascular diseases.

GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

The expression of B23 and EGR1 proteins is functionally linked in tumor cells under stress conditions.

BACKGROUND: The nucleolus is a multi-domain enriched with proteins involved in ribosome biogenesis, cell cycle and apoptosis control, viral replication and differentiation of stem cells. Several authors have suggested a role for the nucleolus also in malignant transformation. We have recently demonstrated that under specific circumstances the transcriptional factor EGR1 is shuttled to the nucleolus where it functions as a negative regulator of RNA polymerase I. Since this activity is hampered in ARF -/- cells, and ARF transcription is regulated by EGR1 while the turnover of ARF protein is under the control of B23, we speculated that some sort of cooperation between EGR1 and B23 might also exist. RESULTS: In this work we identified a canonical EGR1 binding site on the B23 promoter through experiments of transactivation and in vitro DNA binding assay. We then found that the levels of B23 expression are directly correlated with those of EGR1, and that this correlation applies to several cellular types and to different stress conditions. Furthermore, we showed that EGR1 stability and accumulation within the nucleolus is in turn regulated by B23 through proteasome involvement, similarly to ARF turnover. CONCLUSION: Our results highlight EGR1 as a regulator of B23 expression actively playing within the newly discovered nucleolar B23-ARF-EGR1 network.

Direct correlation between double K-RAS mutation and mucinous carcinoma. A case report.

Mutations at the K-RAS locus in colon cancer cells are frequently associated with lack of responsiveness to therapy with EGFR inhibitors, as a consequence of the activation of Ras-dependent intracellular signals. Here we report a colon cancer case carrying a novel combination of K-RAS mutations involving codon 13 (Gly to Asp) and codon 19 (Leu to Phe), on separate alleles. The double mutation was restricted to tumor cells bearing a mucinous-like phenotype.

A case of fatal late vasospasm in a patient with a recurrent, supratentorial rhabdoid primitive neuroectodermal tumor: possible molecular implications.

We report the case of a 44-year-old man who experienced a fatal and untreatable delayed vasospasm after resection of a recurrent temporal IV grade primitive neuroectodermal tumor (PNET). The histological analysis demonstrated a rare rhabdoid variant of the tumor with a diffuse myxoid degeneration; molecular investigations demonstrated an upregulation of IL-1ß and IL-6 expression in the recurrence. We reviewed the pathophysiology of the vasospasm that occurs after tumors resection, and due to the rarity of case, we speculated on the possibility that specific histological and molecular features of the tumor could have contributed to the delayed and fatal complication.

The transcription factor EGR1 localizes to the nucleolus and is linked to suppression of ribosomal precursor synthesis.

EGR1 is an immediate early gene with a wide range of activities as transcription factor, spanning from regulation of cell growth to differentiation. Numerous studies show that EGR1 either promotes the proliferation of stimulated cells or suppresses the tumorigenic growth of transformed cells. Upon interaction with ARF, EGR1 is sumoylated and acquires the ability to bind to specific targets such as PTEN and in turn to regulate cell growth. ARF is mainly localized to the periphery of nucleolus where is able to negatively regulate ribosome biogenesis. Since EGR1 colocalizes with ARF under IGF-1 stimulation we asked the question of whether EGR1 also relocate to the nucleolus to interact with ARF. Here we show that EGR1 colocalizes with nucleolar markers such as fibrillarin and B23 in the presence of ARF. Western analysis of nucleolar extracts from HeLa cells was used to confirm the presence of EGR1 in the nucleolus mainly as the 100 kDa sumoylated form. We also show that the level of the ribosomal RNA precursor 47S is inversely correlated to the level of EGR1 transcripts. The EGR1 iseffective to regulate the synthesis of the 47S rRNA precursor. Then we demonstrated that EGR1 binds to the Upstream Binding Factor (UBF) leading us to hypothesize that the regulating activity of EGR1 is mediated by its interaction within the transcriptional complex of RNA polymerase I. These results confirm the presence of EGR1 in the nucleolus and point to a role for EGR1 in the control of nucleolar metabolism.

Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice.

Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1(-/-)) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1(-/-) mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca(2+)-channel, and PLB mRNA expression were reduced in Egr-1(-/-) mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1(-/-) hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-ß) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1(-/-) mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation-contraction coupling in cardiac myocytes.

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts.

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.

Evaluation of the oncogenic risk of diffuse gastric polyposis. A case report.

Benign polyps of the stomach undergo malignant transformation at a rate correlating to the histological type and size of the proliferative lesion. We report a case of a 50-year-old Caucasian woman, affected by a diffuse gastric polyposis of both hyperplastic and adenomatous type. At endoscopy polyps were more than 1,000, scattered over the entire gastric cavity. The patient underwent total gastrectomy. The perilesional gastric mucosa was characterized by the presence of either atrophic or metaplastic areas and by a mild dysplasia. A single tubulo-villous adenomatous polyp was also present in the ascending tract of the colon. The absence of both high-grade dysplastic lesions and outbreaks of neoplastic transformation well correlated with the histochemical and molecular features, confirming the highly proliferative pattern of the polyps in the lack of signs of malignant progression.

The inhibition of KCa3.1 channels activity reduces cell motility in glioblastoma derived cancer stem cells.

In the present study we evaluated the expression of the intermediate conductance calcium-activated potassium (KCa3.1) channel in human glioblastoma stem-like cells (CSCs) and investigated its role in cell motility. While the KCa3.1 channel is not expressed in neuronal- and glial-derived tissues of healthy individuals, both the KCa3.1 mRNA and protein are present in the glioblastoma tumor population, and are significantly enhanced in CSCs derived from both established cell line U87MG and a primary cell line, FCN9. Consistent with these data, voltage-independent and TRAM-34 sensitive potassium currents imputable to the KCa3.1 channel were recorded in the murine GL261 cell line and several primary human glioblastoma cells lines. Moreover, a significantly higher KCa3.1 current was recorded in U87MG-CD133 positive cells as compared to the U87MG-CD133 negative subpopulation. Further, we found that the tumor cell motility is strongly associated with KCa3.1 channel expression. Blockade of the KCa3.1 channel with the specific inhibitor TRAM-34 has in fact a greater impact on the motility of CSCs (reduction of 75%), which express a high level of KCa3.1 channel, than on the FCN9 parental population (reduction of 32%), where the KCa3.1 channel is expressed at lower level. Similar results were also observed with the CSCs derived from U87MG. Because invasion of surrounding tissues is one of the main causes of treatment failure in glioblastoma, these findings can be relevant for future development of novel cancer therapeutic drugs.

Evaluation of the oncogenic risk of diffuse gastric polyposis. A case report / Evaluación del riesgo oncogénico de la poliposis gástrica difusa. Un caso clínico

Benign polyps of the stomach undergo malignant transformation at a rate correlating to the histological type and size of the proliferative lesion. We report a case of a 50-year-old Caucasian woman, affected by a diffuse gastric polyposis of both hyperplastic and adenomatous type. At endoscopy polyps were more than 1,000, scattered over the entire gastric cavity. The patient underwent total gastrectomy. The perilesional gastric mucosa was characterized by the presence of either atrophic or metaplastic areas and by a mild dysplasia. A single tubulo- villous adenomatous polyp was also present in the ascending tract of the colon. The absence of both high-grade dysplastic lesions and outbreaks of neoplastic transformation well correlated with the histochemical and molecular features, confirming the highly proliferative pattern of the polyps in the lack of signs of malignant progression(AU)

Sporadic diffuse gastric polyposis: Report of a case.

A 50-year-old woman was admitted because of severe sideropenic anemia. The gastrin levels were within normal ranges. Esophagogastroduodenoscopy showed diffuse gastric polyposis with signs of diffuse oozing. Colonoscopy showed the presence of a 3-cm wide pedunculated polyp of the ascending colon, which was removed by diathermy. The patient was treated by total gastrectomy with Roux-Y esophagojejunostomy. Histological examination showed the presence of diffuse gastric polyposis with the contemporary occurrence of hyperplastic polyps and mixed hyperplastic and adenomatous polyps, with a tubular pattern and the focal aspect of serrate adenoma. This is the first case report of sporadic diffuse hyperplastic and adenomatous polyposis of the stomach.

Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression.

BACKGROUND: Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1) predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics. MATERIALS AND METHODS: Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway. RESULTS: We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type p53/INK4a/p16 status. CONCLUSION: Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type p53/INK4a/p16 cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.

DNA instability in replicating Huntington's disease lymphoblasts.

BACKGROUND: The expanded CAG repeat in the Huntington's disease (HD) gene may display tissue-specific variability (e.g. triplet mosaicism) in repeat length, the longest mutations involving mitotic (germ and glial cells) and postmitotic (neurons) cells. What contributes to the triplet mutability underlying the development of HD nevertheless remains unknown. We investigated whether, besides the increased DNA instability documented in postmitotic neurons, possible environmental and genetic mechanisms, related to cell replication, may concur to determine CAG repeat mutability. To test this hypothesis we used, as a model, cultured HD patients' lymphoblasts with various CAG repeat lengths. RESULTS: Although most lymphoblastoid cell lines (88%) showed little or no repeat instability even after six or more months culture, in lymphoblasts with large expansion repeats beyond 60 CAG repeats the mutation size and triplet mosaicism always increased during replication, implying that the repeat mutability for highly expanded mutations may quantitatively depend on the triplet expansion size. None of the investigated genetic factors, potentially acting in cis to the mutation, significantly influence the repeat changes. Finally, in our experiments certain drugs controlled triplet expansion in two prone-to-expand HD cell lines carrying large CAG mutations. CONCLUSION: Our data support quantitative evidence that the inherited CAG length of expanded alleles has a major influence on somatic repeat variation. The longest triplet expansions show wide somatic variations and may offer a mechanistic model to study triplet drug-controlled instability and genetic factors influencing it.

Altered expression of alpha-dystroglycan subunit in human gliomas.

Dystroglycan (DG) is an integral membrane receptor of extracellular matrix proteins, composed of two subunits alpha and beta derived from a common precursor. In brain DG is expressed in neurons, glia limitans, astrocytic endfeet around vessels and endothelial cells. We investigate whether DG may play a role in brain tumors. Western blot and immunofluorescence analysis showed that, while beta-DG subunit was present, the highly glycosylated alpha-DG subunit was strongly reduced in surgically derived human glioblastoma biopsies, in low passage patient-derived cultures and in glioma cell lines, U87MG and A172MG, but not in all glioma cell lines tested. Immunohistochemistry of tumor frozen sections revealed that the loss of alpha-DG was confined in the tumor area but not around blood vessels. Overexpression of DG decreased the growth rate of the glioma cell lines lacking the highly glycosylated alpha-DG subunit and the colony-forming efficiency. Clonogenic assay in presence of temozolomide showed an additive effect between DG overexpression and drug treatment. Our data suggest that DG may be involved in the progression of primary brain tumors.

Severe ultrastructural mitochondrial changes in lymphoblasts homozygous for Huntington disease mutation.

Mutated huntingtin is expressed in nervous and non nervous system included lymphoblasts. Eneregetic metabolism is impaired in Huntington's disease (HD) and other neurodegenerative diseases. Human HD lymphoblasts have provided clear-cut data on mitochondnal disruption. Here we report morphological, morphometric and membrane potential differences in mitochondria from lymphoblasts obtained from patients homozygous and heterozygous for the CAG mutation, and controls. Homozygotes, who despite a similar age at onset show a more aggressive phenotype than heterozygotes, had giant mitochondria and a reduced membrane potential. We argue that early mitochondrial impairment at basal level may affect the severity of HD progression in patients.