Recent evidence indicates that maternal nutrition during pregnancy influences gene expression in offspring through epigenetic alterations. In the present study we evaluated the effect of protein excess and deficiency during porcine pregnancy on offspring hepatic and skeletal muscular expression patterns of key genes of methionine metabolism (DNMT1, DNMT3a, DNMT3b, BHMT, MAT2B and AHCYL1), condensin I subunit genes (NCAPD2, NCAPG and NCAPH), important for chromosome condensation and segregation, global DNA methylation and gene-specific DNA methylation. German Landrace sows were randomly assigned to control (CO), high protein (HP) and low protein (LP) diet groups. Tissue samples of offspring were collected from fetal (dpc95), newborn (dpn1), weanling (dpn28) and finisher pigs (dpn188). Gene expression of DNMT1, DNMT3a and DNMT3b was influenced by both HP and LP diets, indicating an involvement of DNA methylation in fetal programming by maternal protein supply. Moreover, hepatic global methylation was significantly affected by protein restriction at dpc95 (p = 0.004) and by protein excess at dpn188 (p = 0.034). Gene expression in fetal liver was significantly different between CO and LP for NCAPD2 (p = 0.0005), NCAPG (p = 0.0009) and NCAPH (p < 0.0001). In skeletal muscle, LP fetuses had significantly altered gene expression of NCAPD2 (p = 0.020) and NCAPH (p = 0.001), compared with CO. Furthermore, NCAPG was differentially methylated among LP, HP and CO; indeed, a significant positive correlation was detected with transcript amount in fetal pigs (r = 0.47, p = 0.002). These data demonstrate that both restriction and excess dietary protein during pregnancy alters the offspring's epigenetic marks and influences gene expression.

Maternal diet during gestation is known to affect offspring phenotype induction. In the present study the influence of maternal protein restriction and excess during gestation on offspring candidate gene expression was analysed. German Landrace gilts were fed control, low protein (LP) or high protein (HP) diet throughout gestation (n = 18 per diet group). After birth piglets were cross-fostered and lactated by control diet fed nursing sows. Samples of offspring liver tissue were taken at foetal, newborn, weaning and finishing phase (n = 16, respectively). Transcript amount of selected candidate genes related to cell cycle and cell proliferation was estimated by quantitative real-time PCR. Maternal protein restriction influenced gene expression of candidate genes CCND2, GADD45B, GALK1, GSTP1, MARCKS, MGMT, NEAT1, PSEN1, SNX1 and TRPM7 in liver from foetuses, newborn piglets, weaned and/or finisher pigs. In the offspring of mothers fed a HP diet expression of target genes was affected exclusively in finisher pigs showing increased transcript amount of CCND2, GALK1, MARCKS, SNX1 and TRPM7. The results of the present study clearly show a long-lasting impact of the maternal protein supply during gestation on offspring candidate genes. Remarkably, effects of gestational HP diet became evident in finisher pigs while LP supply already alters genes expression in foetal tissue. Thus it is suggested that LP and HP supply affect the offspring in utero by different physiological mechanisms with the consequence of late effects in case of prenatal protein excess in contrast to early effects in case of protein restriction.

ABSTRACT: BACKGROUND: The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox. FINDINGS: In yield reduction assays, EB had an EC50 of 26.7 muM against cowpox and 4.4 muM against monkeypox. The EC50 for plaque reduction was 26.3 muM against cowpox and 48.6 muM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice. CONCLUSIONS: While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.

Mitoxantrone, an FDA-approved therapeutic for the treatment of cancer and multiple sclerosis, was previously reported to exhibit antiviral activity against vaccinia virus. To determine whether this activity extends to other orthopoxviruses, mitoxantrone was tested against cowpox and monkeypox. Mitoxantrone demonstrated an EC(50) of 0.25μM against cowpox and 0.8μM against monkeypox. Intraperitoneal treatment of cowpox virus-challenged C57Bl/6 mice with 0.5mg/kg mitoxantrone resulted in 25% survival and a significant increase in survival time. In an effort to improve its efficacy, mitoxantrone was tested for synergistic activity with cidofovir. In vitro tests demonstrated significant synergy between the two drugs against cowpox; however, no synergistic effect on animal survival or median time-to-death was seen in intranasally-infected BALB/c mice. Significantly fewer animals survived when treated with a combination of 0.5mg/kg mitoxantrone and 100mg/kg cidofovir than with 100mg/kg cidofovir alone. This is, to our knowledge, the first report of limited anti-orthopoxvirus activity by mitoxantrone in an animal model.

Everolimus (RAD001) is an mTOR inhibitor that has been successfully used as immunosuppressant in solid organ transplantation. Data in allogeneic stem cell transplantation (HSCT) is limited. This study aimed to investigate pharmacokinetics, safety and efficacy of RAD001 in a canine allogeneic HSCT model. First, pharmacokinetics of RAD001 were performed in healthy dogs in order to determine the appropriate dosing. Doses of 0.25 mg RAD001 BID in combination with 15 mg/kg cyclosporin A (CsA) BID were identified as appropriate starting doses to achieve the targeted range of RAD001 (3-8 μg/l) when orally administered. Subsequently, 10 dogs were transplanted using 2 Gy total body irradiation (TBI) for conditioning and 0.25 mg RAD001 BID plus 15 mg/kg CsA BID for pre- and posttransplantation immunosuppression. Seven of the 10 transplanted dogs maintained at the starting RAD001 dose throughout the study. For the remaining 3 dogs dose adjustments were necessary. RAD001 accumulation over time did not occur. All dogs initially engrafted. Five dogs eventually rejected the graft (weeks 10, 10, 13, 27, 56). Two dogs died of pneumonia (weeks 8, 72) but were chimeric until then. Total cholesterol rose from median 4.1 mmol/l (3.5-5.7 mmol/l) before HSCT to 6.0 mmol/l (5.0-8.5 mmol/l) at day 21 after HSCT, but remained always within normal range. Changes in creatinine and triglyceride values were not observed. Long-term engraftment rates were inferior to sirolimus/CsA and MMF/CsA regimen, respectively. RAD001/CsA caused a more pronounced reduction of platelet counts to median 2 x 10E9/l (range 0-21 x 10E9/l) and longer time to platelet recovery of 21 days (range 14-24 days) compared to MMF/CsA. CsA c(2h) levels were significantly enhanced in the RAD001/CsA regimen, but c(0h) and AUC(0-12h) values did not differ compared to a MMF/CsA immunosuppression. In summary, immunosuppression consisting of RAD001 and CsA is well tolerated but not as efficient as with other established immunosuppressants in a canine nonmyeloablative HSCT regimen. Hence, our study does not support the application of RAD001/CsA as standard practice in this setting.

Denileukin Diftitox (ONTAK(®), DAB(389) IL-2) is a recombinant DNA-derived fusion protein depleting cells that express high-affinity IL-2 receptor. Important cell targets are CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(reg)). Elimination of immunosuppressive T(reg) by Denileukin Diftitox may provide a way to modulate immune tolerance following stem cell transplantation. Here, we combined T(reg) depletion with a vaccination approach to induce donor-specific immune reactions. To investigate this approach we chose the mixed chimerism canine stem cell transplantation model which represents a high state of tolerance between two hematopoietic systems. The aim was therefore to induce a graft versus hematopoiesis effect thereby converting mixed to full donor chimerism. Dog leukocyte antigen identical siblings that had developed a stable mixed chimerism after non-myeloablative stem cell transplantation received a single dose of Denileukin Diftitox (18μg/kg, i.v.) followed by several cell-lysate vaccinations. Host peripheral blood mononuclear cell lysates combined with CpG-ODN, and Montanide(®) ISA 51 were locally applied. In vitro studies demonstrated that canine T(reg) are a target of Denileukin Diftitox. The suppression of T-cell proliferation by T(reg) was abolished by addition of Denileukin Diftitox (10nM). An increase of proliferation of median 300%(range: 200%-425%) was observed. No change in donor chimerism was observed after administration of Denileukin Diftitox and vaccination. This study highlights that application of Denileukin Diftitox resulted in a depletion of T(reg) followed by an increase of immune response in vitro. This effect could not be confirmed in vivo even if the immune system was stimulated by vaccinations.

Regulatory T cells (T(reg)) are CD4(+) T lymphocytes with constitutive expression of CD25 and FOXP3, as well as the ability to modulate cellular immune responses. In this study, the phenotypic characteristics, function and feasibility of enrichment and expansion of canine T(reg) were examined. Canine peripheral blood mononuclear cells were isolated and enriched by labelling of CD25, and expansion of T(reg) was achieved by adding interleukin (IL)-2 for 1 week. Phenotypic and functional analyses of T(reg) were performed prior to and after expansion. Canine T(reg) could be phenotypically characterized by CD4, CD25, and FOXP3 expression. Isolation and enrichment of canine T(reg) is possible, but high purities are difficult to achieve without significant cell loss. Expansion of canine T(reg) was possible by adding IL-2 without other growth factors. Higher initial cell numbers seeded allow more substantial T(reg) expansion in vitro. Canine T(reg) have the potential to suppress proliferation of effector T cells (T(eff)). By adding expanded T(reg), a higher capability for suppressing T(eff) could be shown in comparison with freshly isolated T(reg). Enrichment and expansion of canine T(reg) is feasible, and canine T(reg) had similar characteristics to T(reg) from other species.

There is growing evidence that maternal nutrition during gestation has an important effect on offspring development as well as on their gene expression with long-term effects on the metabolic state. A potential mechanism forming long-lasting gene expression patterns is DNA methylation of cytosine in CpG dinucleotides within the promoter region of distinct genes. There has been special focus on mitochondrial dysfunction by prenatal malnourishment over the recent years. To this end, we investigated the gene expression of somatic cytochrome c (CYCS), an important member of the respiratory chain, in a porcine model of gestational protein over- and undersupply at 94 d post-conception and 1, 28 and 188 d of age, and analysed the association with the DNA methylation status within the CYCS promoter. Gene expression on day 1 post natum showed a significant increase in the low protein (LP) group (P = 0·0005) and a slight increase in the high protein (HP) group (P = 0·079) compared with the control (CO) group in the liver. The mean of the methylation level over forty-seven CpG sites from nucleotide (nt)- 417 to - 10 was significantly decreased in the LP (P = 0·007) and HP (P = 0·009) groups compared with that in the CO group. Excess and restricted protein supply during pregnancy led to hypomethylation of a number of CpG sites in the CYCS promoter, including those representing putative transcription factor-binding sites, associated with elevated expression levels. However, the impact of the low-protein gestation diet is more pronounced, indicating that the offspring could better adapt to excess rather than restricted protein supply.

BACKGROUND Sarcoidosis is a systemic disorder with unknown etiology, characterized by non-caseating granulomas in numerous organs and tissues. In 90% of patients lung and lymph nodes are involved. The incidence of sarcoidal granulomas in the upper extremities is low. Here we present the case of a primary hand manifestation of sarcoidosis without clinical systemic involvement. OBJECTIVES A young woman presented with a painful swelling in her right hand. There were no signs of inflammation. Normal perfusion, mobility and sensibility were found. Magnetic resonance imaging (MRI) revealed a tumour infiltrating the muscles and flexor tendons of the third digit around the metacarpal bone and with pathological signal enhancement after administration of contrast medium. RESULTS Intraoperatively, nodular masses and fat tissue were seen. Histological examination after radical tumour resection showed sarcoidal granulomas. Postoperative staging diagnostics with computed tomography (CT) demonstrated multiple thoracic lymph node swellings in the mediastinum and bilateral hill. Follow-up after one year we saw normal scars in the palmar hand. There was no sign of local recurrence. The pulmological care is still going on. CONCLUSIONS Sarcoidosis is a rare, often asymptomatic disease. Patients present with dyspnoe and cough caused by the inflammation of the lung. The first clinical manifestation of sarcoidosis as a tumor in the palmar hand is unusual. Extrapulmonary systemic or progressive sarcoidosis is regarded as an indication for therapy with glucocorticosteroids. This case demonstrates that surgical excision enabled complete local cure without necessity of systemic and/or local treatment with steroids.

Abstract Syndromes with focal overgrowth are sporadic diseases and comprise Proteus syndrome and congenital lipomatous overgrowth, vascular malformations, and epidermal naevi (CLOVE) syndrome, and isolated hemihyperplasia. We describe 3 children classified according to standard criteria with dysregulated growth of various tissues that was excised, together with excess toes, and tumours. Correct classification facilitates diagnosis and operations. Interdisciplinary treatment and follow-up are recommended to prevent disfiguration.