BACKGROUND. Angiogenesis is a hallmark of active multiple myeloma. However, two etiologic hypotheses have been proposed:
an angiogenic switch (i.e. differential or de novo expression of ...pro/antiangiogenic genes in MM), and, alternativelyan effect of increased plasma cell number.
AIM of this study was to investigate the angiogenic signature of multiple myeloma cells (MMC), normal bone marrow plasma cells (BMPC), the bone marrow microenvironment (BMME) and cellular subfractions therein.
PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG) / 63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. Whole bone marrow (n=49) and FACSAria sorted subfractions thereof (n=5) were investigated. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). Expression data were gcrma-normalised and the empirical Bayes algorithm used. p-Values were adjusted using the Benjamini-Hochberg method (Bioconductor). iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). HGF expression was verified by real time RT-PCR and western blotting. Based on Medline review, we established a list of 89 pro- and 56 antiangiogenic genes and investigated their expression according to the stage of disease: BMPC vs. MGUS, SD stage I (asymptomatic myeloma) vs. SD stage II/III (symptomatic myeloma requiring therapy).
RESULTS.
BMPC express pro- (e.g. VEGFA) and antiangiogenic genes (e.g. TIMP2).Only one pro-angiogenic gene (hepatocyte growth factor, HGF) is significantly overexpressed in MMC compared to BMPC. HGF has previously been linked with myeloma progression and induction of angiogenesis.Six antiangiogenic genes (TIMP2, SERPINF1, COL18A1, PF4, THBS1, CXCL14) are downregulated in MMC compared with BMPC.Compared to healthy donors, the BMME of MM shows a significant downregulation of PLAU (urokinase, antiangiogenic) and upregulation of TNF(proangiogenic).
CONCLUSION. Upregulation of HGF-expression, downregulation of TIMP2, SERPINF1, COLA18A1, PF4, THBS1 and CXCL14 expression in MMC as well as downregulation of PLAU and upregulation of TNFα in the BMME seem to indicate an “angiogenic switch”. However, given the relatively low number of differentially expressed genes (7/145) and the expression of angiogenic genes by BMPC, an effect caused by an increasing number of plasma cells might be evenly important.
AIM. Expression changes of D-type cyclins are thought to be an early event in the genesis of Multiple Myeloma and are associated with distinct cytogenetic aberrations. These aberrations appear with ...different percentages (“clonal” or “subclonal”) in a given patient. We assessed whether the height of CCND expression assessed by gene expression profiling and quantitative RT-PCR (qRT-PCR) correlates with the presence of clonal or subclonal aberrations of 11q13, t(11;14) and t(4;14).
PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG)/63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus array (VG). CCND1 and CCND2 expression was verified by real time RT-PCR and western blotting. iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). Clonal aberrations were defined as being present in >60%, subclonal aberrations in 20 to 60% of MMC in a given patient. Expression data were gcrma normalised and a Kruskal-Wallis rank sum test used (Bioconductor).
RESULTS. 11q13+. CCND1 (208711_s_at, 208712_at) is significantly higher (p<0.0001), CCND2 (200953_s_at, 200951_s_at) significantly lower (p<0.0001) expressed in MMC harbouring clonal, compared to subclonal, or no gain of 11q13. t(11;14). CCND1 is significantly higher (p<0.0001), CCND2 significantly lower (p<0.0001) expressed in MMC harbouring clonal, compared to subclonal, or no t(11;14). t(4;14). CCND1 is significantly lower (p<0.0001), CCND2 significantly higher (p<0.0001) expressed in MMC harbouring clonal compared to subclonal, or no t(4;14). The expression of CCND3 (201700_at) is not significantly different between the 3 groups for all aberrations investigated. CCND2 and CCND3, but not CCND1 are expressed by normal plasma cells. Results have been verified by qRT-PCR (n=40) for CCND1 and CCND2. Expression of CCND1, CCND2 and CCND3 has been verified by western blotting on selected samples. The expression of CCND2 correlates with short EFS, but not if patients with t(4;14) are excluded. There is no significant difference in EFS for patients harbouring the respective aberrations in a clonal or subclonal pattern.
CONCLUSION.
An additional copy of 11q13 or t(11;14) correlates with increased CCND1 and decreased CCND2 expression, a t(4;14) is associated with an increase of CCND2 and a decrease of CCND1 expression.In each case, the height of the CCND-expression is significantly different whether the respective aberration is clonal or subclonal.Thus, when interpreting expression data in the context of cytogenetic aberrations, it is important to consider if plasma cells carry a respective aberration in a subclonal/clonal pattern.
Background, aim and scope In 2007, the German government approved a national strategy on biological diversity. The strategy requires the integration of its quality targets and action targets into ...other policies. In addition to other pressure factors, area-wide diffuse substance discharges contribute considerably to the loss of biological diversity. Reducing these diffuse substance discharges to long-term ecologically compatible levels is one of the main action targets. This paper aims to provide an overview of preconditions for implementing this objective in terms of content as well as organization and to point out important deficits. Main focus To achieve this, the paper will discuss environmental monitoring and its implementation in Germany drawing on the targets of the national strategy on biological diversity. It will discuss the opportunities for the evaluation of the German and European biodiversity strategies using monitoring required in the execution of environmental laws and regulations as well as international substance-related indicators. Results Environmental monitoring in Germany and in Europe is characterized by a plurality of monitoring networks operated under different administrative competences and environmental sectors. Only a small part of the collected data is evaluated in a cross-media and cross-sectoral perspective even though the Discussion A key problem is that harmonized minimum requirements to monitor the reduction of diffuse substance discharges are missing. Without the implementation of a cooperative and harmonized monitoring approach and organizational guidelines, the reduction of diffuse substance discharges will not be verifiable. Environmental data as well as indicators are essential to evaluate the success of the national biodiversity strategy. Suitable monitoring concepts are available in the German Federal Republic and its Federal States, nevertheless, a political impulse for their implementation is missing. Recommendations and perspectives The national biodiversity strategy may reopen the discussion on the necessity of an integrated monitoring. A coordinated approach that refers both to the international indicator-discussion and to institutional obstacles and aims to develop a proposal for integrated monitoring data is necessary.PUBLICATION ABSTRACT
Summary Background The ideal closure technique of the pancreas after distal pancreatectomy is unknown. We postulated that standardised closure with a stapler device would prevent pancreatic fistula ...more effectively than would a hand-sewn closure of the remnant. Methods This multicentre, randomised, controlled, parallel group-sequential superiority trial was done in 21 European hospitals. Patients with diseases of the pancreatic body and tail undergoing distal pancreatectomy were eligible and were randomly assigned by central randomisation before operation to either stapler or hand-sewn closure of the pancreatic remnant. Surgical performance was assessed with intraoperative photo documentation. The primary endpoint was the combination of pancreatic fistula and death until postoperative day 7. Patients and outcome assessors were masked to group assignment. Interim and final analysis were by intention to treat in all patients in whom a left resection was done. This trial is registered, ISRCTN18452029. Findings Between Nov 16, 2006, and July 3, 2009, 450 patients were randomly assigned to treatment groups (221 stapler; 229 hand-sewn closure), of whom 352 patients (177 stapler, 175 hand-sewn closure) were analysed. Pancreatic fistula rate or mortality did not differ between stapler (56 32% of 177) and hand-sewn closure (49 28% of 175; OR 0·84, 95% CI 0·53–1·33; p=0·56). One patient died within the first 7 days after surgery in the hand-sewn group; no deaths occurred in the stapler group. Serious adverse events did not differ between groups. Interpretation Stapler closure did not reduce the rate of pancreatic fistula compared with hand-sewn closure for distal pancreatectomy. New strategies, including innovative surgical techniques, need to be identified to reduce this adverse outcome. Funding German Federal Ministry of Education and Research.
A novel poxvirus was discovered in
Crocodilurus amazonicus
(Teiidae) presenting with a debilitating skin disease. The generated first genome sequence of a reptilian poxvirus revealed the closest ...phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.
The development and characterization of biomaterials for bone replacement in case of large defects in preconditioned bone (e.g., osteoporosis) require close cooperation of various disciplines. Of ...particular interest are effects observed in vitro at the cellular level and their in vivo representation in animal experiments. In the present case, the material-based alteration of the ratio of osteoblasts to osteoclasts in vitro in the context of their co-cultivation was examined and showed equivalence to the material-based stimulation of bone regeneration in a bone defect of osteoporotic rats. Gelatin-modified calcium/strontium phosphates with a Ca:Sr ratio in their precipitation solutions of 5:5 and 3:7 caused a pro-osteogenic reaction on both levels in vitro and in vivo. Stimulation of osteoblasts and inhibition of osteoclast activity were proven during culture on materials with higher strontium content. The same material caused a decrease in osteoclast activity in vitro. In vivo, a positive effect of the material with increased strontium content was observed by immunohistochemistry, e.g., by significantly increased bone volume to tissue volume ratio, increased bone morphogenetic protein-2 (BMP2) expression, and significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio. In addition, material degradation and bone regeneration were examined after 6 weeks using stage scans with ToF-SIMS and µ-CT imaging. The remaining material in the defects and strontium signals, which originate from areas exceeding the defect area, indicate the incorporation of strontium ions into the surrounding mineralized tissue. Thus, the material inherent properties (release of biologically active ions, solubility and degradability, mechanical strength) directly influenced the cellular reaction in vitro and also bone regeneration in vivo. Based on this, in the future, materials might be synthesized and specifically adapted to patient-specific needs and their bone status.
Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in ...our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases.
During the annual hunt in a privately owned Austrian game population in fall 2019 and 2020, 64 red deer (
), 5 fallow deer (
), 6 mouflon (
), and 95 wild boars (
) were shot and sampled for PCR ...testing. Pools of spleen, lung, and tonsillar swabs were screened for specific nucleic acids of porcine circoviruses. Wild ruminants were additionally tested for herpesviruses and pestiviruses, and wild boars were screened for pseudorabies virus (PrV) and porcine lymphotropic herpesviruses (PLHV-1-3). PCV2 was detectable in 5% (3 of 64) of red deer and 75% (71 of 95) of wild boar samples. In addition, 24 wild boar samples (25%) but none of the ruminants tested positive for PCV3 specific nucleic acids. Herpesviruses were detected in 15 (20%) ruminant samples. Sequence analyses showed the closest relationships to fallow deer herpesvirus and elk gammaherpesvirus. In wild boars, PLHV-1 was detectable in 10 (11%), PLHV-2 in 44 (46%), and PLHV-3 in 66 (69%) of animals, including 36 double and 3 triple infections. No pestiviruses were detectable in any ruminant samples, and all wild boar samples were negative in PrV-PCR. Our data demonstrate a high prevalence of PCV2 and PLHVs in an Austrian game population, confirm the presence of PCV3 in Austrian wild boars, and indicate a low risk of spillover of notifiable animal diseases into the domestic animal population.
During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of ...sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.
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