Ex vivo selection for oncoretrovirally transduced green fluorescent protein-expressing CD34-enriched cells increases short-term engraftment of transduced cells in baboons

In an effort to improve hematopoietic stem cell gene transfer rates using gibbon ape leukemia virus (GALV)-pseudotype retroviral vectors in baboons, we have studied preselection of transduced green fluorescent protein (GFP)-expressing CD34-enriched marrow cells. Three animals were transplanted with GFP-selected (GS) CD34-enriched marrow. To ensure engraftment, preselected GFP-positive cells were infused together with unselected neo-transduced cells. After transduction on fibronectin, cells were cultured for an additional 2 days to allow for expression of GFP. GFP-expressing cells were enriched by fluorescence-activated cell sorting and infused together with cells from the unselected fractions after myeloablative irradiation of the recipient. Three other animals were transplanted with GFP-transduced CD34-enriched cells without prior GFP selection (GU). At 4 weeks after transplant, the percentage of GFP-expressing white blood cells was significantly higher in the GS group (6.6%) than in the GU group (1.3%) (p < 0.002). The higher gene transfer levels in the animals transplanted with GS cells gradually declined, and by day 100 after transplant, gene transfer levels were similar in both groups. PCR analysis performed on genomic DNA isolated from peripheral blood cells demonstrated that the decline in GFP-positive cells was due to the loss of gene-marked cells and not due to loss of expression. These results show that transplantation of CD34-positive marrow cells selected for GFP-positive cells after transduction provides high levels of transduced granulocytes in the short term. However, using this experimental design with concomitant infusion of unselected cells and the use of oncoretroviral vectors, preenrichment of vector-expressing, transduced CD34-enriched cells does not improve long-term persistence and expression.     

Endosonography of pararectal lymph nodes

One hundred thirteen patients with carcinoma of the rectum were evaluated for lymph node metastases by endorectal ultrasound. With the use of 7.5 MHz and based on different echo patterns, two main groups of lymph nodes can be differentiated: hypoechoic and hyperechoic lymph nodes. Compared with pathologic findings, hypoechoic lymph nodes represent metastases, whereas hyperechoic lymph nodes are visualized due to unspecific inflammation. Lymph node metastases can be predicted with a sensitivity of 72 percent and inflammatory lymph nodes with a specificity of 83 percent. The physical basis of the differentiation of lymph nodes was assessed in vitro by the determination of ultrasound parameters (speed of sound, acoustic impedance, attenuation, and backscattered amplitude). The attenuation coefficient of benign lymph nodes [2.5 dB/(MHz×cm)] is significantly higher than the mean value of lymph node metastases [1.3 db/(MHz×cm)]. The results demonstrate that involved nodes can principally be differentiated from not involved nodes. Micrometastases, mixed lymph nodes, and changing echo patterns within inflammatory nodes explain the accuracy rate of 78 percent.Supported by a grant from the Deutsche Forschungsgemeinschaft Hi 385/1-1     

Humans possess two mitochondrial ferredoxins,Fdx1 and Fdx2, with distinct roles in steroidogenesis,heme, and Fe/S cluster biosynthesis

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.     

Five-year evaluation of the influence of keratinized mucosa on peri-implant soft-tissue health and stability around implants supporting full-arch mandibular fixed prostheses

Background: The question of the importance of keratinized mucosa around dental implants for the prevention of peri-implant disease could not be answered in the relevant literature so far.
Objective: To investigate the influence of peri-implant keratinized mucosa on long-term peri-implant soft-tissue health and stability over a period of 5 years.
Material and methods: A total of 386 mandibular dental implants were placed in 73 completely edentulous patients, and subsequently restored with fixed full-arch prostheses. At prosthesis delivery (baseline) and after 3, 6, 12, 18, 24, 36, 48 and 60 months, modified plaque index (mPlI), modified sulcus bleeding index (mBI), distance between implant shoulder and mucosal margin (DIM) and width of peri-implant keratinized mucosa (KM) were recorded. Statistical analysis included multivariate logistic regression, multivariate ordinal logistic regression, generalized estimating equations and Bonferroni's correction.
Results: Fifty-eight patients with 307 implants completed the 5-year study. Statistically significantly higher plaque accumulation on lingual sites (mean mPlI 0.67, SD 0.85), bleeding tendencies on lingual sites (mean mBI 0.22, SD 0.53) and larger soft-tissue recession on buccal sites (mean DIM −0.69 mm, SD 1.11 mm) were found when the width of KM was <2 mm, compared to sites with≥2 mm of KM (mean mPlI 0.40, SD 0.68, P =0.001; mean mBI 0.13, SD 0.41, P <0.01; mean DIM −0.08 mm, SD 0.86 mm, P <0.001). The width of keratinized mucosa had no effect on bleeding tendency or plaque accumulation on buccal sites ( P >0.05).
Conclusion: In patients exercising good oral hygiene and receiving regular implant maintenance therapy, implants with a reduced width of <2 mm of peri-implant keratinized mucosa were more prone to lingual plaque accumulation and bleeding as well as buccal soft-tissue recession over a period of 5 years.     

Pattern of serum autoantibodies allows accurate distinction between a tumor and pathologies of the same organ

PURPOSE: Recent studies impressively showed the diagnostic potential of seroreactivity patterns for different tumor types, offering the prospect for low-cost screening of numerous tumor types simultaneously. One of the major challenges toward this goal is to prove that seroreactivity profiles do not only allow for identifying a tumor but also allow for distinguishing tumors from other pathologies of the same organ. EXPERIMENTAL DESIGN: We chose glioma as a model system and tested 325 sera (88 glioma, 95 intracranial tumors, 60 other brain pathologies, and 82 healthy controls) for seroreactivity on a panel of 35 antigens. RESULTS: We were able to discriminate between glioma and all other sera with cross-validated specificity of 86.1%, sensitivity of 85.2%, and accuracy of 85.8%. We obtained comparably good results for the separation of glioma versus nontumor brain pathologies and glioma versus other intracranial tumors. CONCLUSION: Our study provides first evidence that seroreactivity patterns allow for an accurate discrimination between a tumor and pathologies of the same organ even between different tumor types of the same organ.     

Radiobiological determination of growth kinetics and radiosensitivity of pulmonary micrometastases of the R1H tumour

The aim of the study was to determine the radiosensitivity and the growth kinetics of pulmonary micrometastases of the R1H tumour of the rat. Lung metastases were induced by intravenous injection of viable tumour cells. At different time intervals (3–32 days) after injection, lungs were locally irradiated with 200 kVp X-rays, using 1.5 Gy/fraction. Total doses of 6–33 Gy were administered within 11 days. Endpoints used were survival time, local control rate, and number of metastases in the lungs at autopsy. The data were evaluated using the multi-target model. Beginning in the fifth week after tumour cell inoculation the animals started to exhibit a pronounced dyspnoea and were sacrificed. Sections revealed an extensive metastatic infiltration of the lungs. With increasing total dose a prolongation of survival time as well as an increase in cure rate was observed. The number of metastases found in the lungs decreased with increasing total dose. It is concluded that metastatic growth does not start earlier than 3 days after tumour cell inoculation and accelerates continuously. The doubling time of the tumour cells in the micrometastases decreases continuously and from 5.2 to 1.2 days between day 3 and 40, whereas larger metastases containing more than 10⁶ cells show gompertzian growth kinetics. The cell doubling time in this stage of metastatic growth is longer than 5 days. During the first 4 weeks of metastatic growth the radiosensitivity of metastatic R1H cells in the lungs is the same as in vitro.