[99mTc-MIBI scintigraphy of hypofunctional thyroid nodules. Comparison of planar and SPECT imaging]. / 99mTc-MIBI-Szintigraphie hypofunktioneller Schilddrüsenknoten. Vergleich von planarer und tomographischer Technik.

UNLABELLED: 99mTc-MIBI-scintigraphy allows to assess the dignity of hypofunctional thyroid nodules. A concordant pattern in MIBI- and pertechnetate-scintigraphy excludes malignancy with high accuracy. For increased MIBI-uptake histological evaluation is advised. The assessment of MIBI-isointense nodules is discussed controversially. Objective of our study was to analyse the prevalence of malignancy for isointense nodules and the diagnostic accuracy of image acquisition in SPECT-technique. PATIENTS, METHODS: MIBI-scintigraphies were analysed retrospectively. Imaging was performed 60 min after intravenous injection of 510 MBq 99mTc-MIBI. Thyroid nodules were assessed as hypo-, iso- or hyperintense compared to the paranodular tissue. RESULTS: 83 of 225 patients underwent thyroid surgery (age 48.6 ± 12.6 years, 72% women). In 12 (14.5%) cases a papillary carcinoma was diagnosed. In planar imaging 12, 34 and 37, in tomographical imaging 16, 21 and 46 nodules, respectively, were classified as hypo-, iso- oder hyperintense. Among hypo-, iso- and hyperintense nodules in planar imaging 1, 5 and 6 carcinomas were found, resp. In tomographical imaging no, 4 and 8 carcinomas were found, respectively. Classification of iso- and hyperintense nodules as "suspicious for malignancy" showed for planar imaging a sensitivity, specificity, NPV and PPV of 91.7, 15.5, 91.7 and 15.6%, for tomographical imaging of 100, 22.5, 100 and 17.9%, respectively. CONCLUSION: Hypofunctional thyroid nodules with iso- and hyperintense MIBI-Uptake have a comparable prevalence of malignancy. Image acquisition in SPECT-technique results in improved diagnostic sensitivity and negative predictive value.

Spore-forming bacteria and their utilisation as probiotics.

In this review article, the beneficial application of bacterial spore formers as probiotics in the food industry is discussed based on the knowledge gleaned from current publications. The summary of new scientific results provides evidence of the advantages of the utilisation of Bacillus or Clostridium strains in the food industry. Both bacteria are able to produce a very stable duration form: the endospore. Compared to the widely used lactic acid bacteria, bacterial spores offer the advantage of a higher survival rate during the acidic stomach passage and better stability during the processing and storage of the food product. In many food products, germination of the spores does not occur. Hence the product quality of the food is not affected because of their inactive metabolism. Besides the possible utilisation and functional properties, an overview of the fast-developing knowledge about the mechanisms of the beneficial health effects of spore-forming bacteria is provided.

[Posthepatic obstructive jaundice caused by primary extragonadal germ-cell tumor in a patient with glucose-6-phosphatase dehydrogenase deficiency]. / Posthepatische Cholestase infolge primär-extragonadaler Manifestation eines Keimzelltumors bei einem Patienten mit Favismus.

HISTORY AND ADMISSION FINDINGS: In patients with glucose-6-phosphatase dehydrogenase (G6PD) deficiency (favism) jaundice is usually caused by hemolysis due to stress, infection or following the application of drugs. We report on a 74-year-old Italian with known G6PD deficiency complaining of jaundice, weight loss and abdominal pain. Physical examination revealed jaundice of the eyes. Scrotal examination by palpation and ultrasound showed no abnormalities. INVESTIGATIONS: Serum levels of beta-human chorionic gonadotropin and alpha-fetoprotein were within normal limits, total bilirubin was extremely elevated, with predominant direct bilirubin. Abdominal ultrasound showed posthepatic blockage of bile flow with a dilated ductus hepatocholedochus (DHC) in the absence of gallstones. Enlarged, multiple contrast-stained paraaortic and retroperitoneal lymph nodes were detected by endoscopic ultrasound and magnetic resonance imaging. Due to failed endoscopic retrograde cholangiopancreatography, visualization of the biliary tree by percutaneous transhepatic cholangiography (PTC) was performed showing an occlusion of the DHC. THERAPY AND COURSE: After successful stent-implantation by PTC with decompression of the biliary tree, the jaundice disappeared. Computer tomography-guided percutaneous biopsy of a retroperitoneal lymph node was performed for histological evaluation showing a primary extragonadal nonseminomatous germ cell tumor. According to the histology (embryonic carcinoma) and clinical stage of the tumor systemic chemotherapy was initiated including cisplatin, etoposide and ifosfamide. After the first cycle of chemotherapy the patient suffered from pneumonia leading to septic shock. Twenty-seven days after admission, the patient died of multiple organ failure. CONCLUSION: Extragonadal germ-cell tumor presenting as retroperitoneal lymph nodes with obstructive jaundice has to be considered in the differential diagnosis of cholestasis.

Relevance of microbial coculture fermentations in biotechnology.

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro-organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.

[Dysphagia, heartburn and esophageal bolus impaction in a 43-year old male patient: not always reflux disease]. / Dysphagie, Sodbrennen und Bolusobstruktion bei einem 43-jährigen Mann: Nicht immer Refluxkrankheit.

We report a case of a 43 year old male patient, who was admitted with recurring esophageal bolus impactions. Since his childhood he has been complaining about dysphagia and was unable to swallow medication. He also complained about heartburn. The last esophageal bolus impaction was some weeks ago. After elimination of the bolus impaction with a rigid endoscope we found a high grade stenosis in the proximal esophagus that could not even be passed with a children's endoscope. An initial treatment of eosinophil esophagitis would be the therapy with a local corticoid for 6-9 month. In patients with typical rings or stenosis a dilation therapy might be necessary.

[Erionite-induced pleural plaques. Exposition to urban pollution in a female Turkish migrant in Germany]. / Erionitinduzierte Pleuraplaques. Urbane Belastung bei einer türkischen Migrantin.

Erionite is a zeolite mineral of volcanic origin which contains no asbestos. It is found in different regions of the world, including southeast Turkey in ash and dust, from which it can cause inflammatory pleural plaques or malignant pleural mesothelioma (MPM). We report a female Turkish migrant exposed to urban pollution in her home country who decades later suffered from pleural plaques with a nonspecific chronic inflammatory disease. The differential diagnosis of inflammatory pleural plaques was assumed radiologically and confirmed by video-assisted thoracoscopic biopsy. Short-term clinical and radiologic control of the patient will be necessary because of the risk of MPM. For epidemiologic considerations discussed referring to current literature, a growing incidence of this type of disease in migrants from high-risk areas must be reckoned with in Germany, even without exposition to asbestos.

Expression of calcitonin receptor-like receptor in human vascular tumours.

BACKGROUND: Vascular tumours such as Kaposi's sarcoma and capillary haemangioma are characterised by abnormal vascularisation and proliferation of endothelial cells or neoplastic cells. Adrenomedullin, a potent vasodilative peptide, and its receptor, calcitonin receptor-like receptor (CRLR), play an important part in angiogenesis. AIM: To establish whether this system also plays a part in vascular diseases, showing abnormal proliferation such as vascular tumours. METHODS: CRLR expression was investigated in several specimens of Kaposi's sarcoma and other vascular tumours, using immunohistochemical analysis with a previously described CRLR-specific polyclonal antibody and reverse transcriptase-polymerase chain reaction. RESULTS: Intense and specific CRLR-immunoreactive staining of neoplastic cells was observed in all specimens, which was of greater intensity than similar staining of adjacent normal endothelium. CONCLUSIONS: CRLR is expressed in vascular tumours and, with adrenomedullin, may have a role in neoplastic vascular growth.

[Usefulness of 16S rDNA polymerase-chain-reaction (PCR) in the intraoperative detection of infection in revision of failed arthroplasties]. / Wert der 16S rDNA Polymerase-Ketten-Reaktion (PCR) zur intraoperativen Infektdetektion bei Endoprothesenrevisionseingriffen.

AIM: In this study the accuracy of the 16S DNA polymerase chain reaction (PCR) in revision arthroplasties was compared to that of conventional bacterial culture when correlated to intraoperative histological findings. Furthermore, the influence of antibiotic treatment and different ways of collecting samples was evaluated. METHOD: In a prospective study we collected samples of tissues, aspiration fluids and swabs during revision arthroplasty surgery and examined them with PCR as well as conventional bacterial culturing methods. Also, we correlated these two methods with the histological findings of intraoperative tissue samples. Two independent examiners evaluated the samples according to the criteria of Mirra et al. Sensitivity, specificity, positive and negative prediction value and the accuracy were calculated for the molecular biological and culture methods. Three groups were defined to evaluate the influence of accompanying antibiotic treatment and the way of collecting the microbiological samples. RESULTS: Nine periprosthetic infections could be detected by PCR as well as by conventional bacterial culturing. Correlated with the 25 positive histological findings this resulted in a sensitivity of 0.36, a specificity of 1.0, a negative prediction value of 0.61, a positive prediction value of 1.0 and an accuracy of 0.68 for both methods. Swabs compared to aspiration fluids and tissues samples showed the highest sensitivity with both methods. No higher sensitivity of PCR compared to conventional bacterial culturing could be observed in patients with accompanying antibiotic treatment. CONCLUSION: Although PCR is more rapidly available for the diagnosis of periprosthetic infection, a definite advantage of this more expensive method could not be demonstrated in view of the same low sensitivity of PCR and conventional bacterial culturing.

[Infection diagnosis after knee-TEP-implantation]. / Infektdiagnostik nach Knie-TEP-Implantation.

AIM: In this study, the accuracy of antigranulocyte scintigraphy as a diagnostic means prior to revision in infected total knee replacement was compared to that of preoperative joint aspiration and laboratory parameters. The most efficient combination of all diagnostic methods was calculated and thus a diagnostic algorithm recommended. The value of PCR was compared to commonly used techniques of microbiological culturing. METHODS: Preoperative diagnostic means for infection of 50 total knee replacements in 45 patients requiring revision surgery, were retrospectively analyzed. Inclusion criteria were the intraoperative microbiological and histological verification of infection. Sensitivity, specificity, negative and positive prediction value of C-reactive protein (CRP) and leukocytes, antigranulocyte scintigraphy with (99m)Tc-labeled antibodies, and preoperative joint aspiration were calculated. Furthermore, the accuracy of the different techniques of culturing was compared to that of the polymerase chain reaction (PCR) based on the intraoperative histological findings. Two blinded examiners evaluated specimens taken intraoperatively according to the criteria of Mirra. RESULTS: We observed a sensitivity of 1.0, a specificity of 0.82, a positive prediction value of 0.83 and a negative prediction value of 1.0 for the antigranulocyte scintigraphy. The sensitivity of preoperative joint aspiration was 0.5, the specificity 1.0, and the positive and negative prediction values were 1.0 and 0.5. Correlated to the intraoperative histological findings the accuracy of PCR and culturing was comparable. The highest accuracy was obtained for blood culture samples. CONCLUSION: Compared to preoperative joint aspiration the antigranulocyte scintigraphy proved to be more sensitive in the diagnosis an infected knee replacement while having a high specificity. An advantage of PCR compared to the common microbiological culturing techniques was not observed.

Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate.

L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering. Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase. Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P. Hyperosmotic stress did not affect the L-G3P pool in the gpp1 Delta gpp2 Delta mutant overexpressing GPD1 despite an about 4-fold increase of specific GPD activity. In contrast, oxygen limitation improved intracellular L-G3P concentration by enhancing the availability of cytosolic NADH. The reduction of pyruvate decarboxylase activity by deleting PDC2 led to an additional increase. In fact, the triple mutant gpp1 Delta gpp2 Delta pdc2 Delta overexpressing GPD1 accumulated 17 mg L-G3P/g dry weight during glucose batch fermentation under oxygen limitation. This value corresponds to an about 100-fold increase compared to that found in the wild type.

Antifungal proteins: targets, mechanisms and prospective applications.

All organisms have evolved several defence systems in order to protect themselves against bacteria, fungi and viruses. Higher organisms have developed a complex network of humoral and cellular responses, called adaptive immunity. A second defence system, innate immunity, was discovered in the early 1980s, consisting of small cationic peptides with a broad antimicrobial spectrum. These proteins act immediately at sites of infection or inflammation. The production of proteins with antimicrobial activity was not limited to higher organisms but was also found in insects, plants and microorganisms. During the last 2 decades a broad range of proteins with very different structural features have been isolated and characterised from differing organisms ranging from bacteria to human beings. Over 500 cationic membrane-acting proteins with antimicrobial and antifungal activities have been identified to date. Apart from these proteins, a very large number of antifungal proteins active on the fungal cell wall, on enzymes of the cell wall synthesis machinery, the plasma membrane and on intracellular targets have been characterised.

[Phosphoinositide 3-kinase (PI3-K) expression. Tumorigenesis of epithelial carcinoma of the mouth]. / Phosphoinositid-3-Kinase- (PI3-K) Expression. Tumorgenese von Plattenepithelkarzinomen der Mundhöhle.

Phosphoinositide 3-kinase (PI3-K) is a heterodimeric enzyme involved in the regulation of mitogenesis, apoptosis, cell adhesion, and motility. PI3-K was suggested as a protooncogene in human cancer. To determine the expression of PI3-K during cancerogenesis and tumor invasion of HNSCC, we investigated normal and dysplastic epithelium of the oral cavity, squamous cell carcinoma and lymph node metastasis by immunohistochemistry. The strongest immunoreactivity for p85alpha and p110alpha was found in invasive tumors and their metastases. Carcinomas in situ showed a focal positivity. Dysplasias and normal epithelium reacted predominantly negatively. The PI3-K inhibitor LY294002 inhibited proliferation and invasion of the HNSCC cell line CAL-27 and induced apoptosis in vitro. Our data suggest PI3-K as a marker of malignancy and tumor invasion. We suggest including PI3-K in the multistep carcinogenesis model of HNSCC. In addition, PI3-K is a potential target for pharmacological intervention.

[Stage diagnostics for postinfection revision of hip and knee replacement: value of laboratory parameters and antigranulocyte scintigraphy]. / Stufendiagnostik bei postinfektiöser Hüft- und Knie-TEP-Implantation: Aussagekraft von Laborparametern und Antigranulozytenszintigraphie.

AIM: In the case of two-stage infect revision arthroplasties of hip and knee joint, of primary resection arthroplasties and before implantation of arthroplasties after septic arthritis the accuracy of preoperative laboratory parameters and antigranulocyte scintigraphy was analysed. Furthermore, we stained the intraoperatively taken joint synovial samples with hematoxylin-eosin and also with antibodies against human neutrophil elastase in order to investigate if immunohistological examination provides further or different information. METHOD: In 24 patients with intraoperative verification of infection we calculated sensitivity, specificity, positive and negative predictive values for laboratory tests, antigranulocyte scintigraphy and the most suitable combination of both. The joint synovial samples stained with antibodies against human neutrophil elastase were compared to those stained with hematoxylin-eosin according to the criteria of Mirra. RESULTS: We observed the best results for the combination of C-reactive protein and antigranulocyte scintigraphy with a sensitivity of 1, a specificity of 0.92, a positive predictive value of 0.75 and a negative predictive value of 1. No additional or different information was observed by the immunohistological stained samples. CONCLUSION: Stage diagnostic using C-reactive protein and antigranulocyte scintigraphy provides accurate information to assess the status of infection before hip and knee replacement after infect revision. Additional immunohistological staining besides the routinely taken hematoxylin-eosin staining of joint synovial samples is not recommended.

The antifungal protein from Aspergillus giganteus causes membrane permeabilization.

We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 micro g/ml for Fusarium oxysporum to 200 micro g/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.

Transcriptional regulation of the Antifungal Protein in Aspergillus giganteus.

Regulation of the expression of the afp gene that codes for the Antifungal Protein of Aspergillus giganteus was investigated using a reporter system. For this purpose, the E. coli reporter gene uidA encoding beta-glucuronidase (GUS) was placed under the control of the afp promoter. No homologous integration of the reporter construct into the afp site was observed among 156 transformants tested. In one of the transformants carrying a single, ectopically integrated, copy of the construct, GUS and AFP both displayed exactly the same temporal expression patterns under various cultivation conditions, as assayed by Northern and protein analyses. Thus, this transformant was used to identify factors that are involved in the transcriptional regulation of afp expression. Expression is only detectable in the vegetative mycelium, whereas no expression occurs in aerial hyphae or conidia, indicating that afp expression is developmentally regulated. Transcription of afp is regulated by ambient pH, being suppressed under acidic conditions and strongly induced under alkaline conditions. This observation suggests that PacC regulates the afp gene, which is consistent with the presence of two putative PacC binding sites within the 5' upstream region. Transcription is not subject to carbon catabolite repression or nitrogen metabolite repression. The expression of afp is up-regulated by heat shock, upon growth in the presence of excess NaCl and ethanol, and under conditions of carbon starvation. In contrast, expression decreases slightly in the presence of hydrogen peroxide and under nitrogen starvation. These data are compatible with the presence of a putative heat shock element (NTTCNNGANTTCN) and five putative C(4)T stress-responsive elements within the afp promoter.

[Cellular senescence: a mechanism of the development of osteoporosis?]. / Zelluläre Seneszenz--ein Mechanismus der Osteoporoseentstehung?

Osteoporosis is one of the most common diseases of the elderly. This leads to the hypothesis that the ageing of the organism is reflected as a cytogerontological effect in a specific loss of bone cell function. Three underlying pathogenetic mechanisms need to be considered: (1) cellular aging in general, (2) impairment of the systemic stimulation of bone formation by e.g. decreasing hormone levels, and (3) lower cellular effectiveness of cytokines and growth factors. Cellular aging consists of replicative and postmitotic senescence. While the replicative senescence limits only the number of cell cycles, the postmitotic aging is influenced by endo- and exogenous factors. These lead to genetic alterations known as delayed persistent genomic instability and to an increasing impairment of specific cellular functions. In the postmitotic phase, osteopenia caused by the decrease of systemically available sexual hormones is a major field of research. Osteopenia caused by a decreased activity of locally effective cytokines and growth factors is becoming increasingly understood. New therapeutic strategies, which modulate the local osteoblast activity, e.g. in bone defect healing, are under development. In conclusion, cellular senescence is considered to be one element in the development of bone loss. Potential therapeutic targets may open up an additional path in the treatment of local and systemic osteopenias.

Phase-specific gene expression in Saccharomyces cerevisiae, using maltose as carbon source under oxygen-limiting conditions.

The transcription of ten stress-response genes was investigated under oxygen-limiting conditions with maltose and glucose, respectively. Six of these genes (HSP12, HSP26, HSP30, HSP78, HSP82 and HSP104) showed expression only during the stationary phase. HSP12 and HSP104 were transcribed 10 h earlier with maltose than with glucose. Fermentation in wort yielded similar results to the maltose-based medium. HSP12, HSP26 and HSP30 were highly expressed. Thus, the HSP26 and the HSP30 promoter can be used for late, phase-specific expression of the desired genes with glucose or maltose as carbon source, and HSP12 with glucose only. MET14, a gene important for sulfite formation, was overexpressed in wort, using the HSP26 promoter during the stationary phase.

Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria.

Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5alpha, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.

Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non-artificial conditions.

Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA. These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell. Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB. The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events.

Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts.

BACKGROUND-Intravascular clotting has been implicated in the pathogenesis of cardiac allograft vasculopathy (CAV). We previously identified the expression of tissue factor (TF), the primary cellular initiator of blood coagulation, within the coronary intima, which was associated with neointimal thickening. In the present study, the effect of recombinant hirudin on CAV was assessed in Lewis to Fisher rat heterotopic cardiac allografts. METHODS AND RESULTS-Transplant recipients were randomized to a control group (n=10) and a hirudin-treated group (n=12; 2 mg. kg(-1). d(-1) SC). Histological evaluations of rejection, CAV, and TF staining were performed 120 days after transplantation. No significant differences were observed between the 2 groups with respect to the degree of rejection. Hirudin significantly (P<0.05) suppressed the development of CAV in the graft microvessels, but it was less effective in large coronary arteries. Graft intimal cells, isolated by laser-assisted cell picking, showed a marked upregulation of TF gene transcription, which was prevented by hirudin (P<0.01). As demonstrated by immunohistochemistry and quantitative analyses of TF mRNA levels by real-time polymerase chain reaction, hirudin treatment resulted in a significant reduction of TF protein and mRNA expression (P<0.001). CONCLUSIONS-Treatment with hirudin in this rat cardiac transplant model inhibited TF expression and decreased neointimal hyperplasia. These results suggest that TF inhibition by hirudin, in addition to its direct effect on thrombin, may attenuate the hypercoagulable state and prevent the development of CAV at least in restricted sites of the graft coronary vasculature.