Thin polyester filters as versatile sample substrates for high-pressure freezing of bacterial biofilms, suspended microorganisms and adherent eukaryotic cells.

High-pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high-pressure freezer. We explored the potential of a 10 µm thin polyester filter membrane (PE-filter) as a versatile sample substrate for high-pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE-filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters were used for high-pressure freezing, freeze-substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms. LAY DESCRIPTION: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology-changes due to drying artefacts or the impact of chemical stabilisers, high-pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to -196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology-destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 µm in optimal cases). High-pressure freezing is regarded as the gold-standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high-pressure freezer and their transfer into the high-pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 µm thin polyester filter membrane (PE-filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE-filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface-adhering higher cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters could be directly used for high-pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning-electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms.

[Complementary and alternative medicine in pediatric oncology]. / Komplementäre und alternative Behandlungsmethoden in der Kinderonkologie.

For many families of pediatric cancer patients the use of complementary and alternative medicine (CAM) is an accepted adjunct to conventional therapy, even if data regarding effects and risks are scarse. This report provides information about the prevalence of CAM use and reasons for CAM use among pediatric cancer patients. Frequently used CAM like nutrition (incl. diets and vitamins), homeopathy, anthroposophic medicine, phytotherapy, and acupuncture are highlighted, as were CAM bearing a special risk for their users. Physicians need to be more aware that a substantial percentage of their patients may use CAM without telling them for different reasons. Physicians need to be open-minded and should discuss CAM with parents. A differentiation between potentially useful and potentially dangerous CAM is necessary aiming at minimising the risks for CAM users.

[Recurrent pancreatitis in pediatric ulcerative colitis--a case report]. / Rezidivierende Pankreatitis bei kindlicher Colitis Ulcerosa--ein Fallbericht.

We report on a 12 years old girl with ulcerative colitis, who after a two years course of her disease developed a concomitant idiopathic pancreatitis. We discuss the causes of acute pancreatitis in childhood in view of the presented case summarizing the current literature. In the literature we found only two other children with ulcerative colitis and idiopathic pancreatitis. Both patients were colectomised as our patient did. Six months after colectomy she is still free of symptoms.

Complete remission of a diffuse pontine glioma.

A patient is described in whom a large diffuse glioma of the pons extending into the midbrain was diagnosed at the age of 2 years. Biopsy showed a fibrillary astrocytoma. After shunting of a hydrocephalus, the clinical symptoms abated without conventional therapy. Repeated MRI studies showed a continuous decrease of the tumour which was no longer visible when the patient was 6.6 years old. In reviews on spontaneous remissions of oncologic disorders we were unable to find a case of a biologically benign brain stem tumour. There is one isolated report on a similar case, though without histologic documentation.

Association of class II sequences encoding DR1 and DQ5 specificities with hypersensitivity to chironomid allergen Chi t I.

A panel of 188 unrelated Caucasian subjects who were exposed to the larvae of Chironomus thummi (Diptera, nonbiting midges) was HLA-typed by polymerase chain reaction amplification of the second exons of the DRB, DQA1, and DQB1 genes followed by dot-blot hybridization with sequence-specific oligonucleotide probes. Type I sensitization to the allergen Chi t I and a large number of other inhalant allergens was determined by RAST and skin testing. Sixty-one individuals were found to be sensitized to Chi t I, of whom 24 were sensitive to this allergen and to no other allergens tested. Statistical analyses showed that only in the latter group were the HLA-D genes DRB1*0101, DQA1*0101, and DQB1*0501 associated with IgE-responsiveness to Chi t I. These results suggest that HLA associations with responsiveness to certain allergens may be more striking in monosensitized subjects.

A novel HLA-haplotype containing a DRB5 gene not associated with DRB1*15 or DRB1*16 alleles.

In Caucasians, HLA-DR2 haplotypes usually express two DRB genes encoding the DR specificity. The DRB5 genes are in strong linkage disequilibrium with the respective DRB1*15 or DRB1*16 alleles. So far, no other DRB1 alleles have been found in association with DRB5 genes. Here, we report evidence for a probably recombinant DRB5 haplotype with a DRB1 allele not specific for DR2. From our results the haplotype DQB1*0501, DRB1*0101 and DRB5*0101 seems most likely.

Complete sequences of the rRNA genes of Drosophila melanogaster.

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.

Evolutionary divergence of promoters and spacers in the rDNA family of four Drosophila species. Implications for molecular coevolution in multigene families.

The organization and sequence of the rDNA multigene family of four Drosophila species (melanogaster, orena, virilis and hydei) have been compared in order to understand the quality and quantity of the differences which are involved with interspecific divergence of promoters and the polymerase I complexes (molecular coevolution). Each species has an intergenic spacer (IGS) made up of subrepeats which contain duplications of the promoter. Major structural and point-mutational differences exist, most of which have been spread by unequal crossingover through the family and species. Structural differences involve the types, lengths and copy-number of the IGS subrepeats, and the lengths and position of "unique" regions between blocks of repeats. The 240 base-pair repeat array shared by D. melanogaster and D. orena has been replaced by a 220 base-pair repeat, and the 95 and 330 base-pair arrays are absent altogether in D. virilis and D. hydei. The length of the "unique" region between the 240/220 base-pair arrays and the start of transcription varies, with the unusual situation of the last of the 220 repeats ending at the external transcribed spacer (ETS) boundary in D. virilis. Other structural differences involve regions of high cryptic simplicity arising from slippage in D. virilis and D. hydei IGSs. Sequence analysis of IGS and the ETSs indicates that the rDNA is not uniformly divergent throughout its length. Apart from the genes, there are regions of relatively high conservation covering the promoter regions and at some but not all potential RNA processing sites. The conserved promoter regions are more extensive within each pair of species D. melanogaster versus D. orena and D. virilis versus D. hydei, in keeping with their phylogenetic distances. Slippage-like mechanisms are involved with large numbers of deletions/insertions that make up the ETS differences between the species. Patterns of shared mutations between IGS subrepeats indicate stages of transition during rDNA differentiation by continual homogenization. The simultaneous operation of different turnover mechanisms, at different periodicities and rates, generates a complex picture of reorganization, some of which would influence the process of molecular coevolution in the family.

Hemolytic activity of Serratia marcescens.

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca2+ ions.

[The electrophoresis mobility (em) test in cervical-intra-epithelial neoplasia (c.i.n.) (author's transl)]. / Der Elektrophorese-Mobilitäts-(EM-)Test bei präinvasiven dysplastischen Epithelveränderungen an der Portio vaginalis uteri.

The electrophoresis mobility test (EM) is credited with an accuracy of 70-90% for the diagnosis of gynaecological malignancies. The test was done in 21 patients with dysplasia of carcinoma in situ of the uterine cervix, 34 patients without malignant or pre-malignant gynaecological findings and 27 women who were not in medical care at the time of the test. The correlation between the EM test and cervical intra-epithelial neoplasia was thus tested. In patients with dysplasia or carcinoma in situ of the cervix the test was positive in over 80%. False positive tests were found in 35% of the patients with non-malignant or pre-malignant gynaecological findings and in 11% of the 27 patients not in medical care. Despite the possibility of regression of the cervical intra-epithelial neoplasia an identical precentage of patients to those with invasive carcinomas of the cervix showed a positive EM test. The density of lymphocytic cells in the dysplastic epithelium of the cervix was also investigated. A correlation between the migration and inhibition in the EM test and the number of lymphocytic cells in the dysplastic epithelium was not found.

[Electrophoretic mobility test in the early diagnosis of malignant tumors; findings in the prestages of cervical and breast cancers]. / Der Elektrophorese-Mobilitäts-(EM-)Test in der Frühdiagnostik maligner Tumoren; Befunde bei Vorstufen des Zervixkarzinoms und Mammakarzinoms.

A double-blind pilot study of 130 female patients was carried out to determine the feasibility and significance of the EM test in the early diagnosis of carcinoma of the female genital organs and breast. Early stages of cervical carcinoma (carcinoma in situ) as well as fibroid adenoma, mastopathy and breast tumors were tested and compared with the results of their manifest forms. Positive results were recorded in 87.5% of the cases of middle-grade to severe epithelial dysplasia (Papanicolaou III and IV) (n = 10) and in 90% of the cases of carcinoma in situ (n = 16). A positive result in 90% of the cases of carcinoma in situ (n = 16). A positive result in 90% of the cases of genital carcinoma was also recorded. In the case of fibroid adenoma (n = 10) and breast tumor (Prechtel I and II) (n = 16), negative results were recorded in 80.8%, whereas in the manifest forms of carcinoma of the breast a positive result of over 95% was shown. As a result of this pilot study, it can be seen that immunological in vitro screening has clinical significance in the early diagnosis of tumors as well as for confirmation of their manifest forms.

Cell-mediated immunity in patients with renal adenocarcinoma.

The lymphocytotoxicity test was used to study the existence of immunologically active lymphocyte reactions in patients with renal carcinoma at different stages. In 9 patients with an advanced renal carcioma (stage IV) a low lymphocytotoxicity was found, while the examination of 7 patients with stage I and II renal tumours showed a high value of the lymphocytotoxicity test. The clinical importance of the lymphocytotoxicity test is discussed with special interest in a possible immunotherapy.