The effect of smell training on COVID-19 induced smell loss.

OBJECTIVE: while smell training appears to be effective for post viral smell loss, its effectiveness in COVID-19 induced smell loss is currently not well known. Therefore, we aim to investigate the potential effect of smell training on patients with COVID-19 induced smell loss. METHODS: we conducted a case-control study with two comparable cohorts. One of which (n=111) was instructed to perform smell training twice daily for 12 weeks, therapeutical adherence was monitored on a daily schedule, while the other cohort (n=50) did not perform smell training. The Sniffin' Sticks Test (SST) was used to objectify participants' sense of smell at baseline and after 12 weeks, reported as a Threshold, Discrimination, and Identification (TDI) score. We also determined the association between therapeutical adherence and the TDI scores. RESULTS: we found a significant difference in psychophysical smell function between patients with COVID-19 induced smell disorders who performed 12 weeks of smell training and those who did not. Median TDI difference between groups was 2.00 However, there was no association between the therapeutical adherence and olfactory function. CONCLUSION: we discovered a significant moderate difference in psychophysical smell function between patients with COVID-19-induced smell disorders who performed smell training and those who did not, implying a possible advantage of training. However, no relationship was found between therapeutical adherence of smell training and olfactory function.

Staphylococcus aureus protects its immune-evasion proteins against degradation by neutrophil serine proteases.

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.

Galectin-3 and Cyclin D3 Immunohistochemistry and Tumor Dimensions Are Useful in Distinguishing Follicular Oncocytic Carcinomas from Oncocytic Adenomas of the Thyroid.

Aims. Oncocytic (Hurthle) follicular cell tumors (OTs) of the thyroid are both adenomas (OAs) and follicular carcinomas (OCs). The routine diagnosis of these tumors can be problematic even after an accurate sampling and histological examination. Beside preoperative evaluation due to the tumor's dimension several studies have been performed to find markers able to distinguish malignant from benign follicular tumors in the thyroid, with Galectin-3 being one of the most effective. Recently, some authors suggested cyclin D3 as adjunct to the diagnosis of the oncocytic lesions of the thyroid. Methods and Results. In this paper we assess the role of Galectin-3 and cyclin D3 in a well-selected group of follicular oncocytic tumors (14 OCs and 26 OAs). The diameter of each lesion was also evaluated. The combination of Galectin-3 and cyclin D3 has a good specificity (81%) and sensitivity (100%). Moreover, the maximum diameter (in cm) of OCs is greater than OAs (4.1 versus 2.3). Conclusions. We believe that the use of Galectin-3 and cyclin D3 in OTs of the thyroid can be a helpful panel in daily practice when histology is doubtful.

Bovine Staphylococcus aureus Secretes the Leukocidin LukMF' To Kill Migrating Neutrophils through CCR1.

UNLABELLED: Although Staphylococcus aureus is best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF', exclusively harbored by S. aureus of ruminant origin, is a virulence factor associated with bovine infections. In this study, the molecular basis of the host specificity of LukMF' is elucidated by identification of chemokine receptor CCR1 as its target. Bovine neutrophils, the major effector cells in the defense against staphylococci, express significant cell surface levels of CCR1, whereas human neutrophils do not. This causes the particular susceptibility of bovine neutrophils to pore formation induced by LukMF'. Bovine S. aureus strains produce high levels of LukMF' in vitro. In culture supernatant of the mastitis field isolate S1444, LukMF' was the most important cytotoxic agent for bovine neutrophils. In a fibrin gel matrix, the effects of the in situ secreted toxins on neutrophils migrating toward S. aureus were visualized. Under these physiological ex vivo conditions, bovine S. aureus S1444 efficiently killed approaching neutrophils at a distance through secretion of LukMF'. Altogether, our findings illustrate the coevolution of pathogen and host, provide new targets for therapeutic and vaccine approaches to treat staphylococcal diseases in the cow, and emphasize the importance of staphylococcal toxins in general. IMPORTANCE: This study explains the mechanism of action of LukMF', a bicomponent toxin found in bovine lineages of S. aureus that is associated with mastitis in cattle. At a molecular level, we describe how LukMF' can specifically kill bovine neutrophils. Here, we demonstrate the contribution of toxins in the determination of host specificity and contribute to the understanding of mechanisms of coevolution of pathogen and host. Our study provides new targets that can be used in therapeutic and vaccine approaches to treat staphylococcal diseases in the cow. We also demonstrate the importance of toxins in specific elimination of immune cells, which has broader implications, especially in human infections.

Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.

Staphylococcus aureus community-acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA-MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA-MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α-type and ß-type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time-lapse microscopy. Phagocytosis of the CA-MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increased neutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

BACKGROUND: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. METHODS AND RESULTS: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). CONCLUSION: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

Confocal laser scanning microscopy as a new valuable tool in the diagnosis of onychomycosis - comparison of six diagnostic methods.

Onychomycosis is common and can mimic several different nail disorders. Accurate diagnosis is essential to choose the optimum antifungal therapy. The aim of this study was to evaluate the use of confocal laser scanning microscopy (CLSM) and optical coherence tomography (OCT) as new non-invasive diagnostic tools in onychomycosis and to compare them with the established techniques. In a prospective trial, 50 patients with suspected onychomycosis and 10 controls were examined by CLSM and OCT. Parallel KOH preparation, culture, PAS-staining and PCR were performed. PCR showed the highest sensitivity, followed by CLSM, PAS and KOH preparation. OCT offered the second best sensitivity but displayed the lowest specificity. CLSM and KOH preparation showed a high specificity and CLSM offered the best positive predictive value, similar to KOH preparation and OCT. Fungal culture showed the lowest sensitivity and the worst negative predictive value, yet culture and PCR are the only techniques able to identify genus and species. In summary, CLSM was comparable to PAS staining and superior to KOH preparation. Due to the low specificity we assess OCT not as appropriate. In the differentiation of species PCR outplays the fungal culture in terms of time and sensitivity.

Evasion of Toll-like receptor 2 activation by staphylococcal superantigen-like protein 3.

Toll-like receptors (TLRs) are crucial for our host defense against microbial infections. TLR2 is especially important to fight bacterial infections, as it specifically recognizes bacterial lipoproteins of both Gram-positive and Gram-negative origin. Present on a variety of immune cells, TLR2 is critical for host protection against several bacterial infections, including those caused by Staphylococcus aureus. This major human pathogen causes increasing health care problems due to its increased resistance to antibiotics. S. aureus secretes a wide variety of proteins that inhibit innate immune responses. Recently, several staphylococcal superantigen-like proteins (SSLs) have been described to mediate immune evasive properties. Here, we describe that SSL3 specifically binds and inhibits TLR2 activation on human and murine neutrophils and monocytes. Through binding of the extracellular TLR2 domain, SSL3 inhibits IL-8 production by HEK cells expressing TLR1/2 and TLR2/6 dimers, stimulated with their specific ligands. The SSL3-TLR2 interaction is partially glycan dependent as binding of SSL3 to TLR2 is affected upon removal of sialic acid residues. Moreover, the SSL3(R308A) mutant lacking glycan-binding properties shows lower TLR2 inhibition. An SSL3 mutant, lacking the N-terminal 126 amino acids, still retains full TLR2 inhibiting activity. Of other SSLs tested, only SSL4, which shares the highest homology with SSL3, blocks TLR2 activation. SSL3 is the first-described bacterial protein that blocks TLR2 activation through direct extracellular interaction with the receptor. This unique function of SSL3 adds to the arsenal of immune evasive molecules that S. aureus can employ to subvert both innate and adaptive immunity.

Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ibalpha and alpha IIb beta 3.

OBJECTIVES: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). METHODS AND RESULTS: When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, complete and irreversible aggregation was observed. Proteolysis of the extracellular part of GPIb alpha or the addition of dRGDW abrogated platelet aggregation. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s(-1), stable platelet aggregates were observed, and platelet deposition was substantially reduced after proteolysis of GPIb or after addition of dRGDW. SSL5 was shown to interact with glycocalicin, a soluble GPIb alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3 zeta. In addition, SSL5 was shown to interact with endothelial cell matrix (ECM) and this interaction enhanced aggregation of platelets from whole blood to this ECM. CONCLUSIONS: SSL5 activates and aggregates platelets in a GPIb alpha-dependent manner, which could be important in colonization of the vascular bed and evasion of the immune system by S. aureus.

Molecular evidence for different stages of tuberculosis in ancient bone samples from Hungary.

This paleomicrobiologic study was conducted on osseous tissue specimens from ancient Hungarian skeletal samples from the 7-8th and the 17th centuries AD with typical macromorphologic evidence of osseous tuberculosis (n = 3), morphologic alterations probably due to tuberculosis (n = 6), or with nontypical osseous changes of vertebral bodies suggestive of inflammatory reaction (n = 5). From these bone samples, DNA was extracted and amplified by polymerase chain reaction (PCR) by using various primer pairs recognizing DNA segments of different mycobacterial species. To confirm specificity of the analysis, the amplification products of several samples were subjected to restriction enzyme digestion and/or direct sequencing. Of the analyzed 14 cases, 8 were unambiguously positive for mycobacterial DNA of the Mycobacterium tuberculosis complex, as shown by the amplification of the IS6110 sequence. In 13 cases we found a PCR product with primers specific for the 65-kDa antigen gene, including 2 cases without genomic DNA. We conclude that the application of other mycobacterial DNA primers may reveal contamination of bones with atypical saprophytic mycobacteria. A positive result for typical mycobacteria was seen in 2 of 3 cases with typical morphologic signs of tuberculosis and amplifiable DNA, in 3 of 6 probable cases, but also in 3 of 6 cases with nontypical bone changes. This indicates that minor osseous reactions of the surface of vertebral bodies may be due-at least in several cases-to infections with bacteria of the M. tuberculosis complex. In these cases the disease may have proceeded rapidly, and the morphologic osseous changes may represent "early" stages of tuberculous infection of the vertebrae.

Detection of leprosy in ancient human skeletal remains by molecular identification of Mycobacterium leprae.

We isolated ancient DNA from skeletal remains obtained from a South German ossuary (approximately 1400-1800 AD) and from a 10th century Hungarian cemetery partially indicating macromorphologic evidence of leprosy. In samples taken of 2 skulls from Germany and of 1 hard palate from Hungary, Mycobacterium leprae-specific fragments of RLEP1 and RLEP3 were amplified using polymerase chain reaction (PCR), thereby confirming their specificity by sequencing. In another case, PCR with primers targeting IS6110 of Mycobacterium tuberculosis gave positive results only for a mandibular specimen. No signal for any mycobacterial DNA was observed in samples from 2 Hungarian foot bones. In ancient material, osseous involvement of M leprae may be detected and distinguished from other mycobacterial infections by specific PCR. In the small bones of leprous hands and feet, not enough M leprae DNA seems to be present for detection. This supports the view that rhinomaxillary leprous alterations result from direct bacterial involvement, while osseous mutilations of hands and feet result from a nervous involvement and/or secondary infections due to small lacerations of the overlying soft tissues.

Analysis of lipopolysaccharide (LPS)-binding characteristics of serum components using gel filtration of FITC-labeled LPS.

Lipopolysaccharide (LPS)-binding components in serum play an important role in modifying LPS toxicity. We analyzed the binding characteristics of LPS in the presence of serum using gel filtration of FITC-labeled LPS (FITC-LPS) with on line detection of optical density and fluorescence. FITC-LPS separately behaves as an aggregate resulting in a low, dequenched, fluorescence. Binding of single LPS molecules, segregated from the aggregate, to serum components results in an increase in the fluorescence due to dequenching, and a comigration of fluorescence and optical density signals using gel filtration. This method, in combination with the use of specific antibodies inducing additional shifts, demonstrated that in serum high-density lipoproteins (HDL), albumin and low-density lipoproteins (LDL) were able to monomerize LPS. An ELISA on collected fractions of the gel filtration revealed binding of the recently identified LPS-binding protein, serum amyloid P component (SAP), to the high molecular weight LPS aggregate. In serum, binding of soluble CD14 (sCD14) and LPS-binding protein (LBP) to LPS could not be detected. However, this was probably due to an overshadowing effect of albumin, as an extra addition of recombinant sCD14 to serum clearly monomerized FITC-LPS. Biosensor technology revealed that, of all LPS-binding components tested, only SAP clearly bound to the LPS-coated sensor chip. These results show that gel filtration of FITC-LPS is a quick and reliable method to study the binding characteristics of LPS-binding components.

Serum amyloid P component prevents high-density lipoprotein-mediated neutralization of lipopolysaccharide.

Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.

Serum amyloid P component bound to gram-negative bacteria prevents lipopolysaccharide-mediated classical pathway complement activation.

Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.

New insights into the role of serum amyloid P component, a novel lipopolysaccharide-binding protein.

Serum amyloid P component (SAP) is a highly preserved plasma protein named for its ubiquitous presence in amyloid deposits. Although SAP is described to bind many ligands, no clear biological function has been ascribed to it as yet. This review summarizes the current knowledge about the protein SAP, its ligands and functional properties. Finally, the author focuses on the recent finding of the binding of SAP to lipopolysaccharide (LPS) and Gram-negative bacteria and the possible functional consequences of these interactions.

Microsatellite instability and loss of heterozygosity in prostatic carcinomas: comparison of primary tumors, and of corresponding recurrences after androgen-deprivation therapy and lymph-node metastases.

BACKGROUND: The molecular mechanisms leading to prostate cancer progression are poorly understood. In particular, those changes which are responsible for androgen-independent growth and metastatic spread in prostate cancer are an issue of current investigations. METHODS: To gain more insight into these processes, paired microdissected samples from both untreated, locally advanced primary tumors (n = 20) and recurrences (n = 20) after conventional androgen-deprivation therapy (ADT) were analyzed retrospectively for microsatellite instability (MSI) and loss of heterozygosity (LOH) at nine loci on chromosomes 8, 18, and X by polymerase chain reaction. In parallel, 12 prostatic carcinomas treated by radical prostatectomy and nine corresponding lymph-node metastases were analyzed in the same way. RESULTS: The group treated with ADT showed a total of 10 MSI in 7 of the primary tumors (35%): 4 of these (20%) at one locus, and 3 of these (15%) at two loci. In the recurrences, MSI was observed in 4 cases (20%): 3 of these at one locus (15%), and 1 of these (5%) at two loci. LOH was found in 8 cases (40%) before as well as after ADT. In the radically resected carcinomas, MSI could be detected at two chromosomal loci in one of the primary tumors (8%) and in one of the metastases (11%); LOH was found in 2 primaries (16%) and 3 metastases (33%). CONCLUSIONS: Although MSI can be found in advanced prostatic carcinomas, it apparently does not play a major role in the progression of prostate cancer regarding androgen-independent growth or lymphogenous spread.

Lipopolysaccharide (LPS)-binding synthetic peptides derived from serum amyloid P component neutralize LPS.

Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolonged survival of actinomycin D-sensitized mice treated with LPS to induce septic shock, indicating a potential use of this peptide in the defense against serious gram-negative sepsis in humans.

Microsatellite analysis in serous tumors of the ovary.

Thirty-four serous ovarian neoplasms were analyzed for microsatellite instability (MIN) and loss of heterozygosity (LOH) at 20 chromosomal loci of eight different chromosomes, including the map positions of the six known mismatch repair genes. Twelve of the 20 (60%) serous ovarian tumors of low malignant potential (LMP) and 13 of 15 (87%) samples of serous ovarian carcinomas, taken from 14 patients, showed LOH at one or more of the analyzed microsatellite loci. In serous carcinomas, LOH of the dinucleotide repeat D7S521 was most frequent. Six of 20 (30%) LMP tumors were also affected by MIN at more than one locus, whereas in the carcinomas MIN was found only sporadically (p < 0.025). No correlation between increased occurrence of MIN and LOH at the chromosomal loci of the known mismatch repair genes were discovered for these LMP tumors. Although our observation regarding LOH frequency in serous LMP tumors and serous carcinomas is compatible with the hypothesis that serous LMP tumors might represent precursor lesions of invasive carcinomas, the results concerning the occurrence of MIN suggest that the mechanisms of tumorigenesis of some tumors of LMP are distinct from those in invasive serous carcinomas.

In serous ovarian neoplasms the frequency of Ki-ras mutations correlates with their malignant potential.

We analysed 44 tissue samples from serous ovarian neoplasms of different malignant potential for Ki-ras mutations by denaturing gradient gel electrophoresis (DGGE) and direct sequencing after microdissection. Point mutations at codon 12 were found in 7 of 20 tumours of low malignant potential (LMP) (35%) and in 2 of 6 well-differentiated carcinomas (33%). In contrast, no mutations were detected in the 11 poorly differentiated ovarian carcinoma samples or in the 7 serous cystadenomas. The frequency of Ki-ras mutations in serous ovarian tumours seems to correlate with the malignant potential of the neoplasms. The data favour the hypothesis of a de novo development of poorly differentiated ovarian carcinomas and do not support an evolution from LMP tumours or well-differentiated carcinomas.

Malignant tumors in an ancient Egyptian population.

Since it is still an open debate whether malignant tumors are mainly influenced by environmental factors, the frequency of such malignant tumors in historic populations with different living conditions is of particular interest. In the present study, we investigated the occurrence of malignant tumors affecting bone tissue in a population of mumrnies and skeletons, which had been excavated from the large necropolis of Thebes-West, Upper Egypt. Our study material comprised a series of at least 415 individuals (thereof 325 adults) dating from approx. 1500-500 B.C. All individuals had been mummified, but were severely damaged and partially broken by grave robbers, so that often only parts of the mummies/skeletons were available for investigation. The available specimens were subjected to careful macroscopic examination, while isolated findings were radiologically analyzed. Using this approach, we identified at least 4 cases showing malignant tumors affecting the skeleton. In two cases, multiple mixed osteolytic-osteoblastic lesions suggested multiple metastases from carcinomas. Two further individuals presented with multiple osteolyses (vertebra, pelvis, skull) most suggestive of multiple myeloma. The observation of at least 4 cases of malignant tumors with osseous manifestation in a series of 325 adult individuals provides clear evidence that malignant tumors were not a rare event in the ancient Egyptian study population, particularly when the limitations of a study of tumors manifested only in osseous remnants are taken into consideration. A calculation of the age- and sex-adjusted tumor frequency in our material in comparison with a recent model for such a material by Waldron (1996) indicates that the rate of malignant tumors with bone affection in our series is higher than in an English population from 1901-1905, although lower than in a comparable present day population. This clearly indicates that important factors affecting malignant tumors were effective even in historic populations.