Evaluation of antimicrobial effects of novel implant materials by testing the prevention of biofilm formation using a simple small scale medium-throughput growth inhibition assay.

Staphylococcal colonization of implants is a serious complication of orthopaedic surgery. Anti-infectious modification of implant surfaces may serve to prevent bacterial colonization. The authors set out to develop an in vitro test system for the analysis of prevention of biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus on implant materials. Biofilm growth was monitored over 10 days on titanium disks in order to develop appropriate test parameters. Bacterial cell counts following ultrasonic treatment of the colonized samples were compared with scanning electron microscope images of the specimens. Copper ion containing surfaces (ie copper [Cu] and inter-metallic Ti-Cu films) were used for growth inhibition assays: copper ion releasing specimens led to reduced bacterial numbers in biofilms and decreased bacterial persistence in the model used. The assay used represents an inexpensive and quick in vitro screen for the antibacterial effects of novel implant surface materials.

Intraductal carcinoma is the precursor of carcinoma ex pleomorphic adenoma and is often associated with dysfunctional p53.

AIMS: Although intraductal carcinoma has been demonstrated in intracapsular carcinoma ex pleomorphic adenoma (CEPA), the morphological and genetic stages of transformation of pleomorphic adenoma (PA) to CEPA are not fully understood. The aim of this study was to investigate the morphology of intracapsular CEPA. METHODS AND RESULTS: The largest series of intracapsular CEPA studied was subject to immunohistochemical double-staining to detect p53 protein and cellular proliferation in different types of cell combined with mutational analysis of the p53 gene in laser-microdissected material. Intraductal carcinoma with high-grade cellular atypia and frequent accumulation of p53 protein was found in 15/19 cases. Purely intraductal carcinoma was found in eight cases. Mutation of p53 was found in 7/19 cases, of which it was found in intraductal carcinoma in 5/15 cases. CONCLUSIONS: The frequent demonstration of intraductal carcinoma indicates that this preinvasive lesion is likely to be a constant feature in the malignant transformation of PA to CEPA. It appears to be a feature of CEPA developing from both primary and recurrent PA. The combined immunohistochemical and genetic data show that 14/19 cases of CEPA and 11/15 cases with intraductal carcinoma showed genetic or morphological evidence of dysfunctional p53, indicating that this is an early event in malignant transformation.

AIDS, multicentric Castleman's disease, and plasmablastic leukemia: report of a long-term survival.

Plasmablastic leukemia (PL) as a complication of human herpes virus 8 (HHV8)-associated Castleman's disease is marked by a rapid and fatal outcome. In patients with AIDS, survival of 7 to 14 days after diagnosis has been reported. Prompt splenectomy and chemotherapy might lead to a significant survival benefit. Here we report a case of long-term survival in a patient with AIDS and multicentric Castleman's disease (MCD) complicated by PL.

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines.

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases.

Activation of matrix-metalloproteinase-2 and membrane-type-1-matrix-metalloproteinase in endothelial cells and induction of vascular permeability in vivo by human immunodeficiency virus-1 Tat protein and basic fibroblast growth factor.

Previous studies indicated that the Tat protein of human immunodeficiency virus type-1 (HIV-1) is a progression factor for Kaposi's sarcoma (KS). Specifically, extracellular Tat cooperates with basic fibroblast growth factor (bFGF) in promoting KS and endothelial cell growth and locomotion and in inducing KS-like lesions in vivo. Here we show that Tat and bFGF combined increase matrix-metalloproteinase-2 (MMP-2) secretion and activation in endothelial cells in an additive/synergistic manner. These effects are due to the activation of the membrane-type-1-matrix-metalloproteinase and to the induction of the membrane-bound tissue inhibitor of metalloproteinase-2 (TIMP-2) by Tat and bFGF combined, but also to Tat-mediated inhibition of both basal or bFGF-induced TIMP-1 and -2 secretion. Consistent with this, Tat and bFGF promote vascular permeability and edema in vivo that are blocked by a synthetic MMP inhibitor. Finally, high MMP-2 expression is detected in acquired immunodeficiency virus syndrome (AIDS)-KS lesions, and increased levels of MMP-2 are found in plasma from patients with AIDS-KS compared with HIV-uninfected individuals with classic KS, indicating that these mechanisms are operative in AIDS-KS. This suggests a novel pathway by which Tat can increase KS aggressiveness or induce vasculopathy in the setting of HIV-1 infection.

Inverse relation of Fas-ligand and tumor-infiltrating lymphocytes in angiosarcoma: indications of apoptotic tumor counterattack.

Fas and Fas-L regulate immune responses through the induction of cell death. Fas-L is commonly expressed in activated immune cells and in the endothelium. In the latter it contributes to the inhibition of transvascular cell migration by the induction of apoptosis in Fas-bearing lymphocytes. Here we investigated whether the Fas/Fas-L system may regulate lymphocyte invasion into angiosarcomas. Fas and Fas-L expression was quantitatively determined in different grade angiosarcomas (n = 40) and related to the number of extravasated tumor-infiltrating lymphocytes (TILs). Fas expression was detected in < 50% of the cases. In positive tumors both the number of Fas-positive cells and the staining intensity were highly variable and did not correlate with the number of TILs, the mean time of survival, and the histopathological tumor grade. By contrast, Fas-L expression was detected in >70% of the cases and the relative numbers of Fas-L-positive cells correlated inversely with the numbers of CD3- and CD8-positive TILs (P < or = 0.004). The survival times of patients with high Fas-L-expressing angiosarcomas were significantly reduced as compared to patients with low Fas-L-expressing tumors. Our results show that angiosarcomas with low Fas-L expression are characterized by numerous TILs, whereas sarcomas with high Fas-L expression show significantly reduced numbers of TILs. These results suggest that the Fas/Fas-L system may repress TIL invasion into angiosarcoma and by this may contribute to the evasion of the anti-tumor immune surveillance of angiosarcoma in the course of an apoptotic tumor counterattack mechanism.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Human herpesvirus-8 and Kaposi's sarcoma: relationship with the multistep concept of tumorigenesis.

Kaposi's sarcoma (KS) develops through discrete inflammatory-angiogenic stages of polyclonal nature (early-stage lesions) to monomorphic nodules of spindle-shaped cells that can be clonal (late-stage lesions) and resemble true sarcomas. Molecular and epidemiological studies indicate that development of KS is tightly associated with infection by the human herpesvirus-8 (HHV-8). However, only individuals with specific conditions of immunodysregulation develop KS. In these individuals the systemic and tissue increase of Th-1-type cytokines (IC) reactivate HHV-8 infection, leading to increased viral load, antibody titers, and an expanded cell tropism that precedes the clinical appearance of KS. Recruitment of the virus into tissues by infected monocytes and other cell types is facilitated by the endothelial cell activation due to IC. In clinical lesions, HHV-8 infection increases with lesion stage and in late-stage lesions most of the spindle cells are latently infected, whereas only few lyrically infected cells are present, suggesting that latent genes may have a role in the transformation of the early inflammatory-hyperplastic lesion into a real sarcoma. The development of tumors, however, is regulated through a multistep process based on the acquisition by cells of several different capabilities leading to malignant growth. Here we review the available data on the expression of HHV-8-encoded genes in primary KS lesions and, in view of their biological activity, analyze their potential function in different steps of tumorigenesis. By this pragmatic approach interesting insights into potential key functions of HHV-8-encoded genes are found and steps of potential cooperativity with other viral factors (HIV-1-Tat) in the pathogenesis of KS are identified.

Deregulated expression of cell cycle-associated proteins in solid pseudopapillary tumor of the pancreas.

Solid pseudopapillary tumor of the pancreas was studied in a 20-year-old woman and a 54-year-old woman. In the younger patient, the tumor had metastasized to the liver 8 years after distal pancreatectomy. In both neoplasms, the distinct histologic pattern of solid, pseudopapillary, and degenerative cystic areas was present. Analysis by means of immunohistochemistry revealed a diffuse expression for vimentin, neuron-specific enolase, and a focal positivity for al-antitrypsin, whereas epithelial markers were negative in the tumor of the older patient and only focally expressed in the tumor of the younger patient. Immunohistochemical analysis of cell cycle-associated proteins provided an overexpression of cyclin D1 and cyclin D3 in both tumors, although to varying degrees. In addition, the cyclin-dependent kinase inhibitors p21, and to a lesser extent p27, were up-regulated just as mdm2. There was no accumulation of p53 protein, and Ki67-positive cells were extremely scarce. Analysis of the liver metastases showed an immunoreactive profile similar to that of the primary tumor. The results show a deregulation of the cell cycle with overexpression of cell cycle-activating proteins D1 and D3 and a probably counterbalancing upregulation of the cyclin-dependent kinase inhibitors p21 and p27. The findings may explain the low pool of Ki67-reactive tumor cells and the generally good clinical outcome of these tumors. Whether a more profound dysbalance of the cell cycle regulation is responsible for the development of metastatic disease remains to be clarified.

Gastric outlet obstruction and pulmonary infiltrate in a patient with Crohn's disease: successful treatment by Billroth-II-resection.

We present a 28-year-old women with a 3 yr history of duodenal ulcers. Following four treatment attempts to eradicate helicobacter pylori she was admitted because of gastric outlet obstruction and a weight loss of 20 kg within the last two years. Endoscopy and x-ray showed a circular inflammatory stenosis of the proximal duodenum extending over 8 cm. Additionally, chest x-ray showed a circumscript infiltrate in the third segment of the right lung. Mycobacterial infection could be excluded. Ileocolonoscopy and small intestinal follow-through beyond the duodenum were unremarkable, and Zollinger-Ellison-syndrome was ruled out. Bronchopulmonary histology showed intramucosal epithelioid-cell granulomas and bronchiolitis obliterans. Because the patient did not improve under conservative therapy a Billroth-II-resection was carried out. Histologically the resected specimen showed Crohn-like lesions. Postoperatively, severe peripheral arthritis was treated by steroids over 6 weeks. At follow-up the patient regained 20 kg and was free of symptoms without any medication. The pulmonary infiltrate had subsided almost completely. In summary, this extremely rare coincidence of isolated stenosing duodenal Crohn's disease and pulmonary involvement was successfully treated by Billroth-II-resection. This course of disease is compatible with the hypothesis that Crohn's disease may be maintained by antigens derived from ingested food.

[Differential diagnosis of lymphoepithelial lesions of salivary glands. With particular reference to characteristic duct lesions]. / Zur Differenzialdiagnose lymphoepithelialer Speicheldrüsenläsionen. Mit besonderer Berücksichtigung der charakteristischen Speichelgangveränderungen.

Several salivary gland diseases present with the histomorphological features of a lymphoepithelial lesion with or without cyst formation. Some of the most important differential diagnoses (Sjögren's syndrome, marginal zone B-cell lymphoma, HIV-associated cystic lymphoepithelial lesion) are systemic diseases and require further investigation and therapy. However, in small biopsy specimens and in cases without relevant clinical information an exact diagnosis may be difficult to obtain. We have recently determined that the characteristic lymphoepithelial duct lesions develop by proliferation of basal cells of striated ducts, while we could not confirm the previously postulated participation of myoepithelial cells ("epimyoepithelial lesion/sialadenitis"). Although these duct lesions are typical of Sjögren's syndrome, they manifest in several diseases of salivary glands, exhibiting characteristic patterns concerning frequency and localization. This review discusses the most important lymphoepithelial diseases of salivary glands with respect to clinical presentation and histomorphology. Particular emphasis is placed on the lymphoepithelial duct lesions.

[67-year-old patient with upper gastrointestinal bleeding]

A 67-year-old patient with upper gastrointestinal bleeding. HISTORY AND CLINICAL FINDINGS: A 67-year old patient with melaena and anaemia (haemoglobin 4.8 g/dl) for 14 days had been admitted to hospital under suspicion of upper gastrointestinal bleeding. The patient had a history of 2/3 resection of the stomach for recurrent duodenal ulcer and of a right nephrectomie for hypernephroma 18 years ago. There was no abdominal pain on pressure, nor muscular guarding. INVESTIGATIONS: Endoscopy (oesophago-duodenoscopie, coloscopie), radiography (angiography, gastrografin passage) and ultrasound failed to locate the source of bleeding. TREATMENT AND COURSE: After repeated endoscopic attempts to localize the source of bleeding and growing need of transfusions (12 blood-concentrates in 5 days) an exploratory laparotomie was done. Surprisingly a bleeding fistula between the stump of the right renal arteria and duodenum was found. The defects were excised and directly closed. CONCLUSIONS: Secondary aortoduodenal fistula after nephrectomy is rare. If the source of bleeding can't be found, and there is in any doubt an exploratory laparotomy should be done.

Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi's sarcoma spindle cells.

BACKGROUND: Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues. METHODS: We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay. RESULTS AND CONCLUSIONS: Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions. IMPLICATIONS: These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed.

Expression of human herpesvirus-8 (HHV-8) encoded pathogenic genes in Kaposi's sarcoma (KS) primary lesions.

Transcription of six different HHV-8 specific mRNAs was examined in early- and late-stage KS primary lesions. Expression of the latency-associated T0.7 mRNA and of VP23 mRNA which is a specific marker of lytic/productive infection suggested that HHV-8 is secondarily recruited into the KS lesions by productively infected monocytes, macrophages. From these cells HHV-8 is transmitted to the KS spindle cells, which are latently infected. v-BCL-2, v-MCP-1 and v-IL-6 were not expressed in latently infected KS spindle cells, therefore the impact of these factors in KS pathogenesis appears to be low. By contrast, v-Cyclin D was highly expressed in almost all latently infected spindle cells and may therefore be an important factor triggering progression of late-stage KS lesions.

Reactivation and persistence of human herpesvirus-8 infection in B cells and monocytes by Th-1 cytokines increased in Kaposi's sarcoma.

Patients with Kaposi's sarcoma (KS) have a human herpesvirus-8 (HHV-8) load higher than patients without KS and present a CD8(+) T-cell activation with production of Th1-type cytokines both in tissues and peripheral blood mononuclear cells (PBMC). Because in tissues of KS patients detection of inflammatory cytokines (IC) can precede detection of HHV-8 DNA and because signs of immunoactivation and/or dysregulation can precede KS development, we investigated the effect of IC on HHV-8 infection. To achieve this goal, PBMC and purified cell populations from 45 patients with KS and 45 patients at risk of KS were analyzed for HHV-8 DNA and/or gene expression and for cell survival, growth, and phenotype before or after culture with or without the IC increased in KS. The results indicate that PBMC that are polymerase chain reaction (PCR)-positive at day 0 generally loose the virus upon culture. However, the presence of IC maintains HHV-8 DNA load in cultured cells. In addition, IC increase viral load to detectable levels in PBMC from serologically positive patients that were PCR-negative before culture. gamma Interferon is sufficient for these effects, whereas tumor necrosis factor and interleukin-6 have little or no activity. The increase of HHV-8 DNA by IC is observed after short-term (7 days) or long-term (28 days) culture of the cells and occurs in one or both of the two circulating cell types that are infected in vivo: B cells and monocytes. In both cases it is associated with lytic gene expression, suggesting that virus reactivation is one of the most likely mechanisms for the effect of IC on virus load. However, IC have also effects on the cells target of HHV-8 infection, because they increase B-cell survival and induce the growth and differentiation of monocytes into KS-like spindle cells with markers of endothelial macrophages. Because cells with markers of endothelial macrophages are present in blood and lesions from KS patients and are infected by HHV-8, these data may explain the high HHV-8 load associated with KS development and suggest that infected monocytes may carry the virus to tissues, transmit the infection, or differentiate in loco in spindle cells with endothelial macrophage markers.

Lymphoepithelial duct lesions in Sjögren-type sialadenitis.

It is not clear, whether the so-called basal cells of the salivary striated ducts are an independent cell-type distinct from myoepithelial cells, making characterization of the cell proliferation typical of the duct lesions in Sjögren-type sialadenitis/benign lymphoepithelial lesion (BLEL) difficult. An immunohistochemical investigation including different cytokeratin subtypes, alpha-actin, Ki-67 and Bcl-2 was directed at the epithelial cytoskeleton in normal parotid parenchyma (n=8), BLEL (n=12), HIV-associated lymphoepithelial cysts (n=8) and palatine tonsils (n=8). There are profound morphological and functional differences between basal and myoepithelial cells in the normal salivary duct. Development of duct lesions in BLEL arises from basal cell hyperplasia of striated ducts with aberrant differentiation into a multi-layered and reticulated epithelium, characterized by profound alteration of the cytokeratin pattern. This functionally inferior, metaplastic epithelium is similar to the lymphoepithelial crypt epithelium of palatine tonsils. The often postulated participation of myoepithelial cells in duct lesions of Sjögren disease/BLEL cannot be supported. We regard the designations lymphoepithelial lesion and lymphoepithelial metaplasia as the most appropriate.

Erdheim-Chester disease: a case report and literature overview.

Erdheim-Chester (EC) disease belongs to the group of lipoid granulomatosis. Symmetric sclerosis of the meta- and diaphysis of long tubular bones are pathognomonic radiologic changes. Additionally, other skeletal segments can be affected. Extraskeletal manifestations can occur in almost all organs; lungs, pericardium, retroperitoneum, skin, and orbita play particularly important roles. The last case of 38 cases of Erdheim-Chester disease with an extraordinary mediastinal and perirenal involvement is described. For the second time following the initial description by Chester, an axial skeletal pattern of eburnated vertebra is shown.