[Periodic fever and pancytopenia in a 35-year-old patient]. / Periodisches Fieber und Panzytopenie bei einem 35­jährigen Patienten.

MEDICAL HISTORY AND INITIAL PRESENTATION: A 35-year-old patient with a previous history of persistent episodic fever, sore throat, myalgia, and cephalgia presented for evaluation of pancytopenia. He had no recent travel history, except for a stay in Italy 1 year prior to admission and in Spain several years in the past. DIAGNOSTIC WORKUP: Laboratory evaluation confirmed pancytopenia, agranulocytosis, and elevated infection parameters without indicative serological results en par with lymphadenitis colli. Computed tomography scanning revealed cervical lymphadenopathy, hepatosplenomegaly, and colitis with occult perforation of the sigmoid colon. Bone marrow biopsy showed an infiltration of polyclonal plasma cells. Lymph node biopsy was compatible with necrotizing lymphadenitis. DIAGNOSIS: Polymerase chain reaction analysis of a lymph node specimen confirmed the presence of Leishmania species, thereby enabling the diagnosis of visceral Leishmania. THERAPY COURSE: Treatment with liposomal amphotericin B was initiated. Both fever and lymphadenopathy quickly resolved. CONCLUSION: VL is a clinically pleiotropic, severe disease with fatal outcome if left untreated. It often presents with distinct similarities to hematologic malignancies. Exacerbation can occasionally occur as fulminant macrophage activation syndrome. Disease incidence is globally increasing and has not peaked as yet. A complex interplay between pathogen and the immune system is the key pathophysiological mechanism.

Laser-assisted atom probe tomography of semiconductors: The impact of the focused-ion beam specimen preparation.

This paper demonstrates the increased light absorption efficiency of semiconducting atom probe tips resulting from focused-ion-beam (FIB) preparation. We use transmission electron microscopy to show that semiconducting tips prepared with FIB are surrounded with an amorphized shell. Photomodulated optical reflectance measurements then provide evidence that FIB-induced damage leads to an increase in both sub- and supra-bandgap light absorption efficiency. Using laser-assisted atom probe tomography (La-APT) measurements, we finally show that, for a nanoscale tip geometry, the laser-induced heating of a tip during La-APT is enhanced by the FIB preparation. We conclude that, upon supra-bandgap illumination, the presence of a FIB-amorphized surface dramatically increases the light-induced heat generation inside semiconducting tips during La-APT. Furthermore, we also deduce that, in the intriguing case of sub-bandgap illumination, the amorphization plays a crucial role in the unexpected light absorption.

Wet-chemical etching of atom probe tips for artefact free analyses of nanoscaled semiconductor structures.

We introduce an innovative specimen preparation method employing the selectivity of a wet-chemical etching step to improve data quality and success rates in the atom probe analysis of contemporary semiconductor devices. Firstly, on the example of an SiGe fin embedded in SiO2 we demonstrate how the selective removal of SiO2 from the final APT specimen significantly improves accuracy and reliability of the reconstructed data. With the oxide removal, we eliminate the origin of shape artefacts, i.e. the formation of a non-hemispherical tip shape, that are typically observed in the reconstructed volume of complex systems. Secondly, using the same approach, we increase success rates to ∼90% for the damage-free, 3D site-specific localization of short (250 nm), vertical Si nanowires at the specimen apex. The impact of the abrupt emitter radius change that is introduced by this specimen preparation method is evaluated as being minor using field evaporation simulation and comparison of different reconstruction schemes. The Ge content within the SiGe fin as well as the 3D boron distribution in the Si NW as resolved by atom probe analysis are in good agreement with TEM/EDS and ToF-SIMS analysis, respectively.

Atom probe tomography analysis of SiGe fins embedded in SiO2: Facts and artefacts.

We present atom probe analysis of 40nm wide SiGe fins embedded in SiO2 and discuss the root cause of artefacts observed in the reconstructed data. Additionally, we propose a simple data treatment routine, relying on complementary transmission electron microscopy analysis, to improve compositional analysis of the embedded SiGe fins. Using field evaporation simulations, we show that for high oxide to fin width ratios the difference in evaporation field thresholds between SiGe and SiO2 results in a non-hemispherical emitter shape with a negative curvature in the direction across, but not along the fin. This peculiar emitter shape leads to severe local variations in radius and hence in magnification across the emitter apex causing ion trajectory aberrations and crossings. As shown by our experiments and simulations, this translates into unrealistic variations in the detected atom densities and faulty dimensions in the reconstructed volume, with the width of the fin being up to six-fold compressed. Rectification of the faulty dimensions and density variations in the SiGe fin was demonstrated with our dedicated data treatment routine.

Insights into the nanoscale lateral and vertical phase separation in organic bulk heterojunctions via scanning probe microscopy.

Solution processed polymer (donor) and fullerene (acceptor) bulk heterojunctions are widely used as the photo active layer in organic solar cells. Intimate mixing of these two materials is essential for efficient charge separation and transport. Identifying relative positions of acceptor and donor rich regions in the bulk heterojunction with nanometer scale precision is crucial in understanding intricate details of operation. In this work, a combination of Ar(+)2000 gas cluster ion beam and scanning probe microscopy is used to examine the lateral and vertical phase separation within regio-regular poly(3-hexylthiophene)(P3HT):phenyl-C60-butyric acid methyl ester (PCBM) bulk heterojunction. While the Ar(+)2000 gas cluster ion beam is used as a sputter tool to expose the underneath layers, scanning probe microscopy techniques are used to obtain two-dimensional (2D) electrical maps (with sub-2 nm lateral resolution). The electrical mapping is decoded to chemical composition, essentially producing lateral and vertical maps of phase separation. Thermal stress causes large PCBM-rich hillocks to form, and consequently affecting the balance of P3HT:PCBM heterojunctions, hence a negative impact on the efficiency of the solar cell. We further developed a method to analyze the efficiency of exciton dissociation based on the current maps and a loss of 20% in efficiency is observed for thermally degraded samples compared to fresh un-annealed samples.

Relaxor ferroelectricity and magnetoelectric coupling in ZnO-Co nanocomposite thin films: beyond multiferroic composites.

ZnO-Co nanocomposite thin films are synthesized by combination of pulsed laser deposition of ZnO and Co ion implantation. Both superparamagnetism and relaxor ferroelectricity as well as magnetoelectric coupling in the nanocomposites have been demonstrated. The unexpected relaxor ferroelectricity is believed to be the result of the local lattice distortion induced by the incorporation of the Co nanoparticles. Magnetoelectric coupling can be attributed to the interaction between the electric dipole moments and the magnetic moments, which are both induced by the incorporation of Co. The introduced ZnO-Co nanocomposite thin films are different from conventional strain-mediated multiferroic composites.

Array-based DNA methylation profiling in follicular lymphoma.

Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P<0.05) correlation between FL methylation values and reduced gene expression was shown for up to 28% of loci. Methylation changes occurred predominantly in B cells with variability in the amount of non-malignant tissue between samples preventing conclusive correlation with survival. This represents an important caveat in attributing prognostic relevance to methylation and future studies in cancer will optimally require purified tumour populations to address the impact of methylation on clinical outcome.

Germline mutations in Dok1 do not predispose to chronic lymphocytic leukemia.

The genetic basis of familial CLL is poorly understood and to date no gene which when mutated in the germline has been unambiguously shown to confer susceptibility to the disease. Dok1 maps to chromosome 2p13, a region commonly rearranged in CLL. Dok1 inhibits MAP kinase activity, down-regulates cell proliferation and has a suppressive effect on cellular transformation and B-cell signalling pathways. A recent report has implicated mutation of Dok1 in the aetiology of CLL. To examine the proposition that germline mutations in Dok1 act as high penetrance susceptibility alleles for CLL we screened 140 familial cases for functional sequence variants. No pathogenic mutations were detected. This result indicates that germline mutations in Dok1 are unlikely to cause an inherited predisposition to CLL.

Comparison of whole-body STIR-MRI and 99mTc-methylene-diphosphonate scintigraphy in children with suspected multifocal bone lesions.

The study was performed to compare whole-body short time inversion recovery (STIR) MR imaging and (99m)Tc-methylene diphosphonate planar scintigraphy in the examination of children with suspected multifocal skeletal malignant lesions. Sixteen patients with known or suspected malignant skeletal disease underwent both whole-body STIR MR imaging and bone scintigraphy. The lesions were described and numbered according to scintigraphic evaluation criteria. Thus, 16 regions were analyzed in each patient for the comparison between the two modalities. Histology was proven in the primary malignant regions. Follow-up MRIs were registered. Scintigraphy and MRI follow-up were evaluated as gold standard. A total of 139 different lesions was observed by both modalities. Baseline whole-body MRI revealed 119 bone lesions in 256 possible sites (46.5%); scintigraphy revealed only 58 lesions (22.6%). Congruence was observed in only four patients (25%). According to the location of the lesion, correlation was observed in 39/139 lesions (28%). In all, 57.5% of the lesions were detected only by MRI and 14.5% of the lesions were detected only by scintigraphy. Whole-body MRI was more sensitive (P<0.001). Of all lesions numbered which could be separated in the initial MRI, whole-body MRI detected 178 lesions in the patients. The results suggest that whole-body MRI using a STIR sequence is an effective radiation free method for examination of children with suspected multifocal bone lesions. MRI showed more lesions than conventional (99m)Tc-methylene diphosphonate scintigraphy. Therefore, whole-body MRI may be feasible as a screening modality for metastatic and skip lesions in osteosarcoma, PNET, Ewing sarcoma and Langerhans cell histiocytosis in children.

Beta-myosin heavy chain gene mutations and hypertrophic cardiomyopathy in Austrian children.

Hypertrophic cardiomyopathy occurs in two variants, either as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. Different genes coding sarcomeric proteins of the heart have been identified as causing hypertrophic cardiomyopathy. Missense mutations in the cardiac beta-myosin heavy chain gene are found in 30% of all cases of familial hypertrophic cardiomyopathy. We screened the beta-myosin heavy chain gene of children of nine Austrian families with hypertrophic cardiomyopathy (referred to as group A) and of seven children with sporadic hypertrophic cardiomyopathy (referred to as group B). We were able to find two previously described (V606M, R453C) and two unknown missense mutations (V406M, R663H) in group A. Additionally, in two children of group B we could identify one already known missense mutation, R249Q as well as one previously unknown missense mutation, M877K. The genetically affected children of group A developed no or only mild clinical symptoms, whereas the children of group B with genetically confirmed sporadic hypertrophic cardiomyopathy showed manifest left ventricular hypertrophy and clinical symptoms including chest pain and dyspnoea. Clinical symptoms among the adults of group A, suffering from familial hypertrophic cardiomyopathy, varied significantly. We therefore believe V406M to be a more malignant missense mutation, probably linked with sudden death in the affected family, than R663H, which seems to be more benign causing late-onset hypertrophic cardiomyopathy and mild clinical symptoms in the affected family members.

Orally administered IFN-alpha acts alone and in synergistic combination with intraperitoneally administered IFN-gamma to exert an antitumor effect against B16 melanoma in mice.

Administration of interferons (IFN) via the intranasal route recently has been shown to exert an antitumor activity against a variety of tumors in mice, including B16 melanoma inoculated intravenously. This study confirms the antitumor activity of orally administered IFN-alpha against B16 melanoma challenge using another route of tumor inoculation, the intraperitoneal route. It further demonstrates that orally administered IFN-alpha can synergistically interact with intraperitoneally administered IFN-gamma but not with intraperitoneally administered IFN-alpha. The results support the interpretation that the oral route may provide an effective alternative or supplement to current methods of IFN administration for the control of malignancies.

Interferon regulates expression of mda-6/WAF1/CIP1 and cyclin-dependent kinases independently from p53 in B16 murine melanoma cells.

Interferons (IFNs) induce growth arrest and terminal differentiation through regulation of proliferative genes in a variety of cell types including tumor cells. Growth of melanoma cells is believed to be controlled by the cyclin-dependent kinase inhibitor, mda-6/WAF1/CIP1 gene. IFNs affect the expression of WAF1 in several cell types, including human melanomas. In our earlier reports we demonstrated the antitumor and anticellular activities of different IFN-types on B16 murine melanoma cells. The present study aimed to demonstrate the involvement of mda-6/WAF1 and related cyclin-dependent kinases in antitumor action of different IFN-types in B16 melanoma cells. IFN-alpha has been proven to be a potent inducer of mda-6/WAF1, also inhibiting cyclin-dependent kinases, such as cdc2- and cdk2-kinase. This induction is p53-independent. However, IFN-gamma affects B16 cells differently, it induces p53 activity without inducing WAF1. The combination of IFN-alpha plus IFN-gamma is additive rather than synergistic. Our data demonstrate differential effects of different IFNs on murine B16 melanoma cells which may have relevance in nonsurgical treatment of melanomas.

B16 melanoma cells exposed in vitro to long-term IFN-alpha treatment (B16 alpha cells) as activators of tumor immunity in mice.

Mice inoculated with B16 melanoma cells exposed to long-term in vitro IFN-alpha treatment (> or = 14 days, B16 alpha cells) but not short-term in vitro IFN-alpha treatment (24 h) exhibited an enhanced survival time. Enhanced survival time also occurred when inactivated B16 alpha cells were inoculated at the same time as live B16 cells. Further, an even greater improvement in survival time was observed when the inactivated B16 alpha cells were inoculated before live B16 cell challenge. No enhancement in survival time was observed when mice were inoculated with inactivated, untreated B16 cells. Enhancement of survival time by B16 alpha cells was unrelated to retrovirus surface antigen expression. Long-lasting protective immunity to B16 cells was observed in mice that survived B16 alpha cell, but not normal B16 cell, challenge and subsequent IFN treatment. It is evident that inoculation with inactivated B16 alpha cells, but not with inactivated untreated B16 cells, was able to prolong significantly the survival time of mice either simultaneously or subsequently challenged with live B16 cells. Additionally, survival of B16 alpha-inoculated but not B16-inoculated mice was accompanied by a durable immunity. Inoculation of inactivated B16 alpha cells may serve as a model for the induction of host immunity to a parental primary or secondary tumor.

Lack of mda-6/WAF1/CIP1-mediated inhibition of cyclin-dependent kinases in interferon-alpha resistant murine B16 melanoma cells.

Previously we demonstrated that IFN-alpha augments mda-6/WAF1 and inhibits cyclin-dependent kinases in a p53-independent fashion in B 16 murine melanoma cells. On the other hand, IFN-gamma activates p53 expression without affecting the mda-6/WAF1 system. Combination of the two IFNs is additive. B16 cells acquire IFN-alpha resistant but IFN-gamma sensitive phenotype after long term IFN-alpha treatment (B16alpha cells). Here we demonstrate the absence of mda-6/WAF1-associated repression of cyclin-dependent kinases, but the existence of p53-dependent c-myc inhibition in IFN-gamma-treated B16alpha cells. Clearly, selective desensitization of IFN-alpha related growth regulation does not influence the IFN-gamma associated pathway. Our results further support the coexistence of distinct growth regulatory mechanisms in B16 cells that can be activated by different IFN-types independently of each other.

Enhanced in vivo sensitivity of in vitro interferon-treated B16 melanoma cells to CD8 cells and activated macrophages.

Mouse B16 melanoma cells maintained in vitro in the presence of interferon (IFN)-alpha become resistant to the in vitro antiproliferative effects of IFN-alpha. However, IFN-alpha-treated mice inoculated with these in vitro IFN-treated cells (B16 alpha res cells) have significantly increased life spans (ILS) and significantly higher cure rates than IFN-alpha-treated mice inoculated with B16 cells. This unexpectedly greater sensitivity of B16 alpha res cells to the in vivo antitumor effects of IFN-alpha was evaluated by in vivo cell depletion experiments. Depletion of either activated peritoneal macrophages or cytotoxic T lymphocytes (CTL) reduced the ILS of IFN-treated B16 alpha res-inoculated mice to a level comparable to that of IFN-treated B16-inoculated mice. Depletion of natural killer (NK) cells did not affect the ILS for IFN-treated B16 alpha res-inoculated mice. These studies indicate that activated macrophage and CD8 cell function, but not NK cell function, is important for the enhanced antitumor effects induced by IFN-alpha against B16 alpha res cells. Macrophage killing was unlikely to be mediated by TNF-alpha or IL-1 as B16 and B16 alpha res cells were equally sensitive to TNF-alpha and insensitive to IL-1 in vitro. Further, H-2K antigen expression is significantly more readily inducible on B16 alpha res cells than on B16 cells, consistent with enhanced CD8-mediated killing due to increased MHC class I antigen expression.

Enhanced in vitro macrophage cytotoxicity against interferon-treated B16 melanoma cells.

Resistance to the in vitro antiproliferative effects of INF-alpha rapidly develops in mouse B16 melanoma cells that are maintained in vitro in IFN-alpha (B16 alpha res cells). B16 alpha res cells, however, are significantly more sensitive to the antitumor effects of IFN-alpha when they are injected into mice. This enhanced sensitivity appears to be due, at least in part, to activated macrophages. To investigate enhanced macrophage sensitivity of B16 alpha res cells, macrophage-mediated cytotoxicity assays have been performed using both B16 and B16 alpha res cell targets. Thioglycollate-elicited peritoneal macrophages activated in vitro with IFN-gamma exhibited dose-dependent cytotoxicity against both B16 and B16 alpha res cells, but significantly higher levels of cytotoxicity occurred with B16 alpha res targets. Kinetics experiment results showed that the cytolytic effects against B16 alpha res cells occurred at a very much faster rate than the cytolytic effects against B16 cells (50% cytotoxicity with 2 h of incubation versus 40% cytotoxicity by 24 h, respectively). Finally, peritoneal macrophages from B16-inoculated mice also were significantly more cytotoxic against B16 alpha res cells than against B16 cells. Macrophages from B16 alpha res-inoculated mice were significantly more cytotoxic against B16 alpha res cells than were macrophages from B16-inoculated mice. Taken together, these observations provide in vitro evidence to support the suggestion that peritoneal macrophages are important mediators of the enhanced host-mediated antitumor effects against B16 alpha res cells.

Enhancement of MuIFN-gamma antitumor effects by hyperthermia: sequence dependence and time dependence of hyperthermia.

Heat-induced total body hyperthermia has been shown to synergistically enhance the in vivo antitumor effects of MuIFN-gamma directed against B16 melanoma in mice. This observation was made with an 8-hour exposure to hyperthermia that was administered following MuIFN-gamma treatment. It was not known whether this was the most efficacious treatment protocol. Various treatment protocols exploring MuIFN-gamma treatment at different relative times of exposure to hyperthermia and involving different durations of hyperthermia were evaluated. In a 5-day course of therapy, B16 tumor-bearing mice were injected with MuIFN-gamma or mock interferon and incubated in a dry incubator (resulting in a 2 degrees C rise in body temperature). The antitumor efficacy of MuIFN-gamma administered before, in the middle of, or following 8 hours of hyperthermia was evaluated. The antitumor effects of MuIFN-gamma were synergistically enhanced when the MuIFN-gamma was administered before 8 hours of hyperthermia (3.9-fold greater increased life span than with MuIFN-gamma treatment alone). However, when MuIFN-gamma was administered in the middle or following hyperthermia, the life spans were essentially the same as for MuIFN-gamma treatment alone, indicating that the effect of the hyperthermia exposures according to these treatment protocols were less than additive. Administration of MuIFN-gamma before hyperthermia exposures of 2 hours, 5 hours, and 8 hours showed antagonistic, additive, and synergistic interactions of the MuIFN-gamma treatment and hyperthermia exposure, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced in vivo sensitivity to interferon with in vitro resistant B16 tumor cells in mice.

Mouse B16 melanoma cells rapidly develop resistance to the antiproliferative effects of interferon alpha (IFN alpha) and interferon beta (IFN beta) when they are exposed to the interferons in vitro. This resistance was characterized to be non-genetic and dose-dependent, and does not alter other IFN-induced effects such as antiviral effects and elevation of 2',5'-oligoadenylate synthetase activity in IFN-treated cells. The study of these IFN-resistant cells has been extended to an in vivo tumor model. Resistance, if it occurred in vivo, did not adversely affect the survival of IFN-treated mice. Further, IFN-treated mice inoculated with B16 cells that were resistant in vitro (B16 alpha res cells) survived significantly longer than IFN-treated mice inoculated with B16 cells that were sensitive in vitro. The IFN-treated B16 alpha res-inoculated mice had a significantly higher cure rate as well. The prolonged survival of the mice bearing B16 alpha res cell tumors did not seem to be caused by the slower growth rate of the B16 alpha res cells, since experiments performed with a tenfold higher B16 alpha res cell inoculum and a tenfold lower B16 cell inoculum did not show any change in the survival pattern. It is clear that in vitro resistant B16 alpha res cells are more sensitive to antitumor effects induced by IFN in vivo than in vitro sensitive B16 cells.

Prevention of resistance to IFN-alpha antiproliferative activity: characterization of the effect of IFN-gamma and substitution for IFN-gamma by tumor necrosis factor.

Non-genetic resistance to the antiproliferative effects of interferon-alpha (IFN-alpha) develops in murine and human melanoma cells within 2-4 days of exposure of the cells to IFN-alpha. Simultaneous treatment of murine B16 melanoma cells with MuIFN-gamma and MuIFN-alpha prevents the development of resistance. In this study, the ability of MuIFN-gamma pretreatment to prevent the development of resistance was assessed for varying concentrations of MuIFN-gamma and for varying lengths of time of pretreatment. Pretreatment of the cells for 48 h with MuIFN-gamma using concentrations as low as 5 U/ml prevents the subsequent development of resistance when the cells are cloned in the presence of MuIFN-alpha. Higher concentrations of MuIFN-gamma are more effective in preventing the development of resistance. In addition, short MuIFN-gamma pretreatment times, such as 2-4 h, appeared to be most effective in preventing the development of resistance. In order to determine the mechanism for this biological effect, various second messenger perturbing chemical agents and several other biological agents were screened for ability to prevent the development of resistance. Neither interleukin-2 (IL-2), epidermal growth factor (EGF), nor any of the chemical agents examined could prevent the development of resistance, nor did they alter the ability of MuIFN-gamma to prevent the development of resistance. Tumor necrosis factor (TNF), however, was able to substitute for MuIFN-gamma in preventing the development of resistance, using concentrations of 125 ng/ml and higher.(ABSTRACT TRUNCATED AT 250 WORDS)