Corrigendum to "Hodgkin Lymphoma Cell Lines Are Characterized by a Specific miRNA Expression Profile." Neoplasia 2009, Feb;11(2):167-176.

[This corrects the article DOI: 10.1593/neo.08980.].

Canine Leishmania infantum infection: an imported case in UK after staying in the Canary Islands.

Leishmaniosis is reported in the Iberian Peninsula and the Balearic Islands, but the Canary Islands are deemed free. In the present communication, we report a clinical leishmaniosis due to Leishmania infantum in a dog that was presumptively infected during its stay on Tenerife. The result of Leishmania serology (whole-cell based ELISA with L. infantum antigen) was high positive (test score of 82.2 at a cut-off value of 12.0). This result was further confirmed with quantitative real-time polymerase chain reaction (qPCR) for Leishmania spp. on a blood sample. A medium load of parasites was detected (48 parasites/ml blood). L. infantum was identified by RFLP analysis of the ITS-1 PCR product. Confirmation that leishmaniosis is endemic to the Canary Islands would further require study on local dogs with no travel history as well as reassessment on frequency and distribution of Phlebotomus spp. as well as Leishmania spp. detection in the sand fly vector. However, this case strongly suggests that L. infantum is present on the Canary Islands. Although transmission seems to be still exceptional, preventive measures in dogs travelling to the Canaries should be considered.

CD16⁺ monocytes with smooth muscle cell characteristics are reduced in human renal chronic transplant dysfunction.

In chronic transplant dysfunction (CTD), persistent (allo)immune-mediated inflammation eventually leads to tissue remodeling including neointima formation in intragraft arteries. We previously showed that recipient-derived neointimal α-SMA(+) smooth muscle-like cells are present in human renal allografts with CTD. Human PBMC contain myeloid cells capable of differentiating into α-SMA(+) cells in vitro; the phenotype of the ancestral subset is as yet unknown. This study aimed to investigate whether monocyte subsets contain cells with smooth muscle-like cell differentiation capacity and whether CTD in renal transplant recipients is associated with a shift in these monocyte subsets. To accomplish this goal, monocyte subsets from healthy controls were sorted based on CD14 and CD16 expression to investigate gene expression levels of mesenchymal markers α-SMA and SM22α. CD14(+)/CD16(++) monocytes displayed increased α-SMA and SM22α mRNA expression compared with CD14(++)/CD16(-) monocytes, suggesting increased differentiation potential toward smooth muscle-like cells. Flow cytometry revealed that in non-CTD transplant recipients the percentage of CD14(+)/CD16(++) monocytes was reduced, with an even further reduction in patients with CTD. To determine a potential correlation between CD14(+)/CD16(++) monocytes and α-SMA(+) cell outgrowth potential in vitro, PBMC of healthy controls and transplant recipients with and without CTD were cultured under fibrotic culture conditions, and indeed a significant correlation (p=0.0002, r=0.62) was observed. Finally, double staining for α-SMA and CD16 revealed presence of α-SMA(+)CD16(+) cells in kidney explants from CTD patients, albeit at very low numbers. Our data represent evidence that, compared to CD14(++)CD16(-) monocytes, CD14(+)CD16(++) monocytes have an increased expression of smooth muscle cell-associated genes. This monocyte subpopulation is reduced in renal transplant patients with CTD, possibly due to selective migration into the allograft.

Molecular typing of Giardia duodenalis isolates from German travellers.

Giardia duodenalis isolates from German travellers returning from tropical areas were characterised by PCR amplification and sequencing of fragments of the beta-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. Assignment of isolates to specific G. duodenalis assemblages was found to differ according to the marker used. Indeed, at the bg locus, assemblages A and B were identified, with a higher prevalence of the former over the latter, whereas at the tpi and gdh loci, most samples were classified as assemblage B. In agreement with previous studies, sequence analysis showed that assemblage B isolates have a higher genetic polymorphism than assemblage A isolates, and novel variants were described. The degree of polymorphism was shown in a graphical representation of the polymorphic sites generating a novel sequence, the heterogeneous positions common to assemblages A and B (double peaks), that may represent mixed assemblage infection and the heterogeneous positions detected at random sites. Notably, assemblage D, which is considered to be adapted to dogs, was found at the gdh locus in two samples originating from southern Asia, as novel genotypes. By comparing the geographical origin of the infected cases and the number of German travellers visiting the areas considered, India and west Africa appeared to be the areas associated to the highest risk of acquiring Giardia infection. The analysis of the geographical distribution of the genotypes did not suggest any particular geographical clustering pattern, but it may be useful to evaluate these results with a higher number of isolates. Most of the samples typed at the three markers could not be assigned unequivocally to either assemblage A or B, and this was confirmed also by a real-time PCR assay, using a set of assemblage-specific primers. The results of this study reinforce the notion that genetic exchanges and allelic sequence heterogeneity represent major obstacles towards understanding the epidemiology of giardiasis and that exposure to Giardia parasites in endemic areas often results in mixed infections in returning travellers.

Amoebic liver abscess with negative serologic markers for Entamoeba histolytica: mind the gap!

A 38-year-old male German traveller returning from Asia presented with fever, night sweats and abdominal complaints. Abdominal ultrasonography revealed several fast-growing abscesses of the liver. Three blood cultures as well as serologic investigations for the detection of antibodies to Entamoeba histolytica, performed on day 3 and 7 after the onset of clinical symptoms, remained negative. Stool microscopy revealed the presence of amoeba cysts compatible with E. histolytica infection. Taking both the amoebic and bacterial etiology of the abscesses into consideration, the patient was treated with metronidazole and ciprofloxacin followed by paromomycin. Antibodies to E. histolytica tested positive shortly after anti-amoebic therapy was initiated. The patient fully recovered, and ultrasound follow-up showed complete resolution of the abscesses within 50 days. This case leads to the conclusion that amoebic liver abscess should be considered despite negative amoeba serology and that ultrasonography is an important diagnostic tool for the early diagnosis of extraintestinal amoebiasis.

Quantifying the impact of nevirapine-based prophylaxis strategies to prevent mother-to-child transmission of HIV-1: a combined pharmacokinetic, pharmacodynamic, and viral dynamic analysis to predict clinical outcomes.

Single-dose nevirapine (sd-NVP) and extended NVP prophylaxis are widely used in resource-constrained settings to prevent vertical HIV-1 transmission. We assessed the pharmacokinetics of sd-NVP in 62 HIV-1-positive pregnant Ugandan woman and their newborns who were receiving sd-NVP prophylaxis to prevent mother-to-child HIV-1 transmission. Based on these data, we developed a mathematical model system to quantify the impact of different sd-NVP regimens at delivery and of extended infant NVP prophylaxis (6, 14, 21, 26, 52, 78, and 102 weeks) on the 2-year risk of HIV-1 transmission and development of drug resistance in mothers and their breast-fed infants. Pharmacokinetic parameter estimates and model-predicted HIV-1 transmission rates were very consistent with other studies. Predicted 2-year HIV-1 transmission risks were 35.8% without prophylaxis, 31.6% for newborn sd-NVP, 19.1% for maternal sd-NVP, and 19.7% for maternal/newborn sd-NVP. Maternal sd-NVP reduced newborn infection predominately by transplacental exchange, providing protective NVP concentrations to the newborn at delivery, rather than by maternal viral load reduction. Drug resistance was frequently selected in HIV-1-positive mothers after maternal sd-NVP. Extended newborn NVP prophylaxis further decreased HIV-1 transmission risks, but an overall decline in cost-effectiveness for increasing durations of newborn prophylaxis was indicated. The total number of infections with resistant virus in newborns was not increased by extended newborn NVP prophylaxis. The developed mathematical modeling framework successfully predicted the risk of HIV-1 transmission and resistance development and can be adapted to other drugs/drug combinations to a priori assess their potential in reducing vertical HIV-1 transmission and resistance spread.

Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane.

Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using Total Internal Reflection Microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by Fluorescence Recovery after Photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners.

miRNA analysis in B-cell chronic lymphocytic leukaemia: proliferation centres characterized by low miR-150 and high BIC/miR-155 expression.

Several miRNAs have been reported to be associated with immunoglobulin heavy chain (IgH) mutation and ZAP-70 expression status in blood samples of B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (B-CLL/SLL). In the bone marrow and lymphoid tissues, proliferation centres (PCs) represent an important site of activation and proliferation of the neoplastic cells, suggesting that these tissues better reflect the biology of CLL than circulating blood cells. We collected 33 lymph nodes and 37 blood CLL samples and analysed IgH mutation status and ZAP-70 expression status. Expression of 15 miRNAs was analysed by qRT-PCR and RNA-ISH. Sixty-three per cent of the lymph node cases contained mutated IgH genes and 49% of the lymph node cases were ZAP-70-positive, and a significant correlation was observed between ZAP-70 expression and IgH mutation status. Of the blood CLL samples, 49% contained mutated IgH sequences. The miRNA expression pattern in CLL lymph node and blood samples was very similar. Three of 15 miRNAs (miR-16, miR-21, and miR-150) showed a high expression level in both blood and lymph node samples. No difference was observed between ZAP-70-positive or -negative and between IgH-mutated or unmutated cases. No correlation was found between miR-15a and miR-16 expression levels and 13q14 deletion in the blood CLL samples. RNA in situ hybridization (ISH) revealed strong homogeneous staining of miR-150 in the tumour cells outside the PCs. In reverse BIC/pri-miR-155 expression was observed mainly in individual cells including prolymphocytes of the PCs. This reciprocal pattern likely reflects the different functions and targets of miR-150 and miR-155.

Regulation of pri-microRNA BIC transcription and processing in Burkitt lymphoma.

BIC is a primary microRNA (pri-miR-155) that can be processed to mature miR-155. In this study, we show the crucial involvement of protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) in the regulation of BIC expression upon B-cell receptor triggering. Surprisingly, Northern blot analysis did not reveal any miR-155 expression upon induction of BIC expression in the Burkitt lymphoma-derived Ramos cell line, whereas other microRNAs were clearly detectable. Ectopic expression of BIC in Ramos and HEK293 cells resulted in miR-155 expression in HEK293, but not in Ramos cells, suggesting a specific block of BIC to miR-155 processing in Ramos. In line with the results obtained with Ramos, lack of miR-155 expression after induction of BIC expression was also observed in other Burkitt lymphoma cell lines, indicating a generic and specific blockade in the processing of BIC in Burkitt lymphoma. In contrast, induction of BIC expression in normal tonsillar B cells resulted in very high levels of miR-155 expression and induction of BIC expression in Hodgkin's lymphoma cell lines. It also resulted in elevated levels of miR-155. Our data provide evidence for two levels of regulation for mature miR-155 expression: one at the transcriptional level involving PKC and NF-kappaB, and one at the processing level. Burkitt lymphoma cells not only express low levels of BIC, but also prevent processing of BIC via an, as yet, unknown mechanism.

Gene expression analysis of dendritic/Langerhans cells and Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LC) phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbilical cord blood CD34+ progenitor cells to identify LC-specific genes and the expression of these genes in LCH was investigated. Besides the expression of several genes known to be highly expressed in LCs and LCH such as CD1a, LYZ, and CD207, high expression of genes not previously reported to be expressed in LCs, such as GSN, MMP12, CCL17, and CCL22, was also identified. Further analysis of these genes by quantitative RT-PCR revealed high expression of FSCN1 and GSN in all 12 LCH cases analysed; of CD207, MMP12, CCL22, and CD1a in the majority of these cases; and CCL17 in three of the 12 cases. Immunohistochemistry confirmed protein expression in the majority of cases. The expression of MMP12 was most abundant in multi-system LCH, which is the LCH type with the worst prognosis. This suggests that expression of MMP12 may play a role in the progression of LCH. These data reveal new insight into the pathology of LCH and provide new starting points for further investigation of this clonal proliferative disorder.

Complement dependency of splenic localization of pneumococcal polysaccharide and conjugate vaccines.

The immune response to polysaccharides is initiated when polysaccharides bind complement factor C3d, and these polysaccharide-C3d complexes subsequently localize on splenic marginal zone B cells strongly expressing CD21 (complement receptor 2). Infants and children under the age of 2 years have low or absent expression of CD21 on their marginal zone B cells, and consequently do not adequately respond to polysaccharides. In contrast, polysaccharide-protein conjugate vaccines are able to induce antibodies at this young age. Conjugate vaccines apparently overcome the necessity for CD21-C3d interaction for an antipolysaccharide immune response. We demonstrate in a rat model that localization of pneumococcal polysaccharides on splenic marginal zone B cells indeed is complement dependent. We also show that pneumococcal conjugates do not specifically localize on splenic marginal zone B cells and that splenic localization of polysaccharide conjugates is independent of the presence of complement. Thus, the induction of antipolysaccharide antibodies by conjugate vaccines apparently can occur independently of CD21-C3d interaction. These basic findings may explain the effectiveness of conjugated vaccines in young children and may open the way for their application in other patient groups.

High expression of Mcl-1 in ALK positive and negative anaplastic large cell lymphoma.

AIM: To gain more insight into the genes involved in the aetiology and pathogenesis of anaplastic large cell lymphoma (ALCL). METHODS: Serial analysis of gene expression (SAGE) was undertaken on the CD4+ALK+ (anaplastic lymphoma kinase positive) ALCL derived cell line Karpas299 and as comparison on CD4+ T cells. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were performed on five ALCL derived cell lines and 32 tissue samples to confirm the SAGE data. RESULTS: High expression of Mcl-1 was seen in the Karpas299 cell line, whereas the two other antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-X(L), were not detected in the SAGE library. Quantitative RT-PCR confirmed the high expression of Mcl-1 mRNA and low expression of Bcl-2 and Bcl-X(L) in Karpas299 and in four other ALCL cell lines. To expand on these initial observations, primary tissue samples were analysed for Mcl-1, Bcl-X(L), and Bcl-2 by immunohistochemistry. All 23 ALK+ and nine ALK- ALCL cases were positive for Mcl-1. Bcl-2 and Bcl-X(L) were expressed infrequently in ALK+ ALCL cases, but were present in a higher proportion of ALK- ALCL cases. CONCLUSION: The consistent high expression of Mcl-1 in ALK+ and ALK- ALCL suggests that Mcl-1 is the main antiapoptotic protein in this disease. The high frequency of Mcl-1, Bcl-2, and Bcl-X(L) positive ALCL cases in the ALK- group compared with the ALK+ group indicates that ALK induced STAT3 activation is not the main regulatory pathway in ALCL.

Mother-to-child transmission of HIV and its prevention: awareness and knowledge in Uganda and Tanzania.

Awareness and knowledge about HIV mother-to-child transmission (MTCT) and preventive measures in different population groups and health personnel were analysed in future intervention areas in western Uganda and southwestern Tanzania. In Uganda, a total of 751 persons (440 clients of antenatal and outpatient clinics, 43 health workers, 239 villagers, 29 traditional birth attendants) and in Tanzania, 574 persons (410 clients, 49 health workers, 93 villagers, 18 traditional birth attendants) were interviewed. When given options, knowledge on transmission during pregnancy and delivery in women was 93% and 67% in Uganda and Tanzania respectively, and 86% and 78% for transmission during breastfeeding. In Uganda 59% of male interviewees did not believe that HIV is transmitted during breastfeeding. Expressed acceptance of HIV testing was above 90% in men and women in both countries, but only 10% of the clients in Uganda and 14% in Tanzania had been tested for HIV infection. Health workers' knowledge regarding MTCT was acceptable, while traditional birth attendants' knowledge on both MTCT and preventive measures was extremely poor. Recom endations on infant feeding were not compatible with WHO recommendations for HIV-infected women. If prevention of MTCT (PMTCT) interventions are to be accepted by the population and promoted by health personnel, thorough orientation and training are mandatory.

Molecular assessment of ammonia- and nitrite-oxidizing bacteria in full-scale activated sludge wastewater treatment plants.

Nitrification was assessed in two full-scale wastewater treatment plants (WWTPs) over time using molecular methods. Both WWTPs employed a complete-mix suspended growth, aerobic activated sludge process (with biomass recycle) for combined carbon and nitrogen treatment. However, one facility treated primarily municipal wastewater while the other only industrial wastewater. Real time PCR assays were developed to determine copy numbers for total 16S rDNA (a measure of biomass content), the amoA gene (a measure of ammonia-oxidizers), and the Nitrospira 16S rDNA gene (a measure of nitrite-oxidizers) in mixed liquor samples. In both the municipal and industrial WWTP samples, total 16S rDNA values were approximately 2-9 x 10(13) copies/L and Nitrospira 16S rDNA values were 2-4 x 10(10) copies/L. amoA gene concentrations averaged 1.73 x 10(9) copies/L (municipal) and 1.06 x 10(10) copies/L (industrial), however, assays for two distinct ammonia oxidizing bacteria were required.

[HIV positive patient with pancytopenia and massive splenomegaly]. / HIV-positiver Patient mit Panzytopenie und massiver Splenomegalie.

A 30-year-old homosexual man presented with anemia and a several months history of recurrent fever, night sweats and weakness. His travel history included several stays in mediterranean countries during the recent years. Abdominal ultrasound showed massive splenomegaly, hepatomegaly and abdominal lymphadenopathy. A bone marrow aspirate revealed the presence of numerous Leishmania amastigotes, and bone marrow culture and polymerase chain reaction were also positive for Leishmania. In this case report epidemiological, immunological, diagnostic and therapeutic aspects of HIV-Leishmania coinfection are discussed with special emphasis on the impact of liposomal amphotericin B and highly active antiretroviral therapy on the treatment of HIV-leishmania-coinfection.

Imported schistosomiasis in Europe: sentinel surveillance data from TropNetEurop.

BACKGROUND: Schistosomiasis is a major parasitic disease, increasingly imported into temperate climates by immigrants from and travelers to endemic areas. METHOD: To generate valid data on imported infectious diseases to Europe and to recognize trends over time, the European Network on Imported Infectious Diseases Surveillance (TropNetEurop) was founded in 1999. Three hundred and thirty-three reports of schistosomiasis were analyzed for epidemiologic and clinical features. RESULTS: Male patients accounted for 64% of all cases. The average age of all patients was 29.5 years. The majority of patients were of European origin (53%). Europeans traveled predominantly for tourism (52%). Main reasons for travel for people from endemic areas were immigration and refuge (51%) and visits to relatives and friends (28%). The majority of infections were acquired in Africa; 92 infections were clearly attributable to Schistosoma haematobium, 130 to Schistosoma mansoni, and 4 to Schistosoma intercalatum. Praziquantel was the only treatment used. No deaths were recorded. CONCLUSION: TropNetEurop sentinel provides valuable epidemiologic and clinical data on imported schistosomiasis to Europe.

[Progress in the early diagnosis of breast carcinoma during the years 1981-1990. Results of a longitudinal study]. / Fortschritte in der Früherkennung des Mammakarzinoms in den Jahren 1981-1990. Ergebnisse einer Longitudinalstudie.

BACKGROUND AND OBJECTIVE: Current meta-analyses have left in doubt whether general breast screening increases survival rate. This study investigated whether efforts at early diagnosis of cancer in the 1980s have had an effect on average tumor size at first diagnosis and on survival rate. PATIENTS AND METHODS: From 1981 to 1990, 1656 consecutive patients (average age 56.6 years) at the I. Women's Clinic at the Ludwig-Maximilian University of Munich and the Women's Clinic Berlin-Charlottenburg were operated on for primary breast cancer. In a retrospective analysis, average tumor size at the primary operation and survival rate were determined for two periods: 1981-1985 (n=849) and 1986-1990. Mean follow-up time was 63 months. RESULTS: There was no difference between the two cohorts regarding age (p = 0.77) and axillary node status (p = 0.14). During the follow-up period there was a gradual decrease in the tumor size at first diagnosis. (Pearson's correlation coefficient: -0.79, p < 0.001). Average tumor size in those operated on was 25 mm up to 1985, and 21 mm after 1986 (p < 0.001). Until 1985, the initial reason for mammography was the planned subsequent operation in 19% of patients (n = 164), and in 27% (n = 215; p < 0.001) since 1986. But there was no statistically significant rise in disease-specific survival rate (log rank, p=0.48). Multivariate analysis confirmed the conventional prognostic parameters, such as tumor size (relative risk 2.21) and axillary lymph node metastases (relative risk 3.57), but not the period of follow-up (p=0.90). CONCLUSION: During the stated periods of follow-up there was a significant decrease in average tumor size at initial diagnosis. But this did not result in any demonstrably better disease-specific survival rate.

Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy.

Laser dissection microscopy was applied to isolate endothelial cells from tumors obtained from mice treated with TNF-alpha. RNA integrity was demonstrated from whole sections and dissected cells after acetone fixation and hematoxylin staining. RT-PCR for GAPDH, CD31, VCAM-1, ICAM-1, and E-selectin was successfully performed on these samples. These data demonstrate the feasibility of analyzing local endothelial cell responses in diseased tissues at the level of gene expression.

Epidemiology and clinical features of imported dengue fever in Europe: sentinel surveillance data from TropNetEurop.

Travelers have the potential both to acquire and to spread dengue virus infection. The incidence of dengue fever (DF) among European travelers certainly is underestimated, because few centers use standardized diagnostic procedures for febrile patients. In addition, DF is currently not reported in most European public health systems. Surveillance has commenced within the framework of a European Network on Imported Infectious Disease Surveillance (TropNetEurop) to gain information on the quantity and severity of cases of dengue imported into Europe. Descriptions of 294 patients with DF were analyzed for epidemiological information and clinical features. By far the most infections were imported from Asia, which suggests a high risk of DF for travelers to that region. Dengue hemorrhagic fever occurred in 7 patients (2.4%) all of whom recovered. Data reported by member sites of the TropNetEurop can contribute to understanding the epidemiology and clinical characteristics of imported DF.