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Background: Increased control has produced remarkable reductions of malaria in some parts of sub-Saharan Africa, including Rwanda. In the southern highlands, near the district capital of Butare (altitude, 1,768 m), a combined community-and facility-based survey on Plasmodium infection was conducted early in 2010., Methods: A total of 749 children below five years of age were examined including 545 randomly selected from 24 villages, 103 attending the health centre in charge, and 101 at the referral district hospital. Clinical, parasitological, haematological, and socio-economic data were collected., Results: Plasmodium falciparum infection (mean multiplicity, 2.08) was identified by microscopy and PCR in 11.7% and 16.7%, respectively; 5.5% of the children had malaria. PCR-based P. falciparum prevalence ranged between 0 and 38.5% in the villages, and was 21.4% in the health centre, and 14.9% in the hospital. Independent predictors of infection included increasing age, low mid-upper arm circumference, absence of several household assets, reported recent intake of artemether-lumefantrine, and chloroquine in plasma, measured by ELISA. Self-reported bed net use (58%) reduced infection only in univariate analysis. In the communities, most infections were seemingly asymptomatic but anaemia was observed in 82% and 28% of children with and without parasitaemia, respectively, the effect increasing with parasite density, and significant also for submicroscopic infections., Conclusions: Plasmodium falciparum infection in the highlands surrounding Butare, Rwanda, is seen in one out of six children under five years of age. The abundance of seemingly asymptomatic infections in the community forms a reservoir for transmission in this epidemic-prone area. Risk factors suggestive of low socio-economic status and insufficient effectiveness of self-reported bed net use refer to areas of improvable intervention.

The Department of Veterans Affairs (VA's) mission is to care for those who have borne the battle. As medical technology has advanced, more and more of our returning combat Veterans survive what would have been fatal wounds in previous conflicts ( Gawande, 2004 ). But survival is only the immediate goal-our job is to restore Veterans to the greatest level of health, independence, and quality of life that is medically possible. The VA is achieving this goal through close collaboration with the Department of Defense (DoD) to facilitate a smooth transition and continuum of care that ensures Veterans and Service Members receive the care they deserve. This article describes VA's system of Veteran-centered, post-combat care programs that rely on significant involvement of social workers to support Service Members, Veterans and their families through recovery, rehabilitation, and re-integration into their home communities.

Background: Nevirapine (NVP) single-dose is still a widely used antiretroviral prophylaxis for the prevention of vertical HIV-1 transmission in resource-limited settings. However, the main disadvantage of the Non-nucleoside Reverse Transcriptase Inhibitor (NNRTI) NVP is the rapid selection of NVP-resistant virus with negative implications for subsequent NNRTI-based long-term antiretroviral therapy (ART). Here, we analysed the emergence of drug-resistant HIV-1 including minor variants in the early phase after NVP single-dose prophylaxis and the persistence of drug-resistant virus over time., Methods and Findings: NVP-resistant HIV-1 harbouring the K103N and/or Y181C resistance mutations in the HIV-1 reverse transcriptase gene was measured from 1 week up to 18 months after NVP single-dose prophylaxis in 29 Ugandan women using allele-specific PCR assays capable of detecting drug-resistant variants representing less than 1% of the whole viral population. In total, drug-resistant HIV-1 was identified in 18/29 (62%) women; rates increased from 18% to 38% and 44% at week 1, 2, 6, respectively, and decreased to 18%, 25%, 13% and 4% at month 3, 6, 12 and 18, respectively. The proportion of NVP-resistant virus of the total viral population was significantly higher in women infected with subtype D (median 40.5%) as compared to subtype A (median 1.3%; p = 0.032, Mann-Whitney U test). 33% of resistant virus was not detectable at week 2 but was for the first time measurable 6-12 weeks after NVP single-dose prophylaxis. Three (10%) women harboured resistant virus in proportions >10% still at month 6., Conclusions: Current WHO guidelines recommend an additional postnatal intake of AZT and 3TC for one week to avoid NVP resistance formation. Our findings indicate that a 1-week medication might be too short to impede the emergence of NVP resistance in a substantial proportion of women. Furthermore, subsequent NNRTI-based ART should not be started earlier than 12 months after NVP single-dose prophylaxis.

Background: Since 2008, Tanzanian guidelines for prevention of mother-to-child-transmission of HIV (PMTCT) recommend combination regimen for mother and infant starting in gestational week 28. Combination prophylaxis is assumed to be more effective and less prone to resistance formation compared to single-drug interventions, but the required continuous collection and intake of drugs might pose a challenge on adherence especially in peripheral resource-limited settings. This study aimed at analyzing adherence to combination prophylaxis under field conditions in a rural health facility in Kyela, Tanzania., Methods and Findings: A cohort of 122 pregnant women willing to start combination prophylaxis in Kyela District Hospital was enrolled in an observational study. Risk factors for decline of prophylaxis were determined, and adherence levels before, during and after delivery were calculated. In multivariate analysis, identified risk factors for declining pre-delivery prophylaxis included maternal age below 24 years, no income-generating activity, and enrolment before 24.5 gestational weeks, with odds ratios of 5.8 (P = 0.002), 4.4 (P = 0.015) and 7.8 (P = 0.001), respectively. Women who stated to have disclosed their HIV status were significantly more adherent in the pre-delivery period than women who did not (P = 0.004). In the intra- and postpartum period, rather low drug adherence rates during hospitalization indicated unsatisfactory staff performance. Only ten mother-child pairs were at least 80% adherent during all intervention phases; one single mother-child pair met a 95% adherence threshold., Conclusions: Achieving adherence to combination prophylaxis has shown to be challenging in this rural study setting. Our findings underline the need for additional supervision for PMTCT staff as well as for clients, especially by encouraging them to seek social support through status disclosure. Prophylaxis uptake might be improved by preponing drug intake to an earlier gestational age. Limited structural conditions of a healthcare setting should be taken into serious account when implementing PMTCT combination prophylaxis.

Background: Nevirapine single-dose (NVP-SD) reduces mother-to-child transmission of HIV type-1 (HIV-1), but frequently induces resistance mutations in the HIV-1 genome. Little is known about drug-resistant HIV-1 variants in the breast milk of women who have taken NVP-SD., Methods: Blood and breast milk samples of 39 HIV-1-infected Ugandan women were taken 6-12 weeks after NVP-SD intake. Samples were analysed by population sequencing and allele-specific real-time PCR (AS-PCR) with detection limits for NVP-resistant HIV-1 variants (K103N and Y181C) of < 1% of the total viral population., Results: AS-PCR results for both plasma and breast milk were obtained for 19 women who constituted the final study group (HIV-1 subtype frequencies were A1 n = 11, D n = 5, G n = 2 and C n = 1). A total of 7 (37%) and 10 (53%) women carried NVP-resistant virus in breast milk and plasma, respectively. Overall, 71% (5/7) women with NVP-resistant HIV-1 in breast milk displayed >1 drug-resistant variant. Resistance in breast milk was higher at week 6 (6/13 samples [46%]) compared with week 12 (1/6 samples [17%]). In total, 10 drug-resistant populations harbouring the K103N and/or Y181C mutation were detected in the 19 breast milk samples; 7 (70%) were caused by resistant minorities (< 5% of the total HIV-1 population). In the four women with drug-resistant virus in both plasma and breast milk, the mutation patterns differed between the two compartments., Conclusions: Minor populations of drug-resistant HIV-1 were frequently found in breast milk of Ugandan women after exposure to NVP-SD. Further studies need to explore the role of minor drug-resistant variants in the postnatal transmission of (resistant) HIV-1.

Partner involvement is considered to increase the effectiveness of female-oriented services for sexual and reproductive health (SRH), like those for antenatal care (ANC) or the prevention of mother-to-child transmission of HIV (PMTCT). However, male participation rates remain mostly low, and previous research has identified restrictive provider attitudes among barriers for partner participation in such services. Individual perspectives and experiences of healthcare providers are assumed to significantly influence the quality of delivered services. This study aimed at exploring providers' attitudes regarding partner involvement in ANC/PMTCT and other SRH services. A hundred interviews based on a semi-structured questionnaire were conducted among healthcare providers employed in an ANC-based PMTCT program in Mbeya Region, Tanzania. Interviewees expressed overall approval of male partner integration into the services, but this approval decreased when specifying for different service types, especially in those related to perinatal examinations or labor and delivery. Divergence between general attitudes and self-reported individual behavior was observed, querying the reliability of expressed attitudes. Among providers having at least one child, personally experienced partner attendance and approval of partner involvement were significantly associated for most service types. Although general views on partner involvement in SRH services seem to be mostly supportive, there is a need for health services to strengthen providers' positions toward male involvement, for example by communicating clear policies and job guidelines, and by encouraging partner service attendance among providers themselves.

Nevirapine (single dose), commonly used to prevent the mother-to-child transmission of human immunodeficiency virus (HIV) in developing countries, frequently induces viral resistance. Even mutations which occur only in a minor population of the HIV quasispecies (<20%) are associated with subsequent treatment failure but cannot be detected by population-based sequencing. We developed sensitive allele-specific real-time PCR (ASPCR) assays for two key resistance mutations of nevirapine. The assays were specifically designed to analyze HIV-1 subtype A and D isolates accounting for the majority of HIV infections in Uganda. Assays were evaluated using DNA standards and clinical samples of Ugandan women having preventively taken single-dose nevirapine. Lower detection limits of drug-resistant HIV type 1 (HIV-1) variants carrying reverse transcriptase mutations were 0.019% (K103N [AAC]), 0.013% (K103N [AAT]), and 0.29% (Y181C [TGT]), respectively. Accuracy and precision were high, with coefficients of variation (the standard ratio divided by the mean) of 0.02 to 0.15 for intra-assay variability and those of 0.07 to 0.15 (K103N) and 0.28 to 0.52 (Y181C) for inter-assay variability. ASPCR assays enabled the additional identification of 12 (20%) minor drug-resistant HIV variants in the 20 clinical Ugandan samples (3 mutation analyses per patient; 60 analyses in total) which were not detectable by population-based sequencing. The individual patient cutoff derived from the clinical baseline sample was more appropriate than the standard-based cutoff from cloned DNA. The latter is a suitable alternative since the presence/absence of drug-resistant HIV-1 strains was concordantly identified in 92% (55/60) of the analyses. These assays are useful to monitor the emergence and persistence of drug-resistant HIV-1 variants in subjects infected with HIV-1 subtypes A and D.

MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitt's lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.

Throughout all stages of programmes for the prevention of mother-to-child-transmission of HIV (PMTCT), high dropout rates are common. Increased male involvement and couples' joint HIV counselling/testing during antenatal care (ANC) seem crucial for improving PMTCT outcomes. Our study assessed male attitudes regarding partner involvement into ANC/PMTCT services in Mbeya Region, Tanzania, conducting 124 individual interviews and six focus group discussions. Almost all respondents generally supported PMTCT interventions. Mentioned barriers to ANC/PMTCT attendance included lacking information/knowledge, no time, neglected importance, the services representing a female responsibility, or fear of HIV-test results. Only few perceived couple HIV counselling/testing as disadvantageous. Among fathers who had refused previous ANC/PMTCT attendance, most had done so even though they were not perceiving a disadvantage about couple counselling/testing. The contradiction between men's beneficial attitudes towards their involvement and low participation rates suggests that external barriers play a large role in this decision-making process and that partner's needs should be more specifically addressed in ANC/PMTCT services.

Background: Flat epithelial atypia (FEA) of the breast is characterised by a few layers of mildly atypical luminal epithelial cells. Genetic changes found in ductal carcinoma in situ (DCIS) and invasive ductal breast cancer (IDC) are also found in FEA, albeit at a lower concentration. So far, miRNA expression changes associated with invasive breast cancer, like miR-21, have not been studied in FEA., Methods: We performed miRNA in-situ hybridization (ISH) on 15 cases with simultaneous presence of normal breast tissue, FEA and/or DCIS and 17 additional cases with IDC. Expression of the miR-21 targets PDCD4, TM1 and PTEN was investigated by immunohistochemistry., Results: Two out of fifteen cases showed positive staining for miR-21 in normal breast ductal epithelium, seven out of fifteen cases were positive in the FEA component and nine out of twelve cases were positive in the DCIS component. A positive staining of miR-21 was observed in 15 of 17 IDC cases. In 12 cases all three components were present in one tissue block and an increase of miR-21 from normal breast to FEA and to DCIS was observed in five cases. In three cases the FEA component was negative, whereas the DCIS component was positive for miR-21. In three other cases, normal, FEA and DCIS components were negative for miR-21 and in the last case all three components were positive. Overall we observed a gradual increase in percentage of miR-21 positive cases from normal, to FEA, DCIS and IDC. Immunohistochemical staining for PTEN revealed no obvious changes in staining intensities in normal, FEA, DCIS and IDC. Cytoplasmic staining of PDCD4 increased from normal to IDC, whereas, the nuclear staining decreased. TM1 staining decreased from positive in normal breast to negative in most DCIS and IDC cases. In FEA, the staining pattern for TM1 was similar to normal breast tissue., Conclusion: Upregulation of miR-21 from normal ductal epithelial cells of the breast to FEA, DCIS and IDC parallels morphologically defined carcinogenesis. No clear relation was observed between the staining pattern of miR-21 and its previously reported target genes.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

Hodgkin lymphoma (HL) is derived from preapoptotic germinal center B cells, although a general loss of B cell phenotype is noted. Using quantitative reverse transcription-polymerase chain reaction and miRNA microarray, we determined the microRNA (miRNA) profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas. The two methods showed a strong correlation for the detection of miRNA expression levels. The HL-specific miRNA included miR-17-92 cluster members, miR-16, miR-21, miR-24, and miR-155. Using a large panel of cell lines, we found differential expression between HL and other B-cell lymphoma-derived cell lines for 27 miRNA. A significant down-regulation in HL compared to non-Hodgkin lymphoma was observed only for miR-150. Next, we performed target gene validation of predicted target genes for miR-155, which is highly expressed in HL and is differentially expressed between HL and Burkitt lymphoma. Using luciferase reporter assays, we validated 11 predicted miR-155 target genes in three different HL cell lines. We demonstrated that AGTR1, FGF7, ZNF537, ZIC3, and IKBKE are true miR-155 target genes in HL.

Amodiaquine/artesunate and artemether/lumefantrine are currently implemented as antimalarial drugs in Africa. In this study, we assessed Plasmodium falciparum pfmdr1 alleles suggesting reduced sensitivity to amodiaquine/artesunate (86Y-184Y-1246Y) and artemether/lumefantrine (184F and 86N-184F-1246D) in 111 African isolates imported to Germany between 1997 and 2005. pfmdr1 86Y-184Y-1246Y occurred less frequently in West African isolates (2.5%) than in East African parasites (45.8%) (P<0.001) and less frequently in infections contracted in countries currently implementing amodiaquine/artesunate (5.7%) compared with countries with an artemether/lumefantrine policy (20.8%) (P=0.03). pfmdr1 184F showed an opposite pattern. pfmdr1 alleles reveal significant differences between West and East Africa, supporting the current implementation of first-line antimalarials.

While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin alpha(E)(CD103)beta(7) has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that alpha(E)(CD103)beta(7) also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type alpha(E)(CD103)beta(7) conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by alpha(E)(CD103)beta(7)-directed antibodies or cytochalasin D. The alpha(E)-dependent motility was further increased (P < .001) when point-mutated alpha(E)(CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein-coupled alpha(E)(CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional alpha(E)(CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of alpha(E)(CD103)-deficient mice (P < .001). Thus, integrin alpha(E)(CD103)beta(7) is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes.

It is well documented that reducing blood pressure (BP) in hypertensive individuals reduces the risk of cardiovascular (CV) events. Despite this, many patients with hypertension remain untreated or inadequately treated, and fail to reach the recommended BP goals. Suboptimal BP control, whilst arising from multiple causes, is often due to poor patient compliance and/or persistence, and results in a significant health and economic burden on society. The use of fixed-dose combinations (FDCs) for the treatment of hypertension has the potential to increase patient compliance and persistence. When compared with antihypertensive monotherapies, FDCs may also offer equivalent or better efficacy, and the same or improved tolerability. As a result, FDCs have the potential to reduce both the CV event rates and the non-drug healthcare costs associated with hypertension. When FDCs are adopted for the treatment of hypertension, issues relating to copayment, formulary restrictions and therapeutic reference pricing must be addressed.

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.

Little is known about the gene expression profile and significance of the rosetting CD4+CD26- T cells in classical Hodgkin's lymphoma (cHL). To characterize these T cells, CD4+CD26- and CD4+CD26+ T-cell populations were sorted from lymph node (LN) cell suspensions from nodular sclerosis HL (NSHL) and reactive LNs. mRNA profiles of stimulated and resting cell subsets were evaluated with quantitative RT-PCR for 46 genes. We observed a higher percentage of CD4+CD26- T cells in NSHL than in reactive LNs. The resting CD4+CD26- T cells in NSHL showed higher mRNA levels of CD25, CTLA4, OX40 and CCR4 compared with in LNs, supporting a regulatory T-cell (Treg) type, and this was validated by immunohistochemistry. Moreover, these cells showed low or no expression of the Th1- or Th2-related cytokines IL-2, IFN-gamma, IL-13, IL-12B, IL-4, and IL-5, and the chemoattractant receptor CRTH2. Besides Tregs, Th17 cells may exist in NSHL based on the significantly higher IL-17 mRNA level for both T-cell populations in NSHL. Upon stimulation in vitro, lack of upregulation of mRNA levels of most cytokine genes indicated an anergic character for the CD4+CD26- T-cell subset. Anergy fits with the Treg profile of these cells, probably explaining the immunosuppressive mechanism involved in NSHL.

There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered "true positives," the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

Integrin-bound Src tyrosine kinase mediates alpha(IIb)beta(3) out-side-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during alpha(IIb)beta(3)-mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2-3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Csk-mediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.

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.

Objectives: To evaluate risk factors for treatment denial and loss to follow-up in an antiretroviral treatment (ART) cohort in a rural African setting in western Kenya., Method: Sociodemographic and clinical data of patients enrolled in an ART cohort were collected within 18 months of an observational longitudinal study and analysed by logistic and Cox regression models., Results: Of 159 patients with treatment indication 35 (22%) never started ART. Pregnancy [adjusted odds ratio (AOR) 3.60, 95% confidence interval (CI) 1.10-11.8; P = 0.035] and lower level of education (AOR 3.80, 95% CI 1.14-12.7; P = 0.03) were independently associated with treatment denial. The incidence of total loss of patients under therapy was 43.2 per 100 person years (pys) (mortality rate 19.2 per 100 pys plus drop out rate 24 per 100 pys). Older age [adjusted hazard ratio (AHR) 1.06, 95% CI 1.01-1.12; P = 0.04], AIDS before starting treatment (AHR 5.83, 95% CI 1.15-29.5; P = 0.03) and incomplete adherence to treatment (AHR 1.05, 95% CI 1.03-1.07; P < 0.001) were independent risk factors for death. Incomplete adherence also independently predicted drop out because of other reasons (AHR 1.06, 95% CI 1.04-1.09; P < 0.001)., Conclusion: Pregnancy and lower level of education, higher age, advanced AIDS stage and impaired compliance to ART were identified as risk factors for treatment denial and death, respectively. Adequate counselling strategies for patients with these characteristics could help to improve adherence and outcome of treatment programmes in resource-limited settings.

Background: Intrapartum transmission of HIV has been reported to be associated with HIV in oropharyngeal secretions (OPSs) of the child. In this study, we analyze the frequency of intrapartum transmission after mucosal exposure to HIV after administration of single-dose nevirapine., Methods: Eighty mothers and their children participating in a prevention of mother-to-child transmission of HIV program in Uganda who took a single dose of nevirapine according to the HIVNET012 protocol participated in the study. HIV-1 was quantified by polymerase chain reaction (PCR) in the mothers' and children's plasma, in cervicovaginal secretions (CVSs), and in the children's OPSs. Intrapartum transmission was defined as a positive HIV-1 RNA PCR result at week 1 or 2 after birth and a previously negative PCR result., Results: Ninety-seven percent of children had detectable nevirapine in their OPS (median = 592 ng/mL). Fifty-seven (81%) children had HIV-negative OPSs, and 13 (19%) had HIV-positive OPSs. All children of mothers with HIV-negative CVSs had HIV-negative OPSs. HIV-1 levels of OPSs and CVSs correlated (r = 0.33, P = 0.027). None of the babies with detectable HIV-1 in the OPSs became infected by means of intrapartum transmission., Conclusion: Intrapartum HIV infection was not observed after mucosal exposure to HIV-1 after administration of a single dose of nevirapine to the mother and child.

Tungiasis is a zoonotic ectoparasitosis that causes considerable morbidity in affected populations. The type of microenvironment that facilitates infestation of hosts by Tunga penetrans has not been investigated. In this study, we exposed 30 laboratory-raised Wistar rats, a suitable model for the infestation, at six different places characterized by different microenvironments in a hyperendemic fishing village in northeastern Brazil. During a period of two weeks, the animals were monitored and the number of embedded fleas was documented. The number of lesions varied considerably according to the microenvironment and was highest in a cage placed at the far end of a compound of a household affected by tungiasis. No penetration was observed inside houses. Results indicate that in this endemic area transmission of T. penetrans seems to occur mainly outdoors.

Objective: Upper endoscopy has been suggested as a valuable tool in the diagnosis of giardiasis. The aim of this study was to compare two methods based on endoscopy, i.e. microscopy of duodenal fluid and histology, with a fluorescent-antibody assay for the detection of Giardia lamblia cysts in stool specimens. The role of endoscopy in the identification of other causes of chronic diarrhea acquired during travel abroad was also evaluated., Material and Methods: Thirty-one patients (9 F, 22 M, median age 39 years, range 19-63 years) with persistent diarrhea after returning from tropical or subtropical areas agreed to undergo upper gastrointestinal endoscopy before and after treatment. Lower gastrointestinal endoscopy was subsequently performed. Three stool samples from each patient were examined using the direct fluorescent-antibody assay (DFA) for the detection of G. lamblia, and by routine methods for other protozoal and bacterial enteric pathogens. Each patient underwent upper endoscopy and biopsies and duodenal fluid samples were taken. In 12 patients a further lower endoscopy was performed., Results: In 16 patients G. lamblia was detected in stool samples by DFA (relative sensitivity: 100%). Histology of duodenal biopsies and microscopy of duodenal fluids allowed diagnosis of giardiasis to be made in only 8, and 3 patients, respectively (relative sensitivities: 21% and 44%). Besides giardiasis, upper endoscopic examination revealed an alternative diagnosis (tropical sprue), whereas six additional diagnoses were made by colonoscopy. In six patients the cause of chronic diarrhea remained unclear., Conclusions: Compared to stool examinations using DFA, upper endoscopy is less sensitive for the diagnosis of giardiasis. In patients with negative stool examinations, lower endoscopy yields relevant diagnoses more often than upper endoscopy.

Multiphoton microscopy has become a standard method for noninvasive imaging of thick specimens with subcellular resolution. Higher harmonic generation microscopy (HHGM), based on nonlinear multiphoton excitation, is a contrast mechanism for the structural and molecular imaging of native samples in cell culture and in fixed and live tissues, for both, three-dimensional and four-dimensional reconstructions. HHGM comprises second and third harmonic generation (SHG, THG) of ordered molecules, can be obtained without exogenous labels, and provides detailed real-time optical reconstruction of fibrillar collagen, myosin, microtubules, and membrane potential, as well as cell depolarization. This unit presents the principles of SHG and THG and the basic setup of a HHGM system, and summarizes current applications in cell biology. Multimodal multiphoton microscopy using HHGM together with two-photon excited fluorescence will develop into a key approach to real-time imaging of cell dynamics in the context of live tissues.

To identify genes involved in the pathogenesis of classical Hodgkin lymphoma (cHL), we performed serial analysis of gene expression (SAGE) and array-based comparative genomic hybridization (aCGH). Comparison of SAGE libraries of cHL cell lines L428 and L1236 with that of germinal centre B cells revealed consistent overexpression of only 14 genes. In contrast, 141 genes were downregulated in both cHL cell lines, including many B cell and HLA genes. aCGH revealed gain of 2p, 7p, 9p, 11q and Xq and loss of 4q and 11q. Eighteen percent of the differentially expressed genes mapped to regions with loss or gain and a good correlation was observed between underexpression and loss or overexpression and gain of DNA. Remarkably, gain of 2p and 9p did not correlate with increased expression of the proposed target genes c-REL and JAK2. Downregulation of many genes within the HLA region also did not correlate with loss of DNA. FSCN1 and IRAK1 mapping at genomic loci (7p and Xq) that frequently showed gain were overexpressed in cHL cell lines and might be involved in the pathogenesis of cHL.

Selectins are attractive targets for specific anti-inflammatory therapies. Using human lymphocytes as well as an L-selectin-transfected pre-B-cell line in dynamic flow chamber experiments, we could demonstrate that the small-molecule compound efomycine M blocks L-selectin-mediated lymphocyte rolling on sialylated Lewis(X), an action that was confirmed by plasmon resonance spectroscopy. Recruitment of naive lymphocytes to peripheral lymph nodes depends on L-selectin-mediated adhesion to high endothelial venules. We performed intravital microscopy studying lymphocyte rolling in peripheral lymph nodes and showed a 53% reduction (P=0.0006) of lymphocyte rolling in mice treated with efomycine M or a function-blocking antibody against L-selectin. In addition, the number of lymph node-homing T cells was reduced by >60% using either efomycine M or L-selectin-blocking antibodies. As recruitment of naive lymphocytes is a prerequisite for sensitization in T-cell-mediated immune reactions and allergic responses, mice were treated with efomycine M or an L-selectin-specific antibody during contact sensitization with DNFB. After adoptive transfer of corresponding T cells into non-sensitized recipient mice, the capacity of these cells to induce contact hypersensitivity was significantly reduced (P=0.0002 and P=0.0001, respectively). Our data demonstrate that it is possible, in principle, to diminish T-cell-mediated allergic reactions through interference with L-selectin functions during the early sensitization phase.

Galectin-1 is the homodimeric form of a protein, which is present in a dynamic equilibrium with the beta-galactoside monomeric form and has potent anti-inflammatory and immunomodulating effects. These favorable effects are probably related to the induction of apoptosis in activated T cells and the induction of IL-10, which have been demonstrated to be characteristic for the dimeric form of the protein. Based on these findings it can be speculated that the in vivo effects of galectin-1 can be improved by the generation of stable galectin-1 homodimers (dGal). To test this hypothesis we produced leucine-zipper based stable galectin-1 homodimers and tested its apoptosis inducing effects on MOLT-4 cells and its immunomodulatory effects in vitro on PBMC of five independent donors. Phosphatidylserine exposure and a drop in mitochondrial membrane potential was strongly enhanced on MOLT-4 cells upon treatment with dGal as compared to wtGal. The minimal effective concentration was 20-fold reduced as compared to the minimal effective wtGal concentration. dGal showed enhanced induction of IL-10 on total PBMC as compared to treatment with wild-type protein (wtGal). The minimal effective dGal concentration was 100-fold lower than that of wtGal. Of the purified cell populations monocytes are the strongest IL-10 producers, whereas T cells induce IL-10 at a lower level and no induction is observed in B cells. Besides induction of IL-10, dGal caused an increase in IL-1beta production in all donors and a reduction of IL-2 production in 3 out of 5 donors, whereas no consistent changes were observed for other inflammatory cytokines. In summary, we demonstrated that dGal shows enhanced effects at strongly reduced concentrations. Application of dGal may therefore serve as an improved treatment of chronic inflammatory diseases.

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., (Copyright 2006 Pathological Society of Great Britain and Ireland.)

Background: Cutaneous larva migrans (CLM) is a common but neglected parasitic skin disease in impoverished communities of the developing world., Methods: To describe the clinical features and the morbidity associated with CLM, active-case finding was performed during rainy and dry seasons in a rural community in the state of Alagoas, Northeast Brazil., Results: A total of 62 individuals received a diagnosis for CLM, and among them there were a total of 75 larval tracks. The number of tracks per person ranged from 1 to 3. Children <9 years old were affected significantly more often than other age groups (P<.001). Children had their lesions mainly on the buttocks, genitals, and hands, whereas in older patients, the majority of lesions were located on the feet. Twenty-four percent of the 75 lesions were superinfected. Bacterial superinfection was more common among children. All but 1 patient complained about itching, and 84% of the 62 patients complained about sleep disturbances. Itching was classified as being severe by 61% of patients. Approximately 40% of the 62 patients had attempted to treat their creeping eruption previously. Larvae moved forward 2.7 mm per day. There was a significant correlation between the length of the track and the duration of infestation ( rho =.53; P<.0001)., Conclusions: CLM is a common parasitic skin disease in this rural community, with different topographic patterns of lesions in children and adults. CLM is associated with considerable morbidity. The length of the track can be used to estimate the duration of infestation.

DEV is the only cell line derived from nodular lymphocyte predominance type of Hodgkin's lymphoma (NLPHL); however, a comprehensive report about the genetic and immunophenotypic profile of this unique cell line is lacking. We analyzed DEV with respect to immunophenotype and genetic aberrations. The immunostaining revealed positivity for CD45, CD20, CD22, CD79a, IgA2, CD80, CD86, CD74, and BCL6. Cytogenetically, DEV has complex chromosome 3 translocations involving chromosomes 7, 14, and 22. A detailed analysis of the 3q27 breakpoint of the der(3)t(3;14)(p14;q32)t(3;22)(q27;q11.2) revealed a break in the BCL6 alternative breakpoint region. Using array comparative genomic hybridization, a 3-megabase homozygous deletion at 17q24.1-24.2 was identified. Fluorescence in situ hybridization indicated the presence of 2 chromosome 17 homologues, each of which carried a small interstitial deletion. Eight microsatellite markers flanking the homozygously deleted region all showed a homozygous pattern suggesting loss of one of the parental alleles. D17S1809 and D17S1816 could not be amplified using DEV DNA, in keeping with a location within the homozygously deleted segment. In conclusion, DEV has an immunophenotype that is consistent with the neoplastic cells of NLPHL cases, the lymphocytic and histiocytic cells. We demonstrated involvement of the BCL6 gene based on the presence of a breakpoint in the alternative breakpoint region and nuclear staining for BCL6 protein and identified a homozygously deleted region at 17q24.

Background: In this study we evaluated alternative CD4(+) T-cell counting methods in clients of a PMTCT Programme in rural Uganda., Methods: The Coulter Manual CD4 Count method for CD4(+) T-cell enumeration (Cyto-Spheres) and an automated method (volumetric, single-platform flow cytometry; CyFlow) were compared with a standard, dual-platform flow cytometry protocol (DPFC, FACScan)., Results: Correlation and precision of agreement were higher for the CyFlow method (r = 0.929 and eta = 0.08) when compared to DPFC than for the Cyto-Spheres method (r = 0.725 and eta = 0.3). Multiple linear regression analysis showed that CD4(+) cell counts by the CyFlow method were a stronger predictor for results of DPFC than those of the Cyto-Spheres method (r(2) = 0.864 and r(2) = 0.552, respectively). When compared to DPFC the CyFlow method generated higher CD4(+) cell counts than the Cyto-Spheres method, as expressed by a higher median and mean difference (+70 and +90 cells for CyFlow, +28 and -1.4 cells for Cyto-Spheres)., Conclusion: Both, the manual Cyto-Spheres method and the CyFlow method can be used for the enumeration of CD4(+) cells in resource-limited settings. Under supervised conditions, the CyFlow method produced results more consistent with the reference method than the Cyto-Spheres method., (Copyright 2006 International Society for Analytical Cytology.)

Objective: Prevention-of-mother-to-child transmission (PMTCT) interventions based on single-dose nevirapine (NVP) are widely implemented in Africa, but strategies differ regarding how and when to administer the drug to women and infants. The aim of this study was to analyze the outcome of different strategies with regard to NVP intake in pregnant women and their infants in Tanzania and Uganda., Methods: In an observational study carried out between March 2002 and December 2004, we compared a directly observed NVP administration strategy in Tanzania (supervised NVP intake for women and infants at a health unit) and a semi-observed administration strategy (self-administered NVP for women at home and supervised intake for infants at a health unit) in Uganda., Results: The proportions of HIV-positive women accepting receipt of NVP from the health units were similar in the 2 countries (42.4% in Tanzania vs 45.6% in Uganda; P = .06). NVP intake in infants was significantly higher in Tanzania than in Uganda (43.7% vs 24.1%; P < .001). In a multivariate analysis, maternal age above 25 years, secondary education, Catholic faith, and having undergone PMTCT counseling at a hospital were independently associated with infant NVP intake., Conclusion: In our settings, the directly observed administration strategy resulted in a higher NVP intake in infants. The semi-observed strategy, which implies that, after home delivery, the infant has to be presented to a health unit for NVP administration, was less successful.

Objective: Prevention-of-mother-to-child transmission (PMTCT) interventions based on single-dose nevirapine (NVP) are widely implemented in Africa, but strategies differ regarding how and when to administer the drug to women and infants. The aim of this study was to analyze the outcome of different strategies with regard to NVP intake in pregnant women and their infants in Tanzania and Uganda., Methods: In an observational study carried out between March 2002 and December 2004, we compared a directly observed NVP administration strategy in Tanzania (supervised NVP intake for women and infants at a health unit) and a semi-observed administration strategy (self-administered NVP for women at home and supervised intake for infants at a health unit) in Uganda., Results: The proportions of HIV-positive women accepting receipt of NVP from the health units were similar in the 2 countries (42.4% in Tanzania vs 45.6% in Uganda; P = .06). NVP intake in infants was significantly higher in Tanzania than in Uganda (43.7% vs 24.1%; P > .001). In a multivariate analysis, maternal age above 25 years, secondary education, Catholic faith, and having undergone PMTCT counseling at a hospital were independently associated with infant NVP intake., Conclusion: In our settings, the directly observed administration strategy resulted in a higher NVP intake in infants. The semi-observed strategy, which implies that, after home delivery, the infant has to be presented to a health unit for NVP administration, was less successful.

Anaplastic large cell lymphomas (ALCL) are characterised by the presence of CD30-positive large cells, which usually are of T-cell type. Based on the presence or absence of translocations involving the anaplastic lymphoma kinase (ALK) locus, ALCL cases can be divided into two groups. To gain more insight in the biology of ALCL, we applied serial analysis of gene expression (SAGE) on the Karpas299 cell line and identified 25 up- and 19 downregulated genes. Comparison of the differentially expressed genes with DNA copy number changes in Karpas299 revealed that two overexpressed genes, S100A10 and S100A11, were located in an amplicon suggesting that the increased mRNA levels were caused by DNA amplification. Quantitative reverse transcription polymerase chain reaction on 5 ALCL cell lines and 12 ALCL tissues confirmed the SAGE data for 13 out of 14 up- and one out of four downregulated genes. Immunohistochemical staining confirmed the presence of S100A10, a calcium-binding protein, in three out of five ALK+ and all 7 ALK- ALCL cases. S100A11 staining was confirmed in all ALK+ and six of seven ALK- ALCL cases. Three of the upregulated genes represented calcium-binding proteins, which suggest that altered intracellular signaling might be associated with the oncogenesis of ALCL.

In a previous study we demonstrated high expression of the non-coding BIC gene in the vast majority of Hodgkin's lymphomas (HLs). Evidence suggesting that BIC is a primary microRNA transcript containing the mature microRNA-155 (miR-155) as part of a RNA hairpin is now accumulating. We therefore analysed HL cell lines and tissue samples to determine whether miR-155 is also expressed in HL. High levels of miR-155 could be demonstrated, indicating that BIC is processed into a microRNA in HL. Most non-HL subtypes were negative for BIC as determined by RNA-ISH. However, in diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBL), significant percentages of positive tumour cells were observed in 12/18 and 8/8 cases. A higher proportion of tumour cells were positive for BIC in DLBCL with activated B cell-like phenotype than in DLBCL with germinal centre B cell-like phenotype. Differential BIC expression was confirmed by qRT-PCR analysis. Northern blot analysis showed expression of miR-155 in all DLBCL and PMBL derived cell lines and tissue samples analysed. In summary, we demonstrate expression of primary microRNA BIC and its derivative miR-155 in HL, PMBL and DLBCL., (Copyright (c) 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Anaplastic large cell lymphomas (ALCLs) can be subdivided into two subgroups on the basis of their expression of the ALK protein. ALK protein expression leads to activation of signal transducer and activator of transcription (STAT) 3, which is more commonly expressed in ALK-positive than in ALK-negative tumours. Activated STAT3 leads to the induction of several genes such as Mcl-1, Bcl-2 and Bcl-X(L), and tissue inhibitor of metalloproteinase (TIMP)-1. In this study, we analysed TIMP-1 expression in five ALCL cell lines and 11 tumours by quantitative RT-PCR and immunohistochemistry. We identified high-level TIMP-1 expression by RT-PCR in three ALK-positive ALCL-derived cell lines and in all ALK-positive ALCLs, whereas ALK-negative ALCLs generally demonstrated a lower level of TIMP-1 expression. Concordant with these results, we observed TIMP-1 immunostaining in all ALK-positive ALCLs and in only two of six ALK-negative ALCLs. No relationship was observed between the levels of ALK and TIMP-1 expression in the ALK-positive tumours. STAT3 expression levels were similar in all ALCL samples. Double staining with either CD30 or CD68 demonstrated that TIMP-1 expression was restricted to macrophages in the majority of TIMP-1-positive tumours. Expression of the TIMP-1 substrate MMP-2 was more prominent in ALK-negative tumours, while MMP-9 levels were low in all cases. Expression levels of IL-6 and TGF-beta1, which are cytokines known to induce TIMP-1, were higher in ALK-negative ALCLs and moderate in ALK-positive tumours. No clear relationship was observed between IL-10 expression and ALK positivity. Overall, no correlation was seen in ALCLs between the expression of TIMP-1 and that of cytokines that induce TIMP-1. Lack of TIMP-1 expression in the tumour cells of ALK-positive ALCLs argues against a direct role for ALK-induced activation of STAT3 in the regulation of TIMP-1 expression in ALCL., (Copyright 2005 Pathological Society of Great Britain and Ireland)

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.

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.

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.

HIV and tropical infections affect each other mutually. HIV infection may alter the natural history of tropical infectious diseases, impede rapid diagnosis, or reduce the efficacy of antiparasitic treatment. Tropical infections may facilitate the transmission of HIV and accelerate progression from asymptomatic HIV infection to AIDS. This article reviews data on known interactions for malaria, leishmaniasis, human African trypanosomiasis, Chagas' disease, schistosomiasis, onchocerciasis, lymphatic filariasis, and intestinal helminthiases.

During an epidemic of cutaneous leishmaniais in northeast Brazil a prospective study was conducted in order to assess clinical, parasitological and immunological features of cutaneous leishmaniasis in a representative sample of patients. A general examination was done and demographic and anthropometric data as well as numbers, duration, location, size, consistency and tenderness of enlarged lymph nodes and lesions recorded. Hematologic and biochemical parameters as well as Leishmania-specific antibodies were measured in the blood. Lymph node aspirations were performed to detect Leishmania-specific DNA by PCR. Cellular immunity was assessed by a leishmanin skin test. Ninety-seven patients with cutaneous leishmaniasis presented with a total of 181 lesions and 127 enlarged regional lymph nodes. In 36% the lymph node enlargement had been observed 2 days to 2 months before the development of a lesion. In 59% the lesions were followed by lymphadenopathy by 3 days to 3 months. The skin test was positive in all patients tested. PCR of the lymph node aspirates was positive in 63.2%. No significant correlations were found between positive PCR results, antibody levels or number of lesions. In northeast Brazil cutaneous leishmaniasis due to Leishmania braziliensis is constantly accompanied by an extensive lymphadenopathy. This may help to identify patients when lesions are still small or have not even developed and lymphadenopathy is the only clinical sign.

Tungiasis (caused by the sand flea Tunga penetrans) is hyperendemic in many resource-poor communities in Brazil. To understand transmission dynamics of this parasitic skin disease in a typical endemic area, a longitudinal study was carried out in a slum in Fortaleza in northeastern Brazil. In a door-to-door survey, the population of a randomly selected area (n = 1,460) was examined on four occasions for the presence of embedded sand fleas. Prevalence rates were 33.6% in March (rainy season), 23.8% in June (end of the rainy season), 54.4% in September (peak of the dry season), and 16.8% in January (begin of the rainy season). Tungiasis was more common in males than in females. The intensity of infestation was correlated with the prevalence. The study shows that prevalence of tungiasis and parasite burden vary significantly during the year with a peak in the dry season. These findings have important consequences for the design of control measures.

Since Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) generally have immunoglobulin gene rearrangements, they are considered to be of B-cell origin. One of the characteristics of HRS cells is a prominent production of various cytokines and chemokines. Cytokine production is generally driven by expression of T-cell transcription factors (TFs). Only limited information is available on the expression of T-cell TFs in HL. Expression of four T-cell TFs and the target cytokine spectrum of these TFs were analyzed in six HL and three large B-cell lymphoma (LBCL) cell lines using quantitative PCR. ERM expression was observed in all HL and LBCL cell lines. Out of HL cell lines, T-bet was expressed in five, GATA-3 in four, and c-Maf in two cell lines. Immunohistochemistry in HL tissues revealed that in 11 of 12 (92%) of the classical HL cases HRS cells were GATA-3 and/or T-bet positive. In three of six cases of nodular lymphocyte predominance type of HL, the neoplastic cells were T-bet positive. Overall, the T-cell TF and cytokine profiles of the HL cell lines showed a considerable degree of consistency. The expression of T-cell TFs may explain the production of various cytokines by HL cell lines and HRS cells.

Galectin-1, a beta-galactoside binding protein that can occur as both a monomer and a homodimer, binds to leucocyte membrane antigens such as CD7, CD43, and CD45, and has immune-regulatory functions in several animal models of autoimmune disease. However, its mechanism of action is only partially understood. In this study, a marked increase in IL-10 mRNA and protein levels was demonstrated in non-activated and activated CD4(+) and CD8(+) T-cells, following treatment with a high concentration (dimeric form), but not a low concentration (monomeric form), of recombinant galectin-1 protein. IL-10 is known to suppress TH1 type immune responses and upregulation of IL-10 may thus contribute to the immune-regulatory function of galectin-1. Galectin-1 was strongly expressed on the endothelial cells of human kidney allografts, suggesting a role in the regulation of immune responses in transplantation. Administration of high concentrations of galectin-1 may be a useful tool in the treatment of T-cell-mediated diseases., (Copyright (c) 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Tungiasis is caused by the penetration of the female sand flea Tunga penetrans into the skin of its host. This parasitic skin disease is almost invariably associated with an intense inflammation around embedded fleas, the underlying mechanisms being unknown. A study was undertaken to determine whether Wistar rats can be used as an animal model to assess cytokine kinetics during the natural course of the infection. Laboratory-raised Wistar rats were exposed in cages put on the soil in an area with high human attack rates. Rats were examined daily and blood samples were taken before exposure and at 2, 6, 10, 13, 16 and 20 days after flea penetration. TNF-alpha, IL-1 beta, IFN-gamma, IL-4, IL-10 and CINC (a rat cytokine- induced neutrophil chemoattractant and member of the IL-8 family) were determined by enzyme immunoassay. The results showed an increasing serum concentration of TNF-alpha and IL-1 beta 10-13 days after penetration and a rapid increase in IL-4 2 days after fleas became embedded. During the natural course of the infection, the ratio of the serum concentration of TNF-alpha to that of IL-10 decreased, indicating a relative increase in the secretion of the anti-inflammatory cytokine. The treatment of lesions with silicone oil abrogated the natural disease course and changed the pattern of cytokine secretion. We conclude that the Wistar rat is an appropriate model to study immune responses in tungiasis.

Background: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening., Methods: MultiCode PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in approximately 3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns., Results: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls., Conclusions: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.

The immunochromatographic test (ICT) for the rapid diagnosis of malaria has been marketed for several years. In a study in which three Centres of Tropical Medicine participated and data were pooled, performance of the test varied considerably when comparing the results between each centre. The sensitivity of ICT in 2,343 patients tested in our services was 100% and the specificity 99.74%. Moreover, two patients with a positive ICT would initially have been missed by expert microscopy, with Plasmodium falciparum malaria being confirmed microscopically some hours later. The principal reasons for the better performance of the test in our series appear to be blood collection in EDTA vials and considerable experience with handling and interpreting the ICT test.