Description and phylogenetic analyses of ribosomal transcription units from species of Fasciolidae (Platyhelminthes: Digenea).

Many members of Fasciolidae are common trematodes in cattle, buffaloes, sheep, elephants, pigs, with some capable of infecting humans also. In this study, the complete or near-complete sequences of ribosomal transcription unit (rTU or rDNA), each of Fasciola hepatica (Australia), Fascioloides jacksoni (Sri Lanka), Fasciolopsis buski (Vietnam) and three isolates of F. gigantica (Vietnam), were obtained and characterized. The full length of rDNA for each F. hepatica, 'hybrid' Fasciola sp., Fas. jacksoni and Fa. Buski, was 7657 bp, 7966 bp, 7781 bp and 8361 bp, with the complete intergenic spacer region (IGS) (862 bp, 1170 bp, 987 bp and 561 bp), respectively. The rDNA of two 'pure' F. gigantica isolates from Vietnam was 6794 bp with unsequenced IGS. For 28S rRNA genes the Fasciola spp. are equal, 1958 bp for 18S, 160 bp for 5.8S, 3863 bp and 454 bp for ITS1 but ITS2 differ by one nucleotide (Thymine) (359 or 360 bp). The ITS1 of the sensu lato Fa. buski has some distinguishable features, 286 bp for ITS2, 3862 bp for 28S and four repeat units of 356-361 bp each found in ITS1. The 28S rDNA analysis showed the lowest level of divergence (0-0.57%) between F. hepatica and F. gigantica and higher (2.23-2.62%) and highest (6-6.42%) for Fas. jacksoni and Fasciolopsis, respectively. The tree of 43 strains/species clearly produced a well-supported phylogeny, where 18 fasciolids consistently grouped, forming a discrete Fasciolidae clade, distinct from Philophthalmidae, Echinostomatidae and Echinochasmidae in Echinostomatoidea. Fascioloides jacksoni is outside Fasciola spp.: basal with Fas. magna, as previously demonstrated.

Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines.

Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n=145) and mothers living in an urban area (n=147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45-48%), whereas the majority of the remaining samples had low antibody titers (2-16). Despite these low titers, the breast milk still reduced vaccine strain titers (2×10(6) plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (P<0.05) in rural infants over time suggests a continuous exposure to circulating RV. These results contribute to the understanding of the potential interference of breast milk with RV vaccine efficacy and immunogenicity in Vietnamese infants.

Cost effectiveness of duloxetine in the treatment of fibromyalgia in the United States.

OBJECTIVE: To evaluate the cost effectiveness of duloxetine when considered as an alternative treatment for patients in the United States (US) being treated for fibromyalgia pain. RESEARCH DESIGN AND METHODS: A Markov model was used to evaluate the economic and clinical advantages of duloxetine in controlling fibromyalgia pain symptoms over a 2-year time horizon. A base-case treatment sequence was adopted from clinical guidelines, based on tricyclic antidepressants, serotonin-norepinephrine reuptake inhibitors, anticonvulsants, and opioids. Treatment response was modeled using changes from baseline in pain severity, and response thresholds: full response (at least a 50% change), response (30-49% change), and no response (less than a 30% change). Clinical efficacy and discontinuation data were taken from placebo- and active-controlled trials identified in a systematic literature review and mixed-treatment comparison. Utility data were based on EQ-5D data. MAIN OUTCOME MEASURES: Additional symptom-control months (SCMs), defined as the amount of time at a response level of 30% or less, and quality-adjusted life-years (QALYs) over a 2-year time horizon. RESULTS: For every 1000 patients, first-line duloxetine resulted in an additional 665 SCMs and 12.3 QALYs, at a cost of $582,911 (equivalent to incremental cost-effectiveness ratios [ICERs] of $877 per SCM and $47,560 per QALY). Second-line duloxetine resulted in an additional 460 SCMs and 8.7 QALYs, at a cost of $143,752 (equivalent to ICERs of $312 per SMC and $16,565 per QALY). LIMITATIONS: Response data for TCAs are limited to 30% improvement levels, reported trials are small, and have low placebo response rates. The model necessarily assumes that response rates are independent of placement in the treatment sequence. CONCLUSIONS: The results suggest that the introduction of duloxetine into the standard treatment sequence for fibromyalgia not only provides additional patient benefits, reflected by time spent in pain control, but also is cost effective when compared with commonly adopted thresholds.

Private demand for cholera vaccines in Hue, Vietnam.

OBJECTIVES: This study aims to measure the private demand for oral cholera vaccines in Hue, Vietnam, an area of relatively low endemicity of cholera, using the contingent valuation method. METHODS: Interviews were conducted with either the head of household or spouse in 800 randomly selected households with children less than 18 years old. Respondents were asked whether they would purchase an oral cholera vaccine with different levels of effectiveness and durations of effectiveness (both for themselves and for other household members) at a specified price. RESULTS: The median respondent willingness to pay for 50% effective/3-year vaccine was estimated to be approximately $5, although 17% of the study sample would not pay for a cholera vaccine. The median economic benefit to a household of vaccinating all household members against cholera, as measured by its stated willingness to pay, was estimated to be $40 for a vaccine with these attributes. CONCLUSIONS: The perceived private economic benefits of a cholera vaccine were high, but not evenly distributed across the population. A minority of the people in Hue place no value on receiving a cholera vaccine.

Cost effectiveness of duloxetine in the treatment of diabetic peripheral neuropathic pain in the UK.

OBJECTIVE: The objective of this analysis was to evaluate the cost-effectiveness of duloxetine when considered as an additional treatment option for UK-based patients suffering from diabetic peripheral neuropathic pain. RESEARCH DESIGN AND METHODS: A decision-analytic model was used to represent the sequential management of patients with diabetic peripheral neuropathic pain. The standard UK treatment strategy was defined as first-line tricyclic antidepressants (amitriptyline), second-line anticonvulsants (gabapentin) and lastly an opioid-related treatment. The cost-effectiveness of duloxetine was evaluated as an additional first, second, third or fourth-line therapy over a 6-month treatment period for a cohort of 1000 patients. Treatment response was modelled based on changes from baseline pain severity using a standard 11-point pain scale (0-10); full response (>or= 50% change), partial response (30-49%) and no response (< 30%). The model was populated with efficacy and discontinuation data using indirect comparisons of treatment efficacy based on relative effects to a common placebo comparator. RESULTS: The second-line use of duloxetine resulted in cost savings of pound 77,071 for every 1000 treated patients, with an additional 29 patients achieving a full pain response when compared to standard UK treatment. Additional quality-adjusted life years (QALYs) were achieved at 1.88 QALYs per 1000 patients. CONCLUSIONS: This UK-based economic model suggests that second-line use of duloxetine is a beneficial and cost-effective treatment strategy for diabetic peripheral neuropathic pain.

Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1.

Small-cell lung cancers (SCLCs) initially respond to chemotherapy, but are often resistant at recurrence. The non-steroidal anti-inflammatory drug indomethacin is an inhibitor of multidrug resistance protein 1 (MRP1) function. The doxorubicin-resistant MRP1-overexpressing human SCLC cell line GLC(4)-Adr was highly sensitive for indomethacin compared with the parental doxorubicin-sensitive line GLC(4). The purpose of this study was to analyse the relationship between hypersensitivity to indomethacin and MRP1 overexpression. The experimental design involved analysis of the effect of MRP1 downregulation on indomethacin-induced cell survival and apoptosis in GLC(4)-Adr and GLC(4), using siRNA. In addition the effect of indomethacin on glutathione levels and mitochondrial membrane potential was investigated. Small interfering RNAs directed against MRP1 reduced MRP1 mRNA levels twofold and reduced efflux pump function of MRP1, which was reflected by a 1.8-fold higher accumulation of MRP1 substrate carboxyfluorescein, in si-MRP1 versus si-Luciferase-transfected GLC(4)-Adr cells. Multidrug resistance protein 1 downregulation decreased initial high apoptosis levels 2-fold in GLC(4)-Adr after indomethacin treatment for 24 h, and increased cell survival (IC(50)) from 22.8+/-2.6 to 30.4+/-5.1 microM following continuous indomethacin exposure. Multidrug resistance protein 1 downregulation had no effect on apoptosis in GLC(4) or on glutathione levels in both lines. Although indomethacin (20 microM) for 2 h decreased glutathione levels by 31.5% in GLC(4)-Adr, complete depletion of cellular glutathione by L-buthionine (S,R)-sulphoximine only resulted in a small increase in indomethacin-induced apoptosis in GLC(4)-Adr, demonstrating that a reduced cellular glutathione level is not the primary cause of indomethacin-induced apoptosis. Indomethacin exposure decreased mitochondrial membrane potential in GLC(4)-Adr cells, suggesting activation of the mitochondrial apoptosis pathway. Indomethacin induces apoptosis in a doxorubicin-resistant SCLC cell line through an MRP1-dependent mechanism. This may have implications for the treatment of patients with MRP1-overexpressing tumours.

Estimated prevalence of peripheral neuropathy and associated pain in adults with diabetes in France.

OBJECTIVE: To estimate the point prevalence of diabetic peripheral neuropathy (DPN) and pain associated with DPN (pDPN) in French adults with diabetes and compare severity of symptoms across demographic subpopulations. DESIGN: The participant-administered portion of the Michigan Neuropathy Screening Instrument (MNSI) and selected items of the Brief Pain Inventory (BPI) formed part of a computer-aided telephone survey posed to a representative, random sample of French households from March 1, 2005 to April 30, 2005. Questions from the MNSI and the BPI were used to assess the point prevalence of DPN and pDPN in French adults with self-reported diabetes. RESULTS: The mean age of the study sample was 68 years (SD = 15), the mean duration of diabetes was 15 years (SD = 12) and 56% of participants were female. The prevalence rates of DPN and pDPN in French adults with diabetes were 11 and 8%, respectively. The average age and diabetes duration of participants with DPN and pDPN were not different from participants in the total sample. Among those participants with pDPN, 35% classified their pain as severe, 49% as moderate, and 17% as mild. The prevalence of DPN was higher in participants with type 1 diabetes (14%) than those with type 2 (9%). Among participants with DPN, 88% with severe pain received pain treatment compared to 71% with moderate pain and 58% with mild pain. The most significant limitation of this study is the lack of validation for administering only a portion of the MNSI, but other limitations include the imprecision associated with self-reported questionnaires, a survey sample that does not include participants with undiagnosed diabetes, and a bias toward elderly participants. CONCLUSION: This study concluded that 8% of participants with diabetes in France had pDPN.

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells.

Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC4-Adr and its parental doxorubicin-sensitive line GLC4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC4-Adr. In addition, GLC4-Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC4-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC4-Adr but not in GLC4 cells. Surprisingly, in GLC4-Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC4-Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC4. Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.

Differential modulation of the TRAIL receptors and the CD95 receptor in colon carcinoma cell lines.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L) are potent inducers of apoptosis in various tumour cell types. Death receptors DR4 and DR5 can induce and decoy receptors DcR1 and DcR2 can inhibit TRAIL-mediated apoptosis. The study aim was to investigate whether anticancer agents can modulate similarly TRAIL-receptor and CD95 membrane expression and TRAIL and CD95L sensitivity. Three colon carcinoma cell lines (Caco-2, Colo320 and SW948) were treated with 5-fluorouracil (5-FU), cisplatin or interferon-gamma. TRAIL-receptor and CD95 membrane expression was determined flow cytometrically. Sensitivity to TRAIL or CD95L agonistic anti-CD95 antibody was determined with cytotoxicity and apoptosis assays. SW948 showed highest TRAIL sensitivity. The protein synthesis inhibitor cycloheximide decreased FLICE-like inhibitory protein levels in all cell lines, and the TRAIL-resistant cell lines Caco-2 and Colo320 became sensitive for TRAIL. Exposure of the cell lines to 5-FU, cisplatin and interferon-gamma left TRAIL-receptor membrane expression and TRAIL sensitivity unaffected. CD95 membrane expression and anti-CD95 sensitivity was, however, modulated by the same drugs in all lines. Cisplatin and interferon-gamma raised CD95 membrane levels 6-8-fold, interferon-gamma also increased anti-CD95 sensitivity. These results indicate that the CD95 and TRAIL pathways use different mechanisms to respond to various anticancer agents. Induced CD95 membrane upregulation was associated with increased anti-CD95 sensitivity, whereas no upregulation of TRAIL-receptor membrane expression or TRAIL sensitisation could be established. For optimal use of TRAIL-mediated apoptosis for cancer therapy in certain tumours, downregulation of intracellular inhibiting factors may be required.

Molecular fingerprinting of Mycobacterium tuberculosis strains isolated in Vietnam using IS6110 as probe.

SETTING: Northern and Southern areas of Vietnam. OBJECTIVE: To study the correlation between DNA fingerprinting of 168 Mycobacterium tuberculosis strains isolated from patients with a particular historical past (political separation of Vietnam for 20 years) and data about geographical origin, drug susceptibility, HIV infection and BCG vaccination status. METHODS: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by Southern hybridization of Pvull-digested chromosomal DNA. RESULTS: The number of IS6110 copies for the 168 strains ranges from 0 to 23. Strains originating from the North or the South differ strongly with respect to the number of copies of IS6110. Indeed, the strains originating from the north have predominantly from 3 to 14 IS6110 copies while the southern strains have predominantly from 15 to 23 IS6110 copies. Furthermore, strains isolated in the North are dispersed into 6 groups whereas 80% of the strains isolated in the South form a single group. Moreover, the prevalence of drug resistance is higher in strains isolated in the South than in the North. No noticeable correlation is observed between RFLP patterns, drug susceptibility, or HIV infection. CONCLUSION: The IS6110 fingerprints of 168 M. tuberculosis strains isolated in Vietnam showed a high range of polymorphism. Only a few strains have been found with no IS6110 (1.8%). The differences between the strains from the North and South, having more than six IS6110, suggests that they derived from ancestral strains that would be distinguishable by the number of IS6110 and their transposition sites throughout the genome. The genomic structure of the population of strains from South Vietnam resembles that of the Beijing strain population. This could account for a similar evolution of M. tuberculosis due to a selection by BCG-induced immunity in the two populations.

Long-term follow-up of a modified Anton-Hartrampf nipple reconstruction.

This study was performed to determine the degree of shrinkage over time in nipple projection after reconstruction. Nipple-areolar reconstruction was performed using the modified Anton-Hartrampf technique, and pigmentation was achieved with tattooing. This study looked at 28 consecutive patients with nipple reconstruction performed at The Milton S. Hershey Medical Center of the Penn State Geisinger Health Systems between September 1989 and November 1993. Two patients were lost to follow-up and 3 patients died of breast cancer. Thus, 23 patients and a total of 32 nipples were investigated. Initial measurements of nipple projection were taken 2 weeks postoperatively. Patients were followed an average of 38.7 months (range, 11-66 months). Ten patients (18 nipples) had tissue expansion and implantation for breast mound reconstruction. Thirteen patients (14 nipples) had autologous breast mound reconstruction. The mean decrease in projection of the tissue expansion and implantation group was 76.7+/-9.7%. The mean decrease in projection of the autologous reconstruction group was 64.3+/-12.1%. The mean decrease in projection for the entire group was 71.3+/-21.9%. Comparison between the two groups using a two-sample t-test showed p = 0.0047. The authors concluded that there is a significant reduction in nipple projection over time using the modified Anton-Hartrampf technique regardless of the type of breast mound reconstruction. In addition, their results also indicated that nipple projection on the breast mound reconstructed with an autologous musculocutaneous flap technique achieved a better long-term outcome. This study is potentially helpful in planning the initial size of the reconstructed nipple papule to match the opposite normal nipple. Additional studies need to be performed on other types of nipple-areolar reconstruction.

Combined cytotoxic effects of tumor necrosis factor-alpha with various cytotoxic agents in tumor cell lines that are drug resistant due to mutated p53.

Several studies suggest that tumor necrosis factor-alpha (TNF) is able to overcome drug resistance in tumors. Whether TNF is able to do so in tumor cell lines that are drug resistant due to a mutation in the tumor suppressor gene p53 is unclear. Therefore, we studied the in vitro cytotoxic effects of TNF combined with various cytotoxic agents in a model consisting of a human ovarian cancer cell line containing endogenous wild-type p53 (wtp53) and sublines that were made drug resistant against various cytotoxic agents by transfection of several forms of mutated p53 (mtp53). Using the microculture tetrazolium assay, the cytotoxic effects of TNF alone, the cytotoxic agents VM-26, melphalan, cisplatin, vinblastine, paclitaxel, and mitoxantrone, plus the combined effects of 10 ng/ml TNF added 30 min before various concentrations of the cytotoxic agents were established. Compared with the control cell line (A2780/cmv), two cell lines transfected with mtp53 (A2780/m248 and A2780/m273) showed increased resistance against several cytotoxic agents but also an enhanced sensitivity to TNF. Interaction of TNF with the cytotoxic drugs was additive in the drug-sensitive control cell line as well as in the drug-resistant sublines. However, because of the increased sensitivity of A2780/m248 to TNF at the dose used for the combinations, the combination of TNF with several cytotoxic drugs reduced the level of resistance in A2780/m248 compared with the control cell line A2780/cmv. In conclusion, this study shows that addition of TNF can ameliorate resistance to cytotoxic agents in a subline that is drug-resistant because of mutated p53. This reduction in resistance by TNF is not due to synergistic interaction, but to collateral sensitivity to TNF.

A histological and immunohistochemical study on chronic irritant contact dermatitis.

BACKGROUND: Chronic irritant contact dermatitis (CICD) is characterized by erythema, scaling, hyperkeratosis, chapping and fissures. It may be the result of skin damage evoked by the cumulative effect of a variety of irritant stimuli. The diagnosis of CICD is made on basis of the patient's history and clinical features. No specific diagnostic tests are available. OBJECTIVE: The histopathologic and cell biological features of CICD have not been extensively studied. Here, we describe the histological and immunohistological changes in CICD. METHODS: Punch biopsies were taken from 11 patients with CICD for hematoxylin eosin and immunohistochemical stainings. Four skin biopsies of the palms of the hands of healthy volunteers served as controls. RESULTS: The histopathologic pattern was characterized by different grades of hyperkeratosis, parakeratosis, spongiosis, exocytosis, acanthosis, and mononuclear perivascular infiltrates. Mitotic activity, as measured by Ki-67-staining in the epidermis, was increased fourfold in involved skin as compared with normal skin. Involucrin, a structural protein of the cornified envelope, was expressed from the stratum granulosum throughout the stratum spinosum in all patients with CICD and was upregulated in comparison with normal skin. Epidermal fatty-acid binding protein (E-FABP), a terminal differentiation marker, was proportionally unaltered in the CICD as compared with the normal skin and was localized from the stratum granulosum to the upper layers of the stratum spinosum. Cytokeratin 16, a differentiation marker expressed in hyperproliferative epidermis, was markedly increased from the stratum granulosum throughout the stratum spinosum in 5 out of 11 patients with CICD. Skin-derived antiproteinase (SKALP)/elafin, a proteinase inhibitor expressed in inflamed epidermis, was only detected within the stratum granulosum in 3 out of 11 patients. CONCLUSION: We conclude that CICD is clinically characterized by features of a chronic dermatitis and, at the histological level, by inflammatory changes, epidermal hyperproliferation and altered differentiation.

Effect of a topical corticosteroid, a retinoid and a vitamin D3 derivative on sodium dodecyl sulphate induced skin irritation.

Exposure of the skin to sodium dodecyl sulfate (SDS) leads to disruption of barrier and skin irritation. We used repetitive short exposure to a low molarity SDS solution as an in vivo model to mimic the development of irritant contact dermatitis. In this model, we studied clinical (erythema), functional (transepidermal water loss(TEWL)) and cell biological changes. 24 healthy volunteers were patch tested with SDS (0.2%) for 4 h a day for 5 consecutive days. After removal of the patches, the exposed sites were treated 1 X daily either with a topical corticosteroid (triamcinolon acetonide cream 0.05%), a retinoid (tretinoin cream 0.025%), or a vitamin D3 derivative (calcipotriol ointment 50 micrograms/g). Irritant reactions were assessed by erythema scoring and measurement of barrier function with TEWL up to 14 days after the first challenge. Skin biopsies were taken for cell biological changes at day 4. Vehicle-treated sites served as controls. Repetitive exposure of human skin to SDS resulted in a gradual increase in erythema scoring and TEWL associated with the upregulation of proliferative cells as measured by the expression of Ki-67-antigen and of differentiation markers, visualized by increased expression of involucrin and epidermal-fatty-acid binding protein (E-FABP). Skin irritation as assessed by erythema scoring and TEWL was not significantly suppressed by triamcinolone cream. However, a significant reduction of the number of cycling keratinocytes and a decrease in involucrin positive cell layers was observed in this group. Neither treatment with calcipotriol ointment nor with tretinoin cream induced improvement of skin irritation as judged by visual scoring and TEWL. In contrast to steroid treatment, no significant effect of calcipotriol ointment or tretinoin cream treatment was observed with regard to the number of cycling cells and differentiation markers. Further studies are needed to assess whether treatment with topical corticosteroids is an effective modality in skin irritation and irritant contact dermatitis.

Changes in epidermal proliferation and differentiation in allergic and irritant contact dermatitis reactions.

Allergic and irritant contact dermatitis are similar clinically, histologically and on immunohistochemistry. In the present investigation, we assessed whether study of the recruitment of cycling epidermal cells, and the expression of keratin 16 and involucrin, are of use in differentiating between the response to contact allergens and the response to the irritant detergent sodium lauryl sulphate. Both allergic and irritant challenges induced epidermal proliferation, and the expression of keratin 16 and involucrin, but the dynamics were different. Two and 3 days after challenge, a highly significant difference between the allergic and irritant reactions was observed with respect to involucrin expression assessed by MON-150 staining.