Not Treated Research Articles

Evaluation of antimicrobial and non-steroidal anti-inflammatory treatments for BRD on health and welfare in fattening bulls: a cross-sectional study

Our study aimed to evaluate the effect of different treatments for BRD on health and welfare in fattening bulls. A total of 264 bulls were enrolled. Welfare was assessed on day 2 (T0) and day 15 (T1) after arrival. A decrease in the welfare level was observed from T0 to T1. All bulls were inspected clinically at T0 and T1 revealing an increase of skin lesions and lameness in T1. In both periods, a high incidence of respiratory disease was observed. A prevalence of 79.55% and 95.45% of Mycoplasma bovis using RT-PCR and culture at T0 and T1 respectively was observed. Blood samples were collected for haematology at T0 and T1. At T0, 36 animals were individually treated for BRD with an antimicrobial (IT), 54 received a metaphylactic treatment with tulathromycin (M), 150 received a metaphylactic treatment with tulathromycin plus a second antimicrobial (M + IT) whereas 24 were considered healthy and therefore not treated (NT). Additionally, 128 were treated with a non-steroid anti-inflammatory (NSAID). Neutrophils of M + IT were significantly higher than groups NT and M and the lymphocytes of M + IT were significantly lower than that of IT. White blood cells, neutrophils and N/L ratio of animals treated with an NSAID was significantly higher than that not treated. Lung inspection of 172 bulls at the abattoir indicated that 92.43% presented at least one lung lesion. A statistically significant effect of the NSAID treatment on the lung lesions was observed. Our findings indicate that BRD was a major welfare and health concern and evidence the difficulties of antimicrobial treatment of M. bovis.

Exploiting the microbiota of organic and inorganic acid-treated raw poultry products to improve shelf-life.

Targeted amplicon sequencing of the 16S rRNA delineates the complex microbial interactions that occur during food spoilage, providing a tool to intensively screen microbiota response to antimicrobial processing aids and interventions. The current research determines the microbiota and spoilage indicator (total aerobes and lactic acid bacteria; LAB) response to inorganic and organic antimicrobial intervention use on the shelf-life of fresh, never-frozen, skin-on, bone-in chicken wings. Wings (n=200) were sourced from local processor and either not treated (NT) or treated with 15-s dips of tap water (TW), organic (peracetic acid; PAA), inorganic acids (sodium bisulfate; SBS), and their combination (SBS + PAA). Wings were stored (4°C) and rinsed in neutralizing Buffered Peptone Water (BPW) for 1 min on d 0, 7, 14, and 21 post-treatment. Spoilage indicators, aerobic mesophiles and LAB, were quantified from rinsates. Genomic DNA of d 14 and 21 rinsates were extracted, and V4 of 16S rRNA gene was sequenced. Sequences were analyzed using QIIME2.2019.7. APC and LAB counts were reported as Log10 CFU/g of chicken and analyzed in R Studio as a General Linear Model using ANOVA. Pairwise differences were determined using Tukey's HSD (P£0.05). Spoilage was indicated for all products by day 21 according to APC counts (>7 Log10 CFU/g); however, wings treated with SBS and SBS + PAA demonstrated a 7-day extended shelf-life compared to those treated with NT, TW, or PAA. The interaction of treatment and time impacted the microbial diversity and composition (p < 0.05), with those treated with SBS having a lower richness and evenness compared to those treated with the controls (NT and TW; p < 0.05, Q < 0.05). On d 14, those treated with SBS and SBS + PAA had lower relative abundance of typical spoilage population while having a greater relative abundance of Bacillus spp. (~70 and 50% of population; ANCOM p < 0.05). By d 21, the Bacillus spp. populations decreased below 10% of the population among those treated with SBS and SBS + PAA. Therefore, there are differential effects on the microbial community depending on the chemical intervention used with organic and inorganic acids, impacting the microbial ecology differently.

Safe and Efficient Engraftment of CRISPR-Based ELANE Mono-Allelic Knocked out HSCs in Mice: Evidence for a Novel Treatment for ELANE Neutropenia

ELANE-related severe congenital neutropenia (SCN) is a disorder wherein numerous heterozygous mutations in the ELANE gene lead to misfolding and mislocalization of mutant neutrophil elastase, resulting in the death of myeloid cells and block in neutrophil differentiation.Currently, SCN is treated with daily injections of granulocyte colony-stimulating factor (G-CSF), but patients on this therapy are at risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The only other effective therapy is allogeneic hematopoietic stem cell transplantation that may involve graft versus host disease and risk of serious infections. Thus, there is great interest in safe and efficient therapeutic alternatives.Emendo Biotherapeutics has developed a novel CRISPR-based (OMNI A1) ex-vivo gene editing strategy that involves specific excision of disease-causing ELANE mutant allele. This treatment was tested on CD34 + HSCs from SCN patients and resulted in a significant improvement in the maturation of myeloid cells and their differentiation to normal functional neutrophils.To assess the feasibility and safety of this editing strategy, we conducted an in vivo study using human HSCs derived from two healthy donors that were engrafted into a NOD scid gamma (NSG) immunocompromised mice model. The study involved a single iv injection of HSCs that were either not-treated (NT), electroporated without delivery of CRISPR nuclease (mock) or edited with Emendo's novel nuclease, OMNI A1,(excision). Each group consisted of 6 NSG female mice and an additional group of 4 mice served as vehicle control (no HSCs). All animals were subjected to total body irradiation, 1 day prior to the treatment, at a dose of 250 cGy. Animals were subjected to retro-orbital blood collection, at 4 designated time points during the study (Figure A), for CD45 + cell counts using FACS. All animals were evaluated for systemic reactions, clinical signs and body weight changes during the study period. At the end of the study period, animals were subjected to peripheral blood (PB), bone marrow (BM) and spleen sampling. FACS analysis of CD45 + cells was performed on those samples to determine the engraftment efficiency. Furthermore, excision levels for ELANE were measured in the engrafted cells by droplet digital PCR (ddPCR). Finally, multilineage differentiation capacity of the engrafted cells was evaluated by FACS.Analysis of human CD45 + cells, at 20 weeks, showed comparable levels of engraftment of the not-treated, mock treated and excised HSCs in the mouse PB, BM and spleen (No statistically significant difference, Kruskal Wallis test) (Figure B). Excised cells were detected in the mouse BM and excision levels in some of the mice were comparable to those measured in the HSCs prior to engraftment (about 30-36%). Human-derived cells from the BM and spleen of mice engrafted with excised HSCs showed similar multilineage differentiation capacity as obtained in mice engrafted with either NT or mock-treated HSCs. Engrafted human CD45 + cells differentiated into neutrophils (CD66b +), myeloid cells (CD33 +), Lymphocytes: B-cells (CD19 +) and T-cells (CD3 +). In addition, a population of engrafted hematopoietic CD34 + cells was detected in the BM. Excised HSCs of both donors gave rise to all lineages tested, as efficient as the NT and the mock groups. Finally, engrafted mice showed no systemic reactions, abnormal clinical signs or loss of body weight.These data show that edited cells can be engrafted successfully, maintain their excision profile and populate the bone marrow. This study also supports the safety of the OMNI A1 therapeutic composition. [Display omitted] DisclosuresPovodovski: Emendo Biotherapeutic: Current Employment. Makaryan: Emendo Biotherapeutic: Research Funding. Sabo: Emendo Biotherapeutic: Research Funding. Dicken: Emendo Biotherapeutic: Ended employment in the past 24 months. Herman: Emendo Biotherapeutic: Current Employment. Emmanuel: Emendo Biotherapeutic: Current Employment. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

Chloroquine as a promising adjuvant therapy for type 1 Diabetes Mellitus

Chloroquine (CQ) and hydroxychloroquine, are promising anti-inflammatory drugs for the treatment of Diabetes mellitus (DM) to prevent associated complications. Therefore, this study evaluated the anti-inflammatory effects of CQ-free and CQ-incorporated polylactic acid nanoparticles (NPs) in the peripheral blood mononuclear cells (PBMCs) of patients with type 1 Diabetes mellitus (T1DM). In total, 25 normoglycemic individuals and 25 patients with T1DM aged 10–16 years were selected and glycemic controls evaluated. After cell viability assessed by MTT assay, T1DM PBMCs were subjected to a CQ concentration of 10 µM in three different conditions: not treated (NT), treated with CQ, and treated with CQ NPs. The cells were incubated for 48 h, and the mRNA expressions of cytokines IL1B, IFNG, TNFA, IL12, and IL10 were determined by relative quantification through real-time PCR at 24 h intervals. IL1B expression decreased in CQ and CQ NP-treated cells after 48 h (p < 0.001) and 24 h (p < 0.05) of treatment, respectively. IFNG and IL12 expressions significantly decreased (p < 0.001) in cells treated with CQ and CQ NPs at 24 and 48 h compared to NT. TNFA and IL10 expressions significantly decreased after 48 h (p < 0.001) and 24 h (p < 0.002), respectively, by both CQ and CQ NPs treatment. Despite being a preliminary in vitro study, CQ has anti-inflammatory activity in the primary cells of T1DM patients and could represent an alternative and adjuvant anti-inflammatory therapy to prevent diabetes complications.

Abstract 4410: miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization

Abstract Background: Cancer stem cells (CSCs) have inherent or developed drug resistance for which chemotherapy fails to eliminate all tumor cells, a property that is critically contributing to recurrence and metastasis. Here, we combine miR-34a, a well-known tumor suppressor miRNA that controls proliferation, apoptosis and self-renewal, with Paclitaxel (PTX), a commonly used first-line chemotherapy drug. Based on our preliminary observations (Bonetti et al., manuscript under revision), we hypothesized a combinatorial approach, coupling miRNA- and chemo- treatment, aimed at reducing CSCs in vitro and recurrence in vivo using triple-negative breast cancer (TNBC) as a model. Methodology: As a model, we exploited SUM159pt cells (TNBC mesenchymal-like, with CSC features) to mimic aggressive breast cancer, which harbor a vector for pLUC, which is induced by luciferin in vivo, to monitor tumor growth in real time. Furthermore, we used i) pSlik34a, with tunable expression of miR-34a by doxycycline; ii) treatment with PTX to select for resistant cells (RC); and combined the two, by treating RC with doxycycline. CSC content was evaluated by ALDH (Aldehyde Dehydrogenase) activity, sphere-forming efficiency (SFE) and qRT-PCR of known markers. A similar approach was followed for in vivo studies, with NSG female injected through tail vein injection with 500,000 SUM159/pLUC/pSlik34a cells, and separated into three groups: not treated (NT), PTX alone, and PTX+34a. Results: As observed for aggressive breast cancer, treatment of SUM159 with PTX generated resistant cells (RC) with increased CSC content (ALDH activity and spheres forming ability). When miR-34a was induced in the RC, cells changed morphology, slowed proliferation rate, and decreased CSC content (ALDH activity and spheres). Moreover, a second PTX treatment almost completely abolished the ALDH activity and SFE. The combined effect was evaluated in vivo, following by luminescence the growth of metastases after lung colonization, with PTX+34a mice reducing lung colonization than the PTX treatment or miR-34a treatment alone. Conclusions: Overall, these results indicate that miR-34a sensitize PTX-resistant cells reducing cancer stem-cell capacities, and in this preclinical setting limited the aggressive phenotype of breast cancer, including proliferation, drug resistance, relapse and metastasis causing in delay of lung colonization in vivo. Citation Format: Montserrat Climent, Paola Bonetti, Francesco Nicassio. miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4410.

Altered distribution of peripheral blood dendritic cell subsets in patients with pulmonary paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi from the genus Paracoccidioides in Latin America. PCM-patients (PCM-p) are classified as having acute/subacute or chronic (CF) clinical forms. CF is responsible for 75%–90% of all cases, affects mainly adults over 30 years old and the clinical manifestation are associated mainly with lungs and mucosa of upper airdigestive tract. In addition, the CF patients exhibit fibrosis of the lungs, oral mucous membranes and adrenals, and pulmonary emphysema. Consequently, CF PCM-p with active disease, as well as those that have been apparently cured, seem to be an interesting model for studies aiming to understand the long-term host-fungi relationship and hypoxia. Dendritic cells (DCs) constitute a system that serve as a major link between innate and adaptive immunity composed of several subpopulations of cells including two main subsets: myeloid (mDCs) and plasmacytoid (pDCs). The present study aimed to access the distribution of PBDC subsets of CF PCM-p who were not treated (NT) or treated (apparently cured – AC). CF PCM-p were categorized into two groups, consisting of 9 NTs and 9 ACs. Twenty-one healthy individuals were used as the control group. The determination of the PBDC subsets was performed by FACS (fluorescence-activated cell sorting) and the dosage of serum TNF-α, IL1β, IL-18, CCL3, IL-10 and basic fibroblast growth factor (bFGF) by ELISA (enzyme-linked immunosorbent assay). A high count and percentage of mDCs was observed before treatment, along with a low count of pDCs in treated patients. Furthermore, the mDC:pDC ratio and serum levels of TNF-α was higher in both of the PCM-p groups than in the control group. In conclusion, our findings demonstrated that active PCM influences the distribution of mDCs and pDCs, and after treatment, PCM-p retained a lower count of pDCs associated with pro-inflammatory profile. Therefore, we identified new evidences of persistent immunological abnormalities in PCM-p after treatment. Even these patients showing fungal clearance after successful antifungal treatment; the hypoxia, triggered by the persistent pulmonary sequelae, possibly continues to interfere in the immune response.

Differences in therapeutic effects of topically applied corticosteroid and tacrolimus on atopic dermatitis-like symptoms in NC/Nga mice

BackgroundTopical corticosteroid and calcineurin inhibitor have similar therapeutic benefits in atopic dermatitis (AD), but the differences in therapeutic mechanisms of action of these agents against AD symptoms are not fully understood. ObjectiveThis study was performed to examine the different effects of topical betamethasone valerate (BMV), clobetasol propionate (CBP), and tacrolimus (TAC) on itch-related behavior and dermatitis in NC/Nga mice with AD-like symptoms. MethodsAD-like dermatitis was induced in the dorsal skin of NC/Nga mice by repeated topical application of Dermatophagoides farinae body (Dfb) ointment twice weekly for three weeks. Mice with dermatitis scores over 5 were divided into five groups with equal dermatitis scores and treated with BMV, CBP, TAC, or Vaseline (Vas) once daily for two consecutive days, or were not treated (NT). Scratching behavior was analyzed using a SCLABA®-Real system. Transepidermal water loss (TEWL) before and after treatment was measured using a Tewameter® TM210. Skin collected from each group was analyzed histologically. ResultsAfter the second treatment, dermatitis showed significantly greater improvement in the CBP and TAC-treated groups than in the Vas-treated and NT groups. The numbers of scratching bouts were significantly lower in CBP and TAC-treated mice than in Vas-treated mice. TEWL was significantly lower in TAC-, but not in CBP-, treated mice than in Vas-treated mice. Immunohistochemical examination showed that BMV, CBP and TAC did not reduce the increased densities of epidermal protein gene product 9.5- and substance P-immunoreactive fibers. The numbers of dermal CD4-immunoreactive T cells were significantly lower in BMV and CBP-treated mice than in Vas-treated and NT mice. The numbers of dermal eosinophils were significantly lower in BMV, CBP and TAC-treated mice than in Vas-treated and NT mice, with CBP showing the strongest effect. CBP significantly reduced epidermal thickness compared with Vas and NT. There were no significant differences in the numbers of interleukin-31-immunoreactive cells and mast cells, or in expression of epidermal thymic stromal lymphopoietin among all five groups. ConclusionThe therapeutic potency of TAC against AD-like symptoms, including pruritus, is equal to that of the corticosteroid CBP. Epidermal innervation of sensory nerves itself might not be related to the therapeutic effects of topical tacrolimus and corticosteroids in its early phase.

Long-Term Teduglutide for the Treatment of Patients With Intestinal Failure Associated With Short Bowel Syndrome.

OBJECTIVES:In the pivotal 24-week, phase III, placebo-controlled trial, teduglutide significantly reduced parenteral support (PS) requirements in patients with short bowel syndrome (SBS). STEPS-2 was a 2-year, open-label extension of that study designed to evaluate long-term safety and efficacy of teduglutide.METHODS:Enrolled patients had completed 24 weeks of either teduglutide (TED/TED) or placebo (PBO/TED) in the initial placebo-controlled study or qualified for that study, but were not treated (NT/TED) because of full enrollment. Patients received subcutaneous teduglutide 0.05 mg/kg/day for up to 24 months (NT/TED and PBO/TED) or up to 30 months (TED/TED). Clinical response was defined as 20–100% reduction from baseline in weekly PS volume; baseline was considered the beginning of teduglutide treatment in the initial placebo-controlled study (TED/TED) or STEPS-2 (NT/TED and PBO/TED). Descriptive statistics summarized changes in efficacy and safety variables.RESULTS:Of 88 enrolled patients, 65 (74%) completed STEPS-2. The most common treatment-emergent adverse events were abdominal pain (34%), catheter sepsis (28%), and decreased weight (25%). Mean weight, body mass index, and serum albumin remained stable. In patients who completed the study, clinical response was achieved in 28/30 (93%) TED/TED, 16/29 (55%) PBO/TED, and 4/6 (67%) NT/TED patients. Mean PS volume reductions from baseline were 7.6 (66%), 3.1 (28%), and 4.0 (39%) l/week in the TED/TED, PBO/TED, and NT/TED groups, respectively. Thirteen patients achieved full enteral autonomy.CONCLUSIONS:In patients with SBS, long-term teduglutide treatment resulted in sustained, continued reductions in PS requirements. Overall health and nutritional status was maintained despite PS reductions.

Aged Immature B Cells Resist Immune Depletion and Exhibit an Activated Phenotype.

Introduction: How aging impacts humoral immunity to organ transplantation is unclear. Here, we investigated B cell responses to organ transplantation by employing an experimental murine model. Methods:i) Skin transplant model in which young (2-3 months) and aged (16-18 months) C57BL/6 mice were transplanted with an allogenic skin (young) BALB/c graft. Recipients were either not treated (NT) or received, perioperative anti-CD45RB + anti-CD154. ii) Flow cytometric anaylsis of spleens for B cell phenotyping. iii) ELISA to measure circulating antibodies. Results: Aged recipients exhibited a faster time to allograft rejection than young transplant recipients administered immune therapy (Figure 1A). In young recipients, immunotherapy depleted absolute numbers of follicular (FO), marginal zone (MZ) and immature splenic B cells. Although immunotherapy in aged recipients resulted in depletion of splenic FO and MZ B cells, absolute numbers and frequency of splenic immature B cell remained unchanged in contrast to young recipients (Figure 1B). Characterization of splenic B cells revealed that aged immature B cells had higher expression of MHC-II, TLR2, CD80, and CD86 compared to young immature B cells, indicating that these cells exhibit an activated phenotype with aging. In addition, aged mice had greater levels of circulating total IgM and IgG at baseline and 7-days post skin transplantation compared to young mice.Figure: No Caption available.Conclusions: Aging impairs the efficacy of immune therapy to extend allograft survival. In particular, with aging immature B cells resist immune depletion and exhibit an activated phenotype. Hence, we have potentially uncovered a novel age-specific pathway which enhances humoral immunity to impair immune regulation and accelerate allograft rejection.

Growth and Yield Responses of Two Soybean Cultivars Grown under Controlled Groundwater Level in Southwestern Japan

One of the limiting factors of soybean yield in southwestern Japan is soil moisture fluctuation from flooding to drought. The Farm-Oriented Enhancing Aquatic System (FOEAS), which consists of underdrain and sub-irrigation systems and can keep groundwater levels stable, was developed in 2005 to solve this problem. To obtain basic information on soybean cultivation using the FOEAS in southwestern Japan, we examined the growth response to groundwater level by using two cultivars (cv. Fukuyutaka and Sachiyutaka) in 2008 and 2010. Plant growth, yield, yield components and seed components (protein and oil contents) were analyzed. We also studied the effect of sowing time (normal or late). The groundwater level was controlled at depths of 20 and 35 cm or not treated (NT) as a control. The groundwater level could be kept at the target depths (20 and 35 cm) by FOEAS throughout the experimental period, but that in the NT plot fluctuated greatly. The growth and yield of Sachiyutaka might be increased by FOEAS, especially in late sowing, but the yield of normal sown Sachiyutaka was decreased in the 20-cm plot in 2010. Yield of late-sown Fukuyutaka tended to be decreased in the 20-cm plot. These results suggest that (1) the optimum groundwater level for yield of Sachiyutaka is between 20 and 35 cm, regardless of sowing time and (2) the groundwater level should not be kept at 20 cm for late-sown Fukuyutaka. Further studies are needed to determine the optimum groundwater level for each cultivar.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Epicutaneous immunotherapy on intact skin using a new delivery system in a murine model of allergy

Allergen-specific immunotherapy, subcutaneous immunotherapy (SCIT) or oral, has been used for almost a century to redirect inappropriate immune responses in atopic patients. A new mode of administration through the intact skin [epicutaneous immunotherapy (EPIT)], using an original epicutaneous delivery system, may represent an alternative to these classical methods. Proof of concept of efficacy of EPIT on intact skin in mice sensitized to aeroallergens or food allergens. Mice were sensitized to pollen (n=18), house dust mite (HDM, n=24), ovalbumin (OVA, n=18) or peanut (n=18), and allocated to four groups: EPIT, SCIT, not treated (NT) and control. Specific Ig (sIg)E, sIgG1 and sIgG2a were monitored. After 8 weeks of treatment, plethysmography was performed after aerosol provocation with appropriate allergens. At the highest doses of methacholine, pause enhancement (Penh) values were significantly decreased in the EPIT group vs. the sensitized NT groups (7.5 vs. 12.3 - pollen, 7.6 vs. 8.9 - HDM, 11.5 vs. 14.5 - OVA, 7.6 vs. 12.8 - peanut, respectively) (P<0.05). With all the allergens tested, Penh values were similar in SCIT, EPIT and control. IgG2a for pollen, HDM, OVA and peanuts were significantly increased in the EPIT group vs. NT: 0.97 vs. 0.42 microg/mL, 2.5 vs. 0.46 microg/mL, 0.39 vs. 0.05 microg/mL and 15.0 vs. 5.5 microg/mL, respectively (P<0.05). There were no significant differences between EPIT and SCIT groups. The IgE/IgG2a ratio decreased significantly in the EPIT group for the four allergens from 70 to 58 (pollen), 175 to 26 (HDM), 5433 to 120 (OVA) and 49 to 6 (peanut), respectively (P<0.05). In mice sensitized to the four allergens tested, EPIT was as efficacious as SCIT, considered as the reference immunotherapy. These first results have to be confirmed by clinical studies.

Course of choledochal cysts according to the type of treatment

Objective. To compare natural course and treatment results of various types of choledochal cysts. Material and methods. Complication rate in treated (T) and natural course in not treated (NT) patients with choledochal cysts were compared. The level of bilioenteric anastomosis after cyst resection was categorized and their post-procedure courses were analyzed. Results. A total of 204 patients (T, 174; NT, 30) were included. Patients with initial malignancies were excluded from the outcome analysis (n = 13). Of the remaining 191 patients, the mean follow-up time was 35.9 months (range 6−310.7 months). Biliary stricture was the most troublesome post-operative complication, and occurred in 10 patients (6.1%). It occurred significantly more frequently in the above hilar anastomosis group than in at or below (p-trend = 0.017), and also with older age (p-trend = 0.019). Common bile duct stone was the most frequent adverse event among NT (42.9%). Overall, there were fewer complications among treated patients (T, 17.2%; NT, 67.9%; p < 0.001). Anomalous pancreaticobiliary union (APBU) was present in 69 patients (37.5%), which was associated with higher prevalence of carcinoma (APBU-present, 13%; APBU-absent, 3.5%; p = 0.019). No malignancy developed in both T and NT groups during follow-up. Conclusions. This study suggests, that even asymptomatic choledochal cysts should be treated, not only for prevention of malignancies, but also to lower the future adverse events. Bilioenteric anastomosis at larger caliber duct is recommended for prevention of post-operative biliary strictures.

Non-conventional flow cytometry approaches to detect anti- Trypanosoma cruzi immunoglobulin G in the clinical laboratory

We have recently developed a flow cytometric approach to detect anti-live trypomastigote and anti-fixed epimastigote IgG antibodies (FC-ALTA and FC-AFEA) in sera from individuals infected by Trypanosoma cruzi. Here, we present the first evaluation of the applicability of FC-AFEA-IgG as a diagnostic tool for Chagas disease. Performance analysis demonstrated that FC-AFEA-IgG has a sensitivity of 82% and a specificity of 100%. The assessment for prognosis performed by FC-ALTA-IgG1 and FC-AFEA-IgG, after classification of chagasic patients as belonging to indeterminate (IND), cardiac (CARD) or digestive (DIG) clinical forms, showed that most of IND have higher amounts of IgG than individuals' carrying CARD or DIG Chagas disease. FC-AFEA-IgG was also evaluated as a method to monitor chemotherapy efficacy in individuals classified into three distinct categories: not treated (NT), treated but not cured (TNC), and treated and cured (TC). Performance analysis demonstrated that FC-AFEA-IgG has an extraordinary capacity as a serological criterion to assess cure after therapeutic intervention in Chagas disease. These results represent a great advance in the application of serological techniques for clinical investigations on Chagas disease, and they clearly define new directions and perspectives. We intend to continue this field research focusing our attention on the influence of the degree of clinical damage on the FC-ALTA-IgG1 and FC-AFEA-IgG reactivity.

Electrical therapy for post defibrillatory pulseless electrical activity.

Defibrillation may convert ventricular fibrillation (VF) only to reveal profound mechanical dysfunction. Survival following this dysfunction, known as pulseless electrical activity (PEA) and electromechanical dissociation (EMD), is uncommon. We sought to evaluate an electrical therapy for primary post shock PEA following short duration VF. Forty-eight episodes of VF, lasting 110 +/- 25 s, were induced in 16 anesthetized dogs. Following defibrillation, 35 episodes met PEA criteria (ABP < or = 36 mmHg diastolic and pulse pressure < or = 14 mmHg in the first 20 s post shock). These post defibrillation PEA episodes were either Not Treated (NT) or Treated (T) with packets of 4-20 monophasic 0.2 ms 50-100 Hz pulses of 20-60 V applied across the tip and coil of an integrated bipolar transvenous defibrillation lead positioned in the right ventricle. The therapeutic endpoint was return of spontaneous circulation (ROSC; self-sustained ABP > or = 60 mmHg diastolic and/or > or = 100 mmHg systolic) for over 2 min. In the Not Treated group, only 4 of 19 (21%) episodes spontaneously recovered to ROSC in 123 +/- 49 s while in the Treated group, 11 of 16 (69%) of the PEA episodes achieved ROSC in 102 +/- 92 s. Electrical therapy increased the likelihood of ROSC in primary post defibrillation PEA three-fold (P < 0.01). Recovery occurred in the absence of thoracic compression, mechanical ventilation, or adjunctive drug therapy.

Survival of acid-adapted or nonadapted Escherichia coli O157:H7 in apple wounds and surrounding tissue following chemical treatments and storage

This study evaluated survival/growth of acid-adapted or nonadapted Escherichia coli O157:H7 inoculated (4 log CFU/wound) in wounds (10 mm deep×6 mm diameter) of apples. Wounds were inoculated with a green fluorescent protein (GFP)-expressing derivative of a rifampicin-resistant strain of E. coli O157:H7 ATCC 43895 and allowed to attach (1 h). Apples were dipped (2 min) in solutions (approximately 25 °C) of water (W), 5% acetic acid (AA), 5% hydrogen peroxide (HP), 0.02% sodium hypochlorite (SH), or not treated (NT), and stored at 25 °C. Survivors were determined in cores (10-mm deep) of the apple wounds (12 mm from center of wound; inner core) and surrounding tissue (18 mm from center of wound; outer core) after homogenizing the samples in Dey–Engley (D/E) neutralizing broth and plating on tryptic soy agar (TSA) and TSA supplemented with 100 μg/ml rifampicin (35 °C, 48 h) after 0, 2 and 5 days. Average bacterial populations at day-0 were 4.0 and 2.0 logs in the inner and outer core, respectively. In the inner core of the untreated apples populations increased to 7.0 logs at day-2, while counts did not exceed 3.0 logs in the outer core during storage of all treatments. Previous acid-adaptation of the cultures did not affect survival of the pathogen. Dipping in W, AA and SH did not reduce initial bacterial populations, while at day-2 of storage inner core counts from W, AA and SH reached 7.1, 5.5 and 6.9 logs, respectively. In contrast, HP reduced initial counts in the inner core by approximately 1.5 logs, but they increased to 7.0 logs by day-2. Populations of all treatments reached 6.6–7.2 logs in the inner core by day-5. Thus, sanitizer treatment did not effectively reduce nor inhibit growth of E. coli O157:H7 contamination in apple wounds and surrounding tissue.

Long-Term Outcome, with Monitoring of Platelet Counts, in Patients with Chronic Hepatitis C and Liver Cirrhosis after Interferon Therapy

Objective: Because the determination of the stage of fibrosis depends on rather subjective judgment, more objective parameters are needed. In this study, we followed the long-term outcome, with monitoring of platelet counts, in patients with chronic hepatitis C or liver cirrhosis (LC) who had undergone interferon (IFN) therapy. Methods: 596 patients who were diagnosed at our institute from 1987 to 1998 with chronic hepatitis C and LC were treated with IFNs. A further 58 patients were not treated (NT). The annual rate of changes in platelet counts were calculated and compared for IFN-treated and NT patients. Results: The relationship between the efficacy of IFN therapy and the incidence of hepatocellular carcinoma (HCC) showed that the patients who were virologic sustained responders (VSR) had a significantly lower incidence of HCC than the nonresponders (NR) and NT patients. The change in platelet counts was +4,350/µl/year in the VSR, +1,010/µl/year in the biochemical sustained responders (BSR), –4,540/µl/year in the NR and –6,180/µl/year in the NT patients, indicating a significant platelet increase in the VSR, a decrease of the same magnitude in the NR and NT patients, and no change in the BSR. The cumulative probability of developing HCC and liver failure was significantly higher in groups with decreased platelet counts than in groups with increased platelet counts among patients who had undergone IFN therapy. Multivariate analyses revealed that a decrease in platelet counts was the cardinal risk factor for development of HCC and liver failure in chronic hepatitis C or LC patients. Conclusion: Investigation of platelet counts was useful for determining the long-term outcome of patients who had undergone IFN therapy and for predicting the development of HCC.

Double-blind study to evaluate flow cytometry analysis of anti-live trypomastigote antibodies for monitoring treatment efficacy in cases of human Chagas' disease.

The validation of flow cytometry analysis of anti-live trypomastigote antibodies (FC-ALTA) to monitor cure after treatment of Chagas' disease was evaluated with serum samples from treated and nontreated chagasic patients. After optimization of the original technique, toward better sensitivity and applicability to field surveys, we design a double blind study of 94 coded samples classified into the following categories: patients not treated (NT) and patients treated but not cured (TNC), both presenting positive conventional serology and xenodiagnosis; patients treated and cured (TC), showing negative serology and xenodiagnosis; and patients treated under evaluation (TUE), who presented positive or oscillating conventional serology (CSA) but negative xenodiagnosis. Coded samples, diluted 1:256, were assayed by incubation with live cell culture trypomastigotes, which were subsequently stained with fluorescein isothiocyanate-conjugated anti-human immunoglobulin G, with prior fixation and analysis by flow cytometry. The results were expressed as the percentages of positive fluorescent parasites (PPFP) for each individual sample, establishing 20% PPFP as the cutoff between negative and positive results. Our data demonstrated that all NT and TNC presented positive results while all but one TC had a PPFP lower than 20%. Analysis of TUE demonstrated a wide degree of reactivity, with PPFP values that were negative (PPFP </= 20%), low positive (20% < PPFP </= 50%), and high positive (PPFP > 50%). As TUE with negative PPFP presented negative xenodiagnosis and positive or oscillating CSA, they were classified as dissociated according to the criteria of Krettli and Brener (J. Immunol. 128:2009-2012, 1982) and could indeed be considered cured after chemotherapy. This study demonstrates and validates the use of FC-ALTA to easily identify anti-live trypomastigote membrane-bound antibodies, offering another approach for investigating and monitoring the efficacy of specific chemotherapy in cases of human Chagas' disease.

Influence of parasite presence on the immunologic profile of peripheral blood mononuclear cells from chagasic patients after specific drug therapy.

The aim of this study is to evaluate the effects of parasite clearance on the immunological profile of peripheral blood mononuclear cells (PBMC) from chagasic patients submitted to specific drug therapy. PBMC were examined by flow cytometry and proliferative responsiveness to Trypanosoma cruzi-related stimuli. Three groups of patients were studied: not treated (NT), treated not cured (TNC) and cured (C). All data were compared to values from uninfected individuals (NI). NT displayed a lower percentage of CD3+ cells as compared to NI, while TNC and C had mean values that were between those from NI and NT. Infected patients had double the percent of CD3+ HLA-DR+ cells, independent of the efficacy of the treatment. Thus, absence of circulating parasites did not reduce T cell activation in Chagas' disease. NT displayed a higher percentage of CD5+ B cells as compared to NI, while TNC and C had mean values between those from NI and NT. In contrast to the phenotypic data, the in vitro mean proliferative responses to parasite-related stimuli of PBMC from C were reduced to the low mean levels observed in NI. These striking differences were statistically different from the high responses seen in NT and TNC. Our data suggest that proliferative responses of PBMC from C reflect immunological changes due elimination of parasite. However, successful treatment did not alter the levels of peripheral T cell activation.

A critique of the practice of comparing control data obtained at a single time point to experimental data obtained at multiple time points.

The normal circadian rhythm in DNA synthetic activity (DNA-SA) in the tip of the mouse tongue is presented. When this rhythm, obtained from mice which were not treated (NT) or handled, was compared to the rhythms obtained from mice treated with saline (SAL) or 25 mg/kg isoproterenol (IPR), no alteration in the rhythm was observed after either treatment. The conclusion from this chronobiological, experimental design was that IPR had no effect on DNA-SA in the tip of the tongue. However, when three single time points (08.00, 11.00 or 14.00) are selected from the SAL-treated, control rhythm and compared to the multiple time point data from the IPR-treated mice, three very different, statistically supported conclusions were reached. The common practice of obtaining data at only one time point in control animals and comparing these data to data obtained from drug-treated animals at multiple time points is an example of poor experimental design which results in erroneous conclusions and unnecessary confusion in the literature on in vivo research.