Face Transplantation for Granulomatosis With Polyangiitis (Wegener Granulomatosis): Technical Considerations, Immunological Aspects, and 3-Year Posttransplant Outcome.

BACKGROUND: We report new data for a rare face transplant performed 3 years ago. Granulomatosis with polyangiitis (GPA) (Wegener) is a severe autoimmune necrotizing vasculitis and parenchymal inflammatory disease that can affect any organ including those of the craniofacial region. Skin involvement manifests as malignant pyoderma. This account (1) highlights the technical details of face transplantation for this unique indication, (2) reports the 3-year posttransplant outcome, and (3) describes relevant immunological aspects. METHODS: A Le Fort III near-total face and near-total scalp transplant was performed after extensive trauma and subsequent bone and soft tissue infection in a patient with GPA. Incisions were planned along facial aesthetic subunits. The vascular pedicle comprised the facial and superficial temporal arteries bilaterally. The functioning left eye was preserved and fitted into the donor tissues. RESULTS: The procedure took 21 hours, and transfusion was limited to 4 units of packed red cells. Early medical and surgical complications were successfully treated. At 3 years, acceptable aesthetic outcome was achieved with adequate color match and scalp hair growth. The patient has recovered light touch, temperature, and 2-point discrimination and has evidence of symmetric cheek elevation albeit with limited eyelid and frontalis function. GPA relapse did not occur. Four acute rejections were fully reversed. CONCLUSIONS: This case represents a new underlying disease (trauma + GPA) leading to face transplantation and a unique clinical scenario where allografting was indicated for potentially life-threatening and sight-preserving reasons and not for mere functional and aesthetic concerns. Despite complexity, 3-year clinical outcome is encouraging, and the patient is no longer at risk for dural exposure, meningitis, and related morbidity.

Activation of inflammatory immune gene cascades by lipopolysaccharide (LPS) in the porcine colonic tissue ex-vivo model.

The technique of challenging postmortem tissue explants with inflammation inducer such as lipopolysaccharide (LPS) followed by gene expression analysis is used widely for evaluating the immune-suppressing effect of bioactives. Using porcine colonic tissue as an ex-vivo model of mammalian intestinal gut, this study evaluated the effect of incubation time on the integrity of gene transcripts and activation of inflammatory immune gene cascade by LPS treatment. Post-slaughter colon was removed surgically and explants were incubated for 0, 3, 6 and 12 h and the abundance of mRNA transcripts of a panel of 92 immune genes were evaluated using quantitative polymerase chain reaction (qPCR) arrays. The mRNA transcripts were highly intact after 0 and 3 h of incubation; however, after 6 h the degradation was clearly evident. Following 3 h incubation, 98·8% and 100% mRNA transcripts were detectable in the colonic tissue harvested from weaned and mature pigs, respectively. In the explants of weaned piglets, LPS treatment activated inflammatory signalling pathways [high mobility group B1 (HMGB1), dendritic cell maturation, interleukin (IL)-6, IL-8, IL-17F], while these pathways were inhibited by dexamethasone treatment. Activations of inflammatory genes were also evident in the explants collected from the mature pigs subjected to ex-vivo incubation for 3 h in the absence or presence of LPS. It is concluded that the colonic explant remains physiologically viable and responsive to immunological challenge for up to 3 h ex-vivo.

Effect of dietary seaweed extracts and fish oil supplementation in sows on performance, intestinal microflora, intestinal morphology, volatile fatty acid concentrations and immune status of weaned pigs.

A 2x2 factorial experiment (ten sows per treatment) was conducted to investigate the effect of maternal dietary supplementation with a seaweed extract (SWE; 0 v. 10·0 g/d) and fish oil (FO; 0 v. 100 g/d) inclusion from day 109 of gestation until weaning (day 26) on pig performance post-weaning (PW) and intestinal morphology, selected microflora and immune status of pigs 9 d PW. The SWE contained laminarin (10 %), fucoidan (8 %) and ash (82 %) and the FO contained 40 % EPA and 25 % DHA. Pigs weaned from SWE-supplemented sows had higher daily gain (P=0·063) between days 0 and 21 PW and pigs weaned from FO-supplemented sows had higher daily gain (P<0·05) and gain to feed ratio (P<0·01) between days 7 and 14 PW. There was an interaction between maternal SWE and FO supplementation on caecal Escherichia coli numbers (P<0·05) and the villous height to crypt depth ratio in the ileum (P<0·01) and jejunum (P<0·05) in pigs 9 d PW. Pigs weaned from SWE-supplemented sows had lower caecal E. coli and a higher villous height to crypt depth ratio in the ileum and jejunum compared with non-SWE-supplemented sows (P<0·05). There was no effect of SWE on E. coli numbers and villous height to crypt depth ratio with FO inclusion. Maternal FO supplementation induced an increase in colonic mRNA abundance of IL-1α and IL-6 (P<0·05), while SWE supplementation induced an increase in ileal TNF-α (P<0·01) and colonic TFF3 mRNA expression (P<0·05). In conclusion, these results demonstrate that SWE and FO supplementation to the maternal diet influenced the gastrointestinal environment and performance of the weaned pig.

Effects of dietary seaweed extract supplementation in sows and post-weaned pigs on performance, intestinal morphology, intestinal microflora and immune status.

The present study investigated the effects of dietary supplementation of a seaweed extract (SWE) to sows and weaned pigs on post-weaning growth performance, intestinal morphology, intestinal microflora, volatile fatty acid concentrations and immune status of pigs at days 11 and 117 post-weaning. Gestating sows (n 20) were supplemented with a SWE (0 v. 10·0 g/d) from day 107 of gestation until weaning (day 26). At weaning, pigs (four pigs per sow) were divided into two groups based on sow diet during lactation and supplemented with a SWE (0 v. 2·8 g/kg diet), resulting in four treatment groups: (1) BB (basal sows-basal pigs); (2) BS (basal sows-treated pigs); (3) SB (treated sows-basal pigs); (4) SS (treated sows-treated pigs). Pigs weaned from SWE-supplemented sows had a higher average daily gain (ADG) between days 0 and 21 (P < 0·05) post-weaning compared with pigs weaned from non-SWE-supplemented sows. Pigs offered post-weaning diets (PW) containing SWE had decreased colonic Escherichia coli populations on day 11 (P < 0·01) and decreased colonic Enterobacteriaceae numbers on day 117 (P < 0·05). Pigs offered PW containing SWE had a greater mRNA abundance of MUC2 in the colon at day 11 post-weaning (P < 0·05) compared with pigs offered unsupplemented diets. In conclusion, these results demonstrate that SWE supplementation post-weaning provides a dietary means to improve gut health and to enhance growth performance in starter pigs. Dietary SWE supplementation increased ADG during the grower-finisher (GF) phases. However, there was no growth response to SWE inclusion in GF diets when pigs were weaned from SWE-supplemented sows.

The effects of laminarin derived from Laminaria digitata on measurements of gut health: selected bacterial populations, intestinal fermentation, mucin gene expression and cytokine gene expression in the pig.

The aim of the present study was to establish the optimum inclusion level of laminarin derived from Laminaria digitata on selected microbial populations, intestinal fermentation, cytokine and mucin gene expression in the porcine ileum and colon. A total of twenty-one pigs (mean body weight 17·9 kg) were randomly assigned to one of three dietary treatments: T1 - basal (control) diet, T2 and T3 - basal diets supplemented with laminarin included at 300 and 600 parts per million (ppm), respectively. Selected intestinal bacterial populations and volatile fatty acid (VFA) concentrations were measured in the ileum and colon. Relative gene expression levels for specific cytokine and mucin genes were investigated in ileal and colonic tissue in the absence and presence of a lipopolysaccharide (LPS) challenge. There was an up-regulation of MUC2 gene expression at the 300 ppm inclusion level in the ileum. In the colon, there was a significant reduction in the enterobacteriaceae population at the 300 ppm inclusion level (P = 0·0421). Dietary supplementation of 600 ppm laminarin led to a significant increase in MUC2 (P = 0·0365) and MUC4 (P = 0·0401) expression in the colon, and in the total VFA concentration in the caecum (P = 0·0489). A significant increase was also recorded in IL-6 (P = 0·0289) and IL-8 gene expression (P = 0·0245) in LPS-challenged colonic tissue at both laminarin inclusion levels. In conclusion, dietary inclusion of 300 ppm laminarin appears to be the optimum dose in the present study due to the reduction in the enterobacteriaceae populations and enhanced IL-6 and IL-8 cytokine expression in response to an ex vivo LPS challenge.

Bovine lactoferrin (LTF) gene promoter haplotypes have different basal transcriptional activities.

Genetic polymorphisms present in the bovine lactoferrin (LTF) gene promoter have the potential to affect milk lactoferrin concentrations. The objectives were: (1) to identify, in silico, SNPs in the promoter region of the LTF gene that could affect transcription factor binding activity, (2) to investigate the effects of these SNPs in vitro by measuring promoter transcriptional activities of different bovine LTF promoter haplotypes and (3) to investigate the genetic association between LTF promoter SNPs and milk lactoferrin concentration. Haplotypes were deduced from sequencing of the 2.2-kb bovine LTF promoter in 78 unrelated animals. In silico analysis of the 2.2-kb promoter revealed two major haplotypes (BtLTF_H1a and BtLTF_H2a) that differed at 10 SNP loci that affect transcription factors of both a constitutive (at -28, -1702) and an inducible (at -131, -270, -586, -2047, -2077, -2122, -2140 and -2151) nature. The basal promoter transcriptional activity of BtLTF_H1a was 1.44-fold higher than that of BtLTF_H2a in mammary epithelial cells. Cows with the BtLTF_H1a haplotype had increased lactoferrin protein concentration in milk at various time points over the lactation curves, compared to herdmates with the BtLTF_H2a haplotype. The SNPs c.-28A>C, c.-131T>C, c.-156A>G, c.-270T>C, c.-586C>T, c.-1702A>G, c.-1953G>A, c.-2047A>G, c.-2077A>G, c.-2122C>T, c.-2140A>G and c.-2151G>A were associated (P < 0.001) with milk lactoferrin content in 372 Holstein-Friesian cows. The identification of bovine LTF promoter haplotypes with different basal transcriptional activities in vitro that are associated with lactoferrin levels in milk in vivo may facilitate the identification of designer dairy herds for increased lactoferrin content in milk.

Polymorphisms in the bovine lactoferrin promoter are associated with reproductive performance and somatic cell count.

Lactoferrin plays an important role in the innate immune system, with well-characterized antibacterial, antiviral, and immune modulatory properties. The objective of this study was to determine the allele and haplotype frequency of polymorphisms at positions -586, -190, and -28 of the bovine lactoferrin promoter in Holstein-Friesians and to quantify their association with performance using phenotypic data on progeny from 848 sires. Associations between genotypes and performance were quantified using weighted mixed models with genotyped individuals included as a random effect, and average expected relationships among individuals accounted for through a numerator relationship matrix. The dependent variables were daughter yield deviation for production traits and deregressed predicted transmitting ability for calving interval and functional survival. The C to T polymorphism at -586, which distorts a putative activating protein 2 (AP-2) binding site, was associated with a shorter calving interval and higher somatic cell score. The G to A polymorphism at -190, located in a putative selective promoter factor 1 (SP-1) binding site, was associated with a longer calving interval and decreased functional survival. A third polymorphism (A to C) at position -28, found within the noncanonical TATA box, had a tendency to associate with functional survival. On the basis of the data we proposed a haplotype combination that was associated with improved reproductive performance in the Holstein-Friesian breed. We hypothesized that the observable phenotypic associations with lactoferrin promoter polymorphisms can potentially be explained by allele-specific differences in constitutive or inducible levels of gene expression. The lack of a pleiotropic effect of the single nucleotide polymorphisms studied on both fertility and milk production traits strengthens the importance of these polymorphisms, or at least the lactoferrin promoter, in selection for improved fertility.

Assessment of two glycated hemoglobin immunoassays.

INTRODUCTION: Glycated hemoglobin (HbA1c) level reflects chronic glycemic status if reliable tests are used, however, in some regions worldwide high performing assays might not be readily available. This study aimed to asses two HbA1c immunoassays, comparing them with high-performance liquid chromatography (HPLC) assay, three methods available in Ecuador. MATERIAL AND METHODS: HbA1c were measured in 114 fresh whole blood-samples by DCA-Vantage point-of-care analyzer, I-Chroma portable fluorescent scanner immunoassay and BioRad Variant II Turbo HPLC. Normal and pathological HbA1c ranges were included. Blood samples with variants of hemoglobin were excluded. HbA1c values were expressed in National Glycohemoglobin Standardization Program percentages and mmol/mol, as mean±standard deviation. RESULTS: HbA1c results by HPLC and DCA-Vantage were similar: 6.3±1.7% (45±18.6mmol/mol) vs. 6.3±1.8% (45±19.7mmol/mol), respectively, P=0.057; while HbA1c values by I-Chroma were lower than HPLC, 5.8±1.9% (40±20.8mmol/mol), P<0.001. The coefficient of variation was below 2% for high and low HbA1c levels, in all methods studied. HbA1c values by HPLC and DCA-Vantage were highly correlated (Spearman's Rank Correlation [SRC]: 0.916), while the correlation among HPLC and I-Chroma was weak (SRC: 0.368). The mean bias between DCA-Vantage and HPLC was -0.02±0.29% (-0.2±3.2mmol/mol), while for I-Chroma and HPLC mean bias was -0.50±1.62% (-5.5±17.7mmol/mol). CONCLUSION: HbA1c immunoassays DCA-Vantage was comparable to HPLC assay, showing good correlation, appropriate precision and low bias, whereas I-Chroma assay was precise but inaccurate. Therefore, DCA-Vantage has better performance than I-Chroma. These findings suggest that is convenient to assess the HbA1c immunoassays commercially available in our country, Ecuador.

Effects of different suture materials on wound healing and infection in subcutaneous closure techniques.

BACKGROUND: In this study we aimed to investigate the effects of different suture materials on wound healing and infection in subcutaneous closure techniques. MATERIALS AND METHODS: Twenty-nine rats were randomly allocated to 5 groups. All subjects received 2-cm vertical dermal and subdermal incisions on their backs under sterile and proper surgical conditions. The subdermal cuts were then closed with vicryl (polyglycolic acid), biosyn (monofilament glycomer), prolene (polypropylene) and tissue adhesive (2-octylcyanoacrylate). After 10 days, all sutured areas were examined for seroma, haematoma formation and cosmetic outcome. The incisional lines were excised with 1-cm-wide skin strips and tissue hydroxyproline levels were determined. The specimens obtained were evaluated for the presence of giant cells, mononuclear cells, fibrosis and neutrophils. RESULTS: No differences in tissue hydroxyproline levels were found between any of the suture materials used. Fibrotic process and inflammatory cell infiltration were more prominent in the biosyn and prolene groups than in other groups. Foreign body giant cells were observed in the biosyn group. CONCLUSION: Vicryl and tissue adhesive should be preferred in subcutaneous closure techniques.

Malignant Pyoderma Associated with Granulomatosis with Polyangiitis (Wegener Granulomatosis) as a Unique Indication for Facial Vascularized Composite Allotransplantation: Part I.

BACKGROUND: Granulomatosis with polyangiitis (Wegener granulomatosis) is a rare disease that commonly starts in the craniofacial region and can lead to considerable facial disfigurement. Granulomas and vasculitis, however, can involve many other tissues (especially pulmonary and renal). Dermatologic and subcutaneous components can lead to malignant pyoderma. METHODS: The authors describe a unique pathologic condition, where significant Le Fort type trauma was associated with subsequent development of granulomatosis with polyangiitis and malignant pyoderma. Successive operations to excise necrotic tissue and reconstruct the defects were followed by worsening inflammation and tissue erosions. Trauma and surgery in proximity to the eye and sinuses masked the initial clinical presentation and led to delay in diagnosis and disease progression. The resultant facial disfigurement and tissue loss were substantial. RESULTS: Despite multiple confounding factors, accurate diagnosis was eventually established. This was based on persistence of sinus inflammations in the absence of infective agents, proven sterility of lung lesions, and antineutrophil cytoplasmic antibody positivity with proteinase 3 specificity. Skin lesion biopsy specimens were identified as pyoderma gangrenosum and later as malignant pyoderma. Institution of immunosuppressive therapy allowed successful control of the disease and wound healing. The resulting craniofacial destruction, however, necessitated facial vascularized composite allotransplantation. CONCLUSION: Recognition of this rare pathologic association is essential, to prevent delays in diagnosis and treatment that can lead to major craniofacial tissue loss. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Cyclosporin A and mini short-term methotrexate vs cyclosporin A as graft-versus-host disease prophylaxis in patients with beta thalassemia major undergoing allogeneic blood and marrow transplantation.

We compared the effects of cyclosporin A (CsA) alone as graft-versus-host disease (GVHD) prophylaxis vs cyclosporine with short-course methotrexate (MTX) in patients with thalassemia. In all, 140 patients were enrolled in this study. The first group, of 50 patients, received CsA alone at 3 mg/kg i.v. from day -2 to +5 followed by 12.5 mg/kg p.o., which was tapered according to the patient's condition. The other group, of 90 patients, received the combination of CsA+MTX in which CsA was used with the above-mentioned dose and MTX was on 10 mg/m(2) day +1 and 6 mg/m(2) on days +3 and +6. Incidence of acute GVHD grade II-IV in the CsA group was 78% and in the CsA+MTX group was 52.2%, which was statistically significant (P=<0.001). There were no significant differences in the incidence of chronic GVHD between the two groups. The mean neutrophil engraftment to 0.5 x 10(9)/l was 14 and 23 days for CsA group and CsA+MTX group, respectively (P=<0.001). There were no significant differences for platelet recovery between the two groups. Graft failure in the CsA and CsA+MTX groups was seven (14%) and nine (10%) patients, respectively (P=0.58). Overall survival in the CsA and CsA+MTX groups was 77 and 85%, respectively. Disease-free survival in the CsA and CsA+MTX groups were 58 and 80%, respectively.

The anti-inflammatory potential of a moderately hydrolysed casein and its 5 kDa fraction in in vitro and ex vivo models of the gastrointestinal tract.

Bioactive peptides from milk can impart a wide range of physiological benefits without the allergies and intolerance associated with the consumption of whole milk. The objective of this study was to characterise the anti-inflammatory properties of intact sodium caseinate (NaCAS), a moderately hydrolysed NaCAS enzyme hydrolysate (EH) and its 5 kDa fraction (5kDaR), in both in vitro and ex vivo systems. In vitro, Caco-2 cells were stimulated with tumor necrosis factor (TNF) α and co-treated ± casein hydrolysates or dexamethasone (control). The inflammatory marker interleukin (IL)-8 was measured by ELISA in the supernatant at 24 h. Ex vivo, porcine colonic tissues were stimulated with lipopolysaccharide (LPS) and co-treated with casein hydrolysates for 3 h from which the relative expression of a panel of cytokines was measured in vitro. While the steroid dexamethasone brought about a 41.6% reduction in the IL-8 concentration in the supernatant, the 5kDaR reduced IL-8 by 59% (P < 0.05) when compared to the TNFα stimulated Caco-2 cells. In the ex vivo system, 5kDaR was associated with decreases in IL-1α, IL-1ß, IL-8 and TGF-ß expression and an increase in IL-17 expression (P < 0.05) relative to the LPS challenged tissues. We concluded, that a 5 kDa casein fraction demonstrates potent anti-inflammatory effects both in in vitro and ex vivo models of the gastrointestinal tract.

Clinical correlations of one-carbon metabolism abnormalities.

1. Ninety psychiatric inpatients with a DSM III diagnosis of schizophrenia, mania, or major depression were studied. 2. Upon admission/transfer to the Clinical Studies Unit, and prior to discharge, measurements of symptom severity (BPRS, Ham-D, Young's Mania Scale) and blood samples were obtained. 3. Erythrocytes from these paired (admission and discharge) blood samples were assayed for methionine adenosyltransferase (MAT) activity and phosphatidylcholine (PC) content. 4. Comparisons were made between the changes in MAT Vmax, or % PC, and changes in symptom severity. 5. For the majority of the patients (79.3% of the schizophrenics; 84.6% of the depressives; and 93.8% of the manics), clinical improvement was associated with a "normalization" of enzyme activity. The association between changes in % PC and clinical response did not achieve significant correlation.

Effect on growth parameters of bone marrow transplantation with a chemotherapy-only conditioning regimen.

INTRODUCTION: The increasing use of bone marrow transplantation (BMT) has increased survival among a growing number of children and young adults afflicted by malignant and nonmalignant hematologic disorders. Accordingly, quality of life has emerged as an important issue. Because they are a concern in this group, we assessed growth and development, following chemotherapy-only conditioning regimens. MATERIALS AND METHODS: Twelve prepubertal children (8 boys, G(1)P(1) and 4 girls, B(1)P(1)) with a mean age of 6 +/- 2 years (age range: 3.5 to 10 years) before and at 3, 6, 9, and 12 months post-BMT. RESULTS: Growth velocity at 1 year posttransplant was 10.0 +/- 3.5 cm/y. One year post-BMT, the statistical deviation saturation for growth velocity was 4.31 +/- 4.21. Height standard deviation score was -1.4 +/- 1.2 before and -0.5 +/- 1.3 1 year post-BMT (P <.004). The average weight of our subjects was 20 +/- 6 kg before and 26 +/- 9.5 kg 1 year post-BMT. DISCUSSION: BMT with a chemotherapy-only conditioning regimen not only does not disturb growth in children; it is actually associated with a relative growth spurt afterward.

Thyroid, parathyroid, gonadal, and pancreatic beta-cell function after bone marrow transplantation with chemotherapy-only conditioning.

INTRODUCTION: Radiation and cytotoxic chemotherapy can provoke short- and long-term endocrine dysfunction. We studied the prevalence of thyroid, parathyroid, gonadal, and pancreatic beta-cell function in patients undergoing bone marrow transplantation (BMT). MATERIALS AND METHOD: Forty-six patients (12 women, 34 men), aged 1.5 to 49 years (mean, 15.1 years), were evaluated for thyroid, parathyroid, gonadal, and pancreatic beta-cell function before and 3, 6, and 12 months after BMT with a little busulfan-cyclophosphamide conditioning regimen. RESULTS: Thyroid and parathyroid function was unaltered by BMT. Leydig cell function was normal in 11 adult men (G5P5) before and at 3, 6, and 12 months after BMT, but injury to the germinal epithelium (oligo- or azoospermia) was seen before and 12 months after BMT. There was no relationship between serum FSH and germinal epithelial injury. Maturation was normal in six boys (G2P2 or G3P3 at BMT) 12 months post-BMT. Primary hypogonadism was seen in four adult women (B5P5) after BMT. One 14-year-old girl continued to have regular menstrual periods during the 24 months after BMT. Another girl (P1B1 pre-BMT) developed ovarian failure 12 months post-BMT. Pancreatic beta-cell function was normal pre- and post-BMT in 12 thalassemic patients with serum ferritin > 1000 ng/mL. CONCLUSION: BMT with chemotherapy-only conditioning seems primarily to affect gonadal function, without having any significant effect on thyroid, parathyroid, or pancreatic beta-cell function.

The effects of supplementing varying molecular weights of chitooligosaccharide on performance, selected microbial populations and nutrient digestibility in the weaned pig.

An experiment (complete randomised design) was conducted to investigate the effects of supplementing different molecular weights (MW) of chitooligosaccharide (COS) on pig performance, selected microbial populations and nutrient digestibility post-weaning. A total of 396 weaned piglets (24 days of age, 7.3 kg ± (s.d.) 1.7 kg live weight) were assigned to one of six dietary treatments (22 replicates/treatment) for a 33-day experimental period. The dietary treatments were as follows (1) control diet (0 ppm COS), (2) control diet plus <1 kDa COS, (3) control diet plus 3 to 5 kDa COS, (4) control diet plus 5 to 10 kDa COS, (5) control diet plus 10 to 50 kDa COS and (6) control diet plus 50 to 100 kDa COS. The COS were included at 250 ppm in the diets. There was no significant effect of dietary treatment on piglet performance during the starter period (days 0 to 18; P > 0.05). However, there were quadratic responses in both daily gain (P < 0.05) and gain to feed ratio (P < 0.05) to the increased MW of COS inclusion during the weaner period (days 18 to 33) with all COS-supplemented treatments improving daily gain and gain to feed ratio compared with the control. There was a quadratic response in faecal scoring to the increased MW of COS inclusion from days 0 to 7 (P < 0.001), days 7 to 14 (P < 0.001) and during the overall experimental period (P < 0.01) with all the COS-supplemented treatments having an improved faecal score compared with the control. During the weaner period, there was a cubic response in lactic acid bacteria and Escherichia coli populations as the MW of COS increased (P < 0.05). The 5 to 10 kDa and 10 to 50 kDa COS increased lactic acid bacteria populations compared with the control, whereas lactic acid bacteria populations decreased at 50 to 100 kDa. The 5 to 10 kDa, 10 to 50 kDa and 50 to 100 kDa COS decreased E. coli populations compared with the control. There was a cubic response in the apparent total tract digestibility of dry matter (DM; P < 0.01), organic matter (OM; P < 0.01), ash (P < 0.01), nitrogen (N; P < 0.01) and gross energy (GE; P < 0.01) to the increased MW of COS inclusion during the weaner period. The 5 to 10 kDa COS had a higher apparent total tract digestibility of DM, OM, ash, N and GE in comparison to the control, whereas the apparent total tract nutrient digestibility of these nutrients decreased at 10 to 50 kDa. The current results indicate that the MW ranges of 5 to 10 kDa and 10 to 50 kDa COS decreased E. coli numbers while increasing nutrient digestibility of the diets.

Assessment of RNA integrity in the postmortem pig colonic tissue ex vivo.

Surgical removal of porcine intestinal tissue followed by an ex vivo challenge is an alternative technique of testing the anti-inflammatory effect of bioactive compounds in the intestine of live pigs. We investigated the effects of ex vivo incubation of porcine colonic tissue on the quantity and quality of total RNA over a 12-h time period. Colonic tissue of pig (n = 6) was surgically removed immediately postslaughter and incubated for 0, 3, 6, and 12 h in a humidified cell culture incubator with 5% CO(2) at 37°C. Tissue samples were processed for RNA extraction. The quantity and quality of total RNA were assessed on a NanoDrop Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. Ex vivo incubation had an effect on both the quantity (P < 0.001) and quality (P < 0.001) of total RNA. Relative to the RNA yield at 0 h (505.0 ± 48.64 µg/mg), the yield was significantly reduced after 6 h (227.6 ± 25.52 µg/mg; P < 0.001) and 12 h (159.3 ± 24.19 µg/mg; P < 0.001) of incubation. The 28S and 18S rRNA bands were visibly intact after 0, 3, and 6 h of incubation. However, after 12 h of incubation, a degraded RNA profile was evident. The RNA integrity number (RIN) values for the 0, 3, 6, and 12 h of incubation were 9.4 ± 0.10, 9.0 ± 0.10, 6.7 ± 0.17 (P < 0.001), and 3.3 ± 0.24 (P < 0.001), respectively. The transcript abundances of 4 constitutively expressed genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), beta 2-microglobulin (B2M), and peptidylprolyl isomerase A (PPIA) were reduced at both 6 and 12 h of incubation. It is concluded that ex vivo incubation of porcine colonic tissue up to 3 h postmortem generates good quality total RNA suitable for gene expression studies.

Extracts of brown seaweeds can attenuate the bacterial lipopolysaccharide-induced pro-inflammatory response in the porcine colon ex vivo.

Bioactive compound-rich brown seaweeds are demonstrated to have numerous health benefits including anti-microbial and immunomodulatory bioactivities in the pig intestine. In this study, the immunomodulating effects of extracts of brown seaweed (Ascophyllum nodosum and Fucus serratus) were evaluated on the porcine colon using a bacterial lipopolysaccharide (LPS) ex vivo model. Approximately 1.5 × 1.5 cm of pig colon (n = 6) was stripped of its overlying muscle layer and incubated in 1 mL Dulbecco's Modified Eagle Medium containing bacterial LPS (10 µg) and seaweed extracts (1 mg). Gene expression of interleukin-8 (IL-8) and interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFA) were measured using quantitative real time PCR. In contrast to the low level of expression of IL-8, IL-6, and TNFA genes in the colonic tissue at 0 h, LPS treatment increased (P < 0.05) the expression of IL-8, IL-6, and TNFA genes to 2.38 ± 0.86, 1.90 ± 0.66, and 1.90 ± 0.57 fold, respectively. This pro-inflammatory response induced by the LPS was suppressed by the extracts of Ascophyllum. Ascophyllum extract reduced (P < 0.05) the expression of IL-8, IL-6, and TNFA genes to 0.99 ± 0.53, 0.75 ± 0.33, and 1.01 ± 0.17 fold, and Fucus extract reduced (P < 0.05) the expression of the corresponding genes to 0.70 ± 0.32, 0.69 ± 0.38, and 1.15 ± 0.25 fold, respectively. It is concluded that the extracts of Ascophyllum and Fucus seaweeds have potential to suppress the pro-inflammatory response induced by the bacterial LPS in the pig colon.

Effect of maternal seaweed extract supplementation on suckling piglet growth, humoral immunity, selected microflora, and immune response after an ex vivo lipopolysaccharide challenge.

The present study was conducted to investigate the effect of maternal dietary supplementation (n = 10 sows/treatment) with seaweed extract (SWE: 0 vs. 10.0 g/d) from d 107 of gestation until weaning (d 26) on neonatal piglet growth, humoral immunity, intestinal morphology, selected intestinal microflora, and VFA concentrations. Furthermore, this study examined the effect of dietary treatment on the immune response after an ex vivo Escherichia coli lipopolysaccharide (LPS) tissue challenge at weaning in a 2 × 2 factorial arrangement. The main factors consisted of sow dietary treatment (SWE or control) and immunological challenge (yes or no). The SWE supplement (10.0 g/d) contained laminarin (1.0 g), fucoidan (0.8 g), and ash (8.2 g) and was extracted from a Laminaria spp. The SWE-supplemented sows had greater colostrum IgA (P < 0.01) and had a trend for greater IgG (P = 0.062) concentrations compared with non-SWE-supplemented sows. Piglets suckling SWE-supplemented sows had greater serum IgG (P < 0.05) concentrations on d 14 of lactation compared with those suckling non-SWE-supplemented sows. Dietary SWE supplementation decreased fecal Enterobacteriaceae populations in sows at parturition (P < 0.05), and piglets suckling SWE-supplemented sows had a decreased colonic E. coli population at weaning (P < 0.01) compared with non-SWE-supplemented sows. Lipopolysaccharide challenge increased the mRNA abundances of the pro-inflammatory cytokines IL-1α and IL-6 (P < 0.01) in ileal tissue and tumor necrosis factor (TNF)-α in colonic (P < 0.01) tissue. There was a treatment × LPS challenge interaction for ileal TNF-α mRNA expression (P < 0.05). Piglets suckling SWE-supplemented sows had greater TNF-α mRNA expression after ex vivo LPS challenge compared with non-SWE-supplemented sows (P < 0.05). However, there was no effect of sow dietary treatment on TNF-α mRNA expression in the unchallenged ileal tissue. Piglet BW at birth and weaning, and small intestinal morphology were unaffected by sow dietary treatment under current experimental conditions. In summary, these results demonstrate an important immunomodulatory role of SWE supplementation characterized by enhanced colostral IgA and IgG concentrations, greater piglet circulatory IgG concentrations on d 14 of lactation, and enhanced TNF-α mRNA expression in the ileum after an ex vivo LPS challenge. These results indicate that SWE supplementation enhanced piglet immune function and colonic microflora at weaning.

The microbiological and immunomodulatory effects of spray-dried versus wet dietary supplementation of seaweed extract in the pig gastrointestinal tract.

Seaweeds and seaweed extract (SWE) possess antimicrobial, anti-inflammatory, prebiotic, and growth-promoting properties. Extracts can be prepared in different ways including wet, spray-dried, and freeze-dried forms. The aim of this study was to determine if spray drying of laminarin and fucoidan derived from Laminaria digitata had an effect on the microbiological and cytokine profile of the gastrointestinal tract (GIT) compared to the wet SWE in newly weaned pigs. No differences in cytokine expression were observed between wet and spray dried SWE formulation in either the ileum or colon. Bifidobacteria counts were greater (P < 0.05) in the wet SWE formulation relative to both spray dried SWE and the basal diet in the ileum. In conclusion, neither of the SWE formulations had significant effects on the cytokine profile in the ileum or colon. However, a prebiotic effect observed in the ileum of piglets in response to the wet SWE formulation was lost following spray drying of the SWE.