Biliary atresia in Uganda: Current ethical challenges and advancement of public policy.

Biliary atresia is a progressive cholangiopathy in neonates, which often results in liver failure. In high-income countries, initial treatment requires prompt diagnosis followed by Kasai portoenterostomy. For those with a late diagnosis, or those in whom Kasai portoenterostomy fails, liver transplantation is the only lifesaving treatment. Unfortunately, in low- and middle-income countries, timely diagnosis is a challenge and liver transplantation is rarely accessible. Here, we discuss the ethical dilemmas surrounding treatment of babies with biliary atresia in Uganda. Issues that require careful consideration include: risk of catastrophic health expenditure to families, ethical dilemmas of transplant tourism, medical risks of maintaining the transplant in a low-resourced health system, and difficult decisions encountered by the surgeon caring for these patients. Four distinct models of the patient-physician relationship are applied to biliary atresia in Uganda. These models describe differences in patient and physician roles, and patient values and autonomy. Solid organ transplantation is a rapidly evolving segment of healthcare in Uganda and ongoing policy advancements may shift ethical considerations in the future.

T Cells Promote Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase-9 via a C-C Chemokine Receptor Type 2 Pathway: Implications for Chronic Lung Allograft Dysfunction.

Chronic lung allograft dysfunction (CLAD) is the major limitation of long-term survival after lung transplantation. CLAD manifests as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Alloimmune reactions and epithelial-to-mesenchymal transition have been suggested in BOS. However, little is known regarding the role of allogenicity in epithelial cell differentiation. Primary human bronchial epithelial cells (BECs) were treated with activated T cells in the presence or absence of transforming growth factor (TGF)-ß. The expression of epithelial and mesenchymal markers was investigated. The secretion of inflammatory cytokines and matrix metalloproteinase (MMP)-9 was measured in culture supernatants and in plasma from lung transplant recipients (LTRs): 49 stable, 29 with BOS, and 16 with RAS. We demonstrated that C-C motif chemokine 2 secreted by T cells supports TGF-ß-induced MMP-9 production by BECs after binding to C-C chemokine receptor type 2. Longitudinal investigation in LTRs revealed a rise in plasma MMP-9 before CLAD onset. Multivariate analysis showed that plasma MMP-9 was independently associated with BOS (odds ratio [OR] = 6.19, p = 0.002) or RAS (OR = 3.9, p = 0.024) and predicted the occurrence of CLAD 12 months before the functional diagnosis. Thus, immune cells support airway remodeling through the production of MMP-9. Plasma MMP-9 is a potential predictive biomarker of CLAD.

TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/ß-catenin signaling.

BACKGROUND: Airway epithelial cells (AEC) act as the first line of defence in case of lung infections. They constitute a physical barrier against pathogens and they participate in the initiation of the immune response. Yet, the modalities of pathogen recognition by AEC and the consequences on the epithelial barrier remain poorly documented. METHOD: We investigated the response of primary human AEC to viral (polyinosinic-polycytidylic acid, poly(I:C)) and bacterial (lipopolysaccharide, LPS) stimulations in combination with the lung remodeling factor Transforming Growth Factor-ß (TGF-ß). RESULTS: We showed a strong production of pro-inflammatory cytokines (Interleukin (IL)-6, Tumor Necrosis Factor α, TNFα) or chemokines (CCL2, CCL3, CCL4, CXCL10, CXCL11) by AEC stimulated with poly(I:C). Cytokine and chemokine production, except CXCL10, was Toll Like Receptor (TLR)-3 dependent and although they express TLR4, we found no cytokine production after LPS stimulation. Poly(I:C), but not LPS, synergised with TGF-ß for the production of matrix metalloproteinase-9 (MMP-9) and fibronectin. Mechanistic analyses suggest the secretion of Wnt ligands by AEC along with a degradation of the cellular junctions after poly(I:C) exposure, leading to the release of ß-catenin from the cell membrane and stimulation of the Wnt/ß-catenin pathway. CONCLUSION: Our results highlight the cross talk between TGF-ß and TLR signaling in bronchial epithelium and its impact on the remodeling process.

Characterization of newly revealed sequences in the infectious myonecrosis virus genome in Litopenaeus vannamei.

Infectious myonecrosis virus (IMNV) causes significant economic losses in farmed shrimp, where associated mortality in ponds can reach 70 %. To explore host/pathogen interactions, a next-generation sequencing approach using lymphoid organ tissue from IMNV-infected Litopenaeus vannamei shrimp was conducted. Preliminary sequence assembly of just the virus showed that there were at least an additional 639 bp at the 5' terminus and 23 nt at the 3' terminus as compared with the original description of the IMNV genome (7561 nt). Northern blot and reverse transcription-PCR analysis confirmed the presence of novel sequence at both ends of the genome. Using 5' RACE, an additional 4 nt were discovered; 3' RACE confirmed the presence of 22 bp rather than 23 bp of sequence. Based on these data, the IMNV genome is 8226 bp in length. dsRNA was used to trigger RNA interference (RNAi) and suppress expression of the newly revealed genome sections at the 5' end of the IMNV genome in IMNV-infected L. vannamei. An RNAi trigger targeting a 376 bp length of the 5' UTR did not improve survival of infected shrimp. In contrast, an RNAi trigger targeting a 381 bp sequence in ORF1 improved survival to 82.2 % as compared with 2.2 % survival in positive control animals. These studies revealed the importance of the new genome sections to produce high-titre infection, and associated disease and mortality, in infected shrimp.

Osteomyelitis associated with Nocardiopsis composta in a dog.

We report the first detection of Nocardiopsis composta in association with osteomyelitis in a young male miniature Australian shepherd dog. Findings included suppurative osteomyelitis containing intralesional Fite's acid fast bacilli, aerobic culture of branching Gram-positive rods, and positive identification via phenotypic analysis and 16S rDNA sequencing.

Ostéomyélite associée àNocardiopsis compostachez un chien. Nous signalons la première détection de Nocardiopsis composta en association avec l'ostéomyélite chez un jeune chien berger Australien miniature mâle. Les résultats incluaient une ostéomyélite suppurative contenant des bacilles alcoolo-acido résistants à la coloration de Fite, une culture aérobie de bâtonnets à Gram positif embranchés et l'identification positive par une analyse phénotypique et le séquençage de l'ADNr 16S.(Traduit par Isabelle Vallières).

Sequence-optimized and targeted double-stranded RNA as a therapeutic antiviral treatment against infectious myonecrosis virus in Litopenaeus vannamei.

Infectious myonecrosis virus (IMNV) is a significant and emerging pathogen that has a tremendous impact on the culture of the Pacific white shrimp Litopenaeus vannamei. IMNV first emerged in Brazil in 2002 and subsequently spread to Indonesia, causing large economic losses in both countries. No existing therapeutic treatments or effective interventions currently exist for IMNV. RNA interference (RNAi) is an effective technique for preventing viral disease in shrimp. Here, we describe the efficacy of a double-stranded RNA (dsRNA) applied as an antiviral therapeutic following virus challenge. The antiviral molecule is an optimized dsRNA construct that targets an IMNV sequence at the 5' end of the genome and that showed outstanding antiviral protection previously when administered prior to infection. At least 50% survival is observed with a low dose of dsRNA administered 48 h post-infection with a lethal dose of IMNV; this degree of protection was not observed when dsRNA was administered 72 h post-infection. Additionally, administration of the dsRNA antiviral resulted in a significant reduction of the viral load in the muscle of shrimp that died from disease or survived until termination of the present study, as assessed by quantitative RT-PCR. These data indicate that this optimized RNAi antiviral molecule holds promise for use as an antiviral therapeutic against IMNV.

dsRNA provides sequence-dependent protection against infectious myonecrosis virus in Litopenaeus vannamei.

Viral diseases are significant impediments to the sustainability of shrimp aquaculture. In addition to endemic disease, new viral diseases continue to emerge and cause significant impact on the shrimp industry. Disease caused by infectious myonecrosis virus (IMNV) has caused tremendous losses in farmed Pacific white shrimp (Litopenaeus vannamei) since it emerged in Brazil and translocated to Indonesia. There are no existing antiviral interventions, outside of pathogen exclusion, to mitigate disease in commercial shrimp operations. Here, we describe an iterative process of panning the genome of IMNV to discover RNA interference trigger sequences that initiate a robust and long-lasting protective response against IMNV in L. vannamei. Using this process, a single, low dose (0.02 µg) of an 81 or 153 bp fragment, with sequence corresponding to putative cleavage protein 1 in ORF1, protected 100 % of animals from disease and mortality caused by IMNV. Furthermore, animals that were treated with highly efficacious dsRNA survived an initial infection and were resistant to subsequent infections over 50 days later with a 100-fold greater dose of virus. This protection is probably sequence dependent, because targeting the coding regions for the polymerase or structural genes of IMNV conferred lesser or no protection. Interestingly, non-sequence specific dsRNA did not provide any degree of protection to animals as had been described for other shrimp viruses. Our data indicate that the targeted region for dsRNA is a crucial factor in maximizing the degree of protection and lowering the dose required to induce a protective effect against IMNV infection in shrimp.

Nucleic-acid based antivirals: augmenting RNA interference to 'vaccinate'Litopenaeus vannamei.

The Pacific white shrimp, Litopenaeus vannamei (Penaeidae: Litopenaeus) has emerged as the dominant farmed shrimp species globally in tropical countries. Rearing animals at high density in semi-intensive or intensive culture systems, and translocating animals across the globe, have created optimum conditions for devastating epizootics. Of the various pathogens that impact shrimp culture, viruses are arguably the most important infectious disease agents that exact devastating economic losses to the industry. Augmenting the RNA interference (RNAi) capacity of shrimp is a promising, emerging solution to prevent disease caused by a variety of highly pathogenic shrimp viruses. Indeed RNAi functions as a primary mechanism of antiviral RNA in arthropods, as was revealed initially in studies of mosquito-virus interactions. Double-stranded RNA (dsRNA) or small interfering RNA (siRNA) can be used as RNAi triggers in vivo in L. vannamei to reduce the pathology associated with virus infection. We explored the efficacy of those triggers as a function of the target gene in the virus genome and show that efficacy is virus-specific and cannot be predicted based on the target gene function or transcript level in an infected cell. Further, we show that carefully designed RNAi triggers provide an immune stimulus that results in specific, long-term protection and therefore suggest that these dsRNA antivirals can function as vaccines in controlling disease.

A pilot study of intensive intervention using a novel trismus device.

PURPOSE: Trismus secondary to head and neck neoplasm treatment impacts upon quality of life, nutrition, oral hygiene, and dentition. Current treatment options for trismus apply unquantified force to the jaw, and in many cases, the device costs are prohibitive. This study aimed to prospectively evaluate the impact of a novel trismus device. METHOD: This single arm cohort study prospectively evaluated the impact of a novel trismus device on maximal incisal opening (MIO), trismus-related function and quality of life scores. Seventeen patients diagnosed with trismus were recruited to undergo a 10-week program using a novel device. The effect of the intervention was assessed by comparing pre- vs post-intervention validated measures. RESULT: A significant improvement in MIO was observed post the 10-week intervention period (12.6 mm). This was associated with an improvement in patient reported trismus symptomology including quality of life, swallowing, speech, and jaw pain. CONCLUSIONS: This pilot study demonstrates the feasibility of a novel device in the treatment of trismus. Further evaluation of this device is warranted to assess efficacy, safety, and cost-effectiveness in a larger cohort with appropriate controls.

Osteopetrosis in mice lacking NF-kappaB1 and NF-kappaB2.

The nfkb1 and nfkb2 genes encode closely related products regulating immune and inflammatory responses. Their role during development and differentiation remains unclear. The generation of nfkb1 null mice (p50-/-) resulted in altered immune responses, but had no effect on development. Similarly, nfkb2 knockout mice (p52-/-) did not show developmental defects (J.C. et al., manuscript submitted). We have investigated the potential for in vivo compensatory functions of these genes by generating double-knockout mice. The surprising result was that the animals developed osteopetrosis because of a defect in osteoclast differentiation, suggesting redundant functions of NF-kappaB1 and NF-kappaB2 proteins in the development of this cell lineage. The osteopetrotic phenotype was rescued by bone marrow transplantation, indicating that the hematopoietic component was impaired. These results define a new mouse osteopetrotic mutant and implicate NF-kappaB proteins in bone development, raising new directions in the treatment of bone disorders.

Comparative analysis of hemocyte phagocytosis between six species of arthropods as measured by flow cytometry.

Phagocytosis of pathogens by hemocytes is a rapid-acting immune response and represents a primary means of limiting microbial infection in some species of arthropods. To survey the relative capacity of hemocyte phagocytosis as a function of the arthropod immune response, we examined the extent of phagocytosis among a wide taxonomic range of arthropod species including a decapod crustacean (Litopenaeus vannamei), three ixodid tick species (Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis), a mosquito species (Aedes aegypti), and a larval moth (Manduca sexta). Injected fluorescent beads were used as a model to elicit phagocytosis and were measured by flow cytometry, a technique provided in detail that may be adapted for use with any species of arthropod. The data indicated that smaller arthropods generally had a higher proportion of phagocytic cells than larger arthropods.

Isolation of Moraxella spp. from horses with conjunctivitis in Southern Brazil.

Infectious keratoconjunctivitis (IKC) is the most frequent ocular disease in livestock worldwide and is primarily caused by Moraxella bovis, M. ovis, and/or M. bovoculi. The economic impact of IKC is mainly due to ocular damage, which leads to weight loss, management difficulties, pain and discomfort, and cost of treatments. In horses, limited information is available on the association of Moraxella spp. with keratoconjunctivitis. The present report describes two cases of equine keratoconjunctivitis caused by members of the genus Moraxella. Both animals presented with lacrimation, conjunctivitis, photophobia, mucoid or purulent secretions, blepharitis, and conjunctival hyperemia. The diagnosis of IKC was based on the epidemiological and clinical findings; the etiological agent was identified through bacteriological (culture and biochemistry assays) and molecular testing (PCR and nucleotide sequencing). Our study reports the isolation of Moraxella bovoculi (SBP 88/19) and a putative new species/mutant of Moraxella (SBP 39/19) recovered from ocular secretions in horses. Thus, we suggest the inclusion of Moraxella spp. infection in the differential diagnosis of conjunctivitis in horses in Southern Brazil.

Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.

Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking. The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases. One C-C chemokine receptor, CCR2, has been identified that mediates in vitro responses to MCP-1 and its close structural homologues. CCR2 has also recently been demonstrated to be a fusion cofactor for several HIV isolates. To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene. Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities. However, ccr2(-/-) mice failed to recruit macrophages in an experimental peritoneal inflammation model. In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes. These results suggest that CCR2 has a nonredundant role as a major mediator of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and other CCR2 ligands are effectors of those functions.

Decrease in Hatchability of Pheasant Eggs Associated with Enterococcus faecalis.

A game bird producer in the North Central region of the United States submitted unhatched ring-neck pheasant (Phasianus colchicus) eggs for diagnostic evaluation. The submitting complaint was a drastic drop in hatchability. This operation has its own breeder birds that are housed in outside pens. This hatch occurred in the latter third of the production cycle. Typical hatchability for this operation is around 75% (± 3%). The hatchability of this hatch was between 14%-15%. Approximately 30,000 eggs were set with an expected hatchability of about 23,000 birds. The number of birds from this hatch was less than 4500, with a net loss approaching 20,000 chicks. All unhatched eggs submitted were in late stage development. The chick embryos had pipped through the shell but died before hatching. Approximately 5000 eggs originating from an outside breeder source were also set at the same time in the same machines and experienced a normal hatch. The exterior surfaces of the eggshells of the unhatched eggs experiencing low hatchability were swabbed and submitted for bacteriologic evaluation. Additionally, embryos from some of the unhatched eggs were removed aseptically from their eggshells, and their internal organs were harvested and submitted for bacteriologic evaluation. The bacteriology results identified no pathogenic bacteria from the eggshells. However, the embryo samples revealed large quantities of Enterococcus faecalis. In discussions with the producer, the only factor identified was an unusually warm period followed by an atypically cold and wet period during the time of egg collection for those eggs experiencing low hatchability.

Benefits of barbed suture utilisation in gastrointestinal anastomosis: a systematic review and meta-analysis.

INTRODUCTION: Anastomosis formation constitutes a critical aspect of many gastrointestinal procedures. Barbed suture materials have been adopted by some surgeons to assist in this task. This systematic review and meta-analysis compares the safety and efficacy of barbed suture material for anastomosis formation compared with standard suture materials. METHODS: An electronic search of Embase, Medline, Web of Science and Cochrane databases was performed. Weighted mean differences were calculated for effect size of barbed suture material compared with standard material on continuous variables and pooled odds ratios were calculated for discrete variables. FINDINGS: There were nine studies included. Barbed suture material was associated with a significant reduction in overall operative time (WMD: -12.87 (95% CI = -20.16 to -5.58) (P = 0.0005)) and anastomosis time (WMD: -4.28 (95% CI = -6.80 to -1.75) (P = 0.0009)). There was no difference in rates of anastomotic leak (POR: 1.24 (95% CI = 0.89 to 1.71) (P = 0.19)), anastomotic bleeding (POR: 0.80 (95% CI = 0.29 to 2.16) (P = 0.41)), or anastomotic stricture (POR: 0.72 (95% CI = 0.21 to 2.41) (P = 0.59)). CONCLUSIONS: Use of barbed sutures for gastrointestinal anastomosis appears to be associated with shorter overall operative times. There was no difference in rates of complications (including anastomotic leak, bleeding or stricture) compared with standard suture materials.

Crystal structure of the catalytic domain of human plasmin complexed with streptokinase.

Streptokinase is a plasminogen activator widely used in treating blood-clotting disorders. Complexes of streptokinase with human plasminogen can hydrolytically activate other plasminogen molecules to plasmin, which then dissolves blood clots. A similar binding activation mechanism also occurs in some key steps of blood coagulation. The crystal structure of streptokinase complexed with the catalytic unit of human plasmin was solved at 2.9 angstroms. The amino-terminal domain of streptokinase in the complex is hypothesized to enhance the substrate recognition. The carboxyl-terminal domain of streptokinase, which binds near the activation loop of plasminogen, is likely responsible for the contact activation of plasminogen in the complex.

Whole genome sequencing of Moraxella bovoculi reveals high genetic diversity and evidence for interspecies recombination at multiple loci.

Moraxella bovoculi is frequently cultured from the ocular secretions and conjunctiva of cattle with Infectious Bovine Keratoconjunctivitis (IBK). Previous work has shown that single nucleotide polymorphism (SNP) diversity in this species is quite high with 81,284 SNPs identified in eight genomes representing two distinct genotypes isolated from IBK affected eyes (genotype 1) and the nasopharynx of cattle without clinical IBK signs (genotype 2), respectively. The goals of this study were to identify SNPs from a collection of geographically diverse and epidemiologically unlinked M. bovoculi strains from the eyes of IBK positive cattle (n = 183) and another from the eyes of cattle (most from a single population at a single time-point) without signs of IBK (n = 63) and to characterize the genetic diversity. Strains of both genotypes were identified from the eyes of cattle without IBK signs. Only genotype 1 strains were identified from IBK affected eyes, however, these strains were isolated before the discovery of genotype 2, and the protocol for their isolation would have preferentially selected genotype 1 M. bovoculi. The core genome comprised ~74% of the whole and contained >127,000 filtered SNPs. More than 80% of these characterize diversity within genotype 1 while 23,611 SNPs (~18%) delimit the two major genotypes. Genotype 2 strains lacked a repeats-in-toxin (RTX) putative pathogenesis factor and any of ten putative antibiotic resistance genes carried within a genomic island. Within genotype 1, prevalence of these elements was 0.85 and 0.12 respectively in strains from eyes that were IBK positive. Recombination appears to be an important source of genetic diversity for genotype 1 and undermines the utility of ribosomal-locus-based species identification. The extremely high genetic diversity in genotype 1 presents a challenge to the development of an efficacious vaccine directed against them, however, several low-diversity pilin-like genes were identified. Finally, the genotype-defining SNPs described in this study are a resource that can facilitate the development of more accurate M. bovoculi diagnostic tests.

Neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes.

Chemokines (pro-inflammatory chemoattractant cytokines) are expressed in pathological conditions of the central nervous system (CNS). Previous studies suggested that the CNS is relatively resistant to leukocyte diapedesis after chemokine injection, leaving their functional role unresolved. The CNS function of N51/KC, a neutrophil-selective chemokine, was addressed by expressing N51/KC under control of the myelin basic protein (MBP) promoter in transgenic (tg) mice (MBP-N51/KC mice). CNS-specific N51/KC expression produced remarkable neutrophil infiltration into perivascular, meningeal, and parenchymal sites, demonstrating that this chemokine exerts the multiple functions in vivo required to recruit leukocytes into the CNS. MBP-N5 1/KC mice represent an incisive model for the molecular dissection of neutrophil entry into the CNS. Unexpectedly, MBP-N51/KC mice developed a neurological syndrome of pronounced postural instability and rigidity at high frequency beginning at 40 days of age, well after peak chemokine expression. 68/182 mice in one tg fine were found dead before one year of age, with prominent neurological symptoms premortem in 26 (38%). Florid microglial activation and blood-brain barrier disruption without dysmyelination were the major neuropathological alterations. Late-onset neurological symptoms in MBP-N51/KC mice may indicate unanticipated consequences of CNS chemokine expression.

Estimating the rate of re-expansion of spontaneous pneumothorax by a formula derived from computed tomography volumetry studies.

BACKGROUND: Available evidence suggesting that the rate of re-expansion of spontaneous pneumothorax is 1.25%/day is based on a small sample and mathematical modelling-based estimates. AIM: To estimate the rate of re-expansion of spontaneous pneumothoraces by a formula derived from computed tomography volumetry studies. METHODS: This retrospective study included adult patients with spontaneous pneumothorax, identified from patient management databases, who were treated conservatively. Medical records were reviewed to confirm that no intervention such as aspiration or catheter drainage had occurred. Radiographs were reviewed independently by two researchers and measured according to the method described by Collins et al. Their formula was used to estimate pneumothorax size on each date. The rate of re-expansion was defined as the change in size (%)/number of days between radiographs. Patients were excluded if they did not have at least two radiographs taken, at least 1 day apart. Data were analysed using cluster analysis by patient to minimise the effect of repeated measures from an individual patient. RESULTS: 88 episodes were identified in 57 patients. 82% were men and the patients had a median age of 22 years. The average rate of re-expansion was 2.2%/day (95% confidence interval 1.4% to 3.0%), but varied between -7.5% and 13.4%/day. CONCLUSION: Spontaneous pneumothoraces treated conservatively re-expand at an average rate of 2.2%/day.

Note on Ehrlichia chaffeensis, Ehrlichia ewingii, and "Borrelia lonestari" infection in lone star ticks (Acari: Ixodidae), Nebraska, USA.

The lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae), is established in southeastern Nebraska yet the prevalence of tick-associated microorganisms is not known. An initial PCR-based analysis for Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia infection in host-seeking adult ticks collected in southeast Nebraska was conducted. A total of 251 adult ticks collected in six sites in southeast Nebraska were tested. E. chaffeensis, E. ewingii, and Borrelia spp. were present, and the prevalence of each was approximately 1.6%. This study demonstrates that Ehrlichia spp. are present in Nebraska lone star tick populations.