The Effect of Metronidazole versus a Synbiotic on Clinical Course and Core Intestinal Microbiota in Dogs with Acute Diarrhea.

The usefulness of antibiotics in dogs with acute diarrhea (AD) is controversial. It is also unclear what effect metronidazole has on potential enteropathogens such as Clostridium perfringens and Escherichia coli. Thus, the aim of this study was to evaluate the effect of metronidazole vs. a synbiotic on the clinical course and core intestinal bacteria of dogs with AD. Twenty-seven dogs with AD were enrolled in this prospective, randomized, blinded clinical trial and treated with either metronidazole (METg) or a synbiotic (SYNg; E. faecium DSM 10663; NCIMB 10415/4b170). The Canine Acute Diarrhea Severity (CADS) index was recorded daily for eleven days. Bacteria were quantified using qPCR. Data were analyzed using mixed models with repeated measures. A higher concentration of E. coli was observed in the METg group vs. the SYNg group on Day 6 (p < 0.0001) and Day 30 (p = 0.01). Metronidazole had no effect on C. perfringens. C. hiranonis was significantly lower in the METg group than in the SYNg group on Days 6 and 30 (p < 0.0001; p = 0.0015). No significant differences were observed in CADS index, fecal consistency, or defecation frequency between treatment groups (except for the CADS index on one single day). In conclusion, metronidazole negatively impacts the microbiome without affecting clinical outcomes. Thus, synbiotics might be a preferred treatment option for dogs with AD.

1- and 2-Tetrazolylacetonitrile as Versatile Ligands for Laser Ignitable Energetic Coordination Compounds.

1- and 2-Tetrazolylacetonitrile (1- and 2-TAN) have been synthesized by the reaction of chloroacetonitrile with 1H-tetrazole under basic conditions. They further were reacted with sodium azide in the presence of zinc(II) chloride to form 5-((1H-tetrazol-1-yl)methyl)-1H-tetrazole (1-HTMT) and 5-((2H-tetrazol-2-yl)methyl)-1H-tetrazole (2-HTMT). The nitrogen-rich compounds have been applied as ligands for Energetic Coordination Compounds (ECCs) and show interesting coordinative behavior due to different bridging modes. The structural variability of the compounds has been proved by low-temperature X-ray analysis. The ECCs were analyzed for their sensitivities to provide information about the safety of handling and their capability to serve as primary explosives in detonator setups to replace the commonly used lead styphnate and azide. All colored ECCs were evaluated for their ignitability by laser initiation in translucent polycarbonate primer caps. In addition, the spin-crossover characteristics of [Fe(1-TAN)6](ClO4)2 were highlighted by the measurement of the temperature-dependent susceptibility curve.

Prevalence of Clostridioides difficile in Canine Feces and Its Association with Intestinal Dysbiosis.

The role of Clostridioides (C.) difficile as an enteropathogen in dogs is controversial. In humans, intestinal bile acid-dysmetabolism is associated with C. difficile prevalence. The relationship between fecal qPCR-based dysbiosis index (DI) and especially the abundance of bile acid-converting Clostridium hiranonis with the presence of C. difficile in dogs was explored across the following 4 cohorts: 358 fecal samples submitted for routine diagnostic work-up, 33 dogs with chronic enteropathy, 14 dogs with acute diarrhea, and 116 healthy dogs. Dogs that tested positive for C. difficile had significantly higher DI (median, 4.4 (range from 0.4 to 8.6)) and lower C. hiranonis (median, 0.1 (range from 0.0 to 7.5) logDNA/g) than dogs that tested negative for C. difficile (median DI, -1 (range from -7.2 to 8.9); median C. hiranonis abundance, 6.2 (range from 0.1 to 7.5) logDNA/g; p < 0.0001, respectively). In 33 dogs with CE and 14 dogs with acute diarrhea, the treatment response did not differ between C. difficile-positive and -negative dogs. In the group of clinically healthy dogs, 9/116 tested positive for C. difficile, and 6/9 of these had also an abnormal DI. In conclusion, C. difficile is strongly linked to intestinal dysbiosis and lower C. hiranonis levels in dogs, but its presence does not necessitate targeted treatment.

Correlation between Targeted qPCR Assays and Untargeted DNA Shotgun Metagenomic Sequencing for Assessing the Fecal Microbiota in Dogs.

DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI < 0 with any targeted taxa outside the reference interval, RI), mild-moderate changes (R = 0.24, 0 < DI < 2), and significant dysbiosis (R = 0.54, 0.73, and 0.91 for DI > 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs.

Comparison of the respiratory bacterial microbiome in cats with feline asthma and chronic bronchitis.

Objectives: While feline chronic bronchitis (CB) is known as neutrophilic bronchial inflammation (NI), feline asthma (FA) is defined as an eosinophilic airway inflammation (EI). Feline chronic bronchial disease refers to both syndromes, with similar clinical presentations and applied treatment strategies. Recent studies described alterations of the microbiota composition in cats with FA, but little is known about the comparison of the lung microbiota between different types of feline bronchial disease. The study aimed to describe the bacterial microbiota of the lower respiratory tracts of cats with FA and CB and to identify potential differences. Methods: Twenty-two client-owned cats with FA (n = 15) or CB (n = 7) confirmed via bronchoalveolar-lavage (BALF)-cytology were included. Next-generation sequencing analysis of 16S rRNA genes was performed on bacterial DNA derived from BALF samples. QIIME was used to compare microbial composition and diversity between groups. Results: Evenness and alpha-diversity-indices did not significantly differ between cats with FA and CB (Shannon p = 0.084, Chao 1 p = 0.698, observed ASVs p = 0.944). Based on a PERMANOVA analysis, no significant differences were observed in microbial composition between animals of both groups (Bray-Curtis metric, R-value 0.086, p = 0.785; unweighted UniFrac metric, R-value -0.089, p = 0.799; weighted Unifrac metric, R-value -0.072, p = 0.823). Regarding taxonomic composition, significant differences were detected for Actinobacteria on the phylum level (p = 0.026), Mycoplasma spp. (p = 0.048), and Acinetobacteria (p = 0.049) on the genus level between cats with FA and CB, with generally strong interindividual differences seen. There was a significant difference in the duration of clinical signs before diagnosis in animals dominated by Bacteriodetes (median 12 months, range 2-58 months) compared to animals dominated by Proteobacteria (median 1 month, range 1 day to 18 months; p = 0.003). Conclusions and relevance: Lung microbiota composition is very similar in cat populations with spontaneous FA and CB besides small differences in some bacterial groups. However, with disease progression, the lung microbiome of cats with both diseases appears to shift away from dominantly Proteobacteria to a pattern more dominated by Bacteriodetes. A substantial proportion of cats tested positive for Mycoplasma spp. via sequencing, while none of them tested positive using classical PCR.

Utility of diagnostic tests in vomiting dogs presented to an internal medicine emergency service.

Introduction: Vomiting is a common sign in dogs presenting to emergency services. It can be self-limiting, a sign of a life-threatening extraintestinal, or intestinal disorder. Reasonable diagnostics should be performed to determine the underlying cause. This study aimed to assess the utility of diagnostic tests in vomiting dogs, and its correlation with patient history, and physical examination results. Additionally, parameters to differentiate uncomplicated vomiting from complicated vomiting were investigated. Methods: In this prospective, observational, clinical study, data from 99 client-owned dogs with vomiting, presenting as first opinion cases, were evaluated. History, physical examination, duration of clinical signs, overall number of episodes of vomiting, appetite, and additional clinical signs were recorded. The standardized diagnostic evaluation of all patients included venous blood gas analysis, complete blood count, serum biochemistry profile, canine pancreatic lipase, abdominal radiographs, ultrasound, and urinalysis. Follow-up was performed 4-5 days later. Based on severity of disease and clinical course, dogs were categorized to "uncomplicated vomiting" (UN), or "complicated vomiting" (COM). The utility of each test for diagnosing the cause of vomiting was evaluated. Spearman correlation coefficient, Chi-squared-, unpaired t-, and Mann-Whitney U-test were used. Statistical significance was defined as p ≤ 0.05. Results: Out of the 99 dogs, 34 had uncomplicated courses of disease (UN). In 60/99 cases, a diagnosis was obtained, and in 39/99 cases, the cause for vomiting remained unknown. Longer duration of clinical signs, and reduced appetite were associated with higher utility of abdominal ultrasound. A poor mentation was associated with a higher utility of blood examinations and abdominal radiographs. Dogs presenting with an impaired mentation or with additional clinical signs other than diarrhea, were more likely to be in the COM group. Discussion: Based on this investigation, general recommendations concerning the diagnostic approach for patients with vomiting could not be provided. For dogs who have exclusively vomiting as a clinical sign, and present in good mentation, further investigations might not be beneficial, and these dogs may recover with symptomatic treatment alone. Additional diagnostics could be indicated in dogs with additional clinical signs other than diarrhea.

Antiviral Toll-like Receptor Signaling in Non-Parenchymal Liver Cells Is Restricted to TLR3.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Frequency of signs of chronic gastrointestinal disease in dogs after an episode of acute hemorrhagic diarrhea.

BACKGROUND: Acute enteropathy is a trigger of chronic gastrointestinal (GI) disease in humans. OBJECTIVE: To report the prevalence of and explore possible risk factors for signs of chronic GI disease in dogs after an episode of acute hemorrhagic diarrhea (AHD). ANIMALS: One hundred and fifty-one dogs, 80 dogs with a historical diagnosis of AHD, 71 control dogs with no history of AHD. METHODS: In this retrospective longitudinal study, data were collected from dogs with a historical diagnosis of AHD and healthy controls matched by breed, age and sex, aged between 1 year and 15 years of age, for which a follow-up of at least 12 months after enrolment was available. Dog owners responded to a questionnaire to determine the history of signs of chronic GI disease. RESULTS: There was a higher prevalence of signs of chronic GI disease in the dogs with a previous episode of AHD compared to control dogs (AHD 28%; controls 13%; P = .03; odds ratio = 2.57; confidence interval [CI] 95% 1.12-6.31) over a similar observation time (median 4 years; range, 1-12 years). CONCLUSIONS AND CLINICAL IMPORTANCE: Severe intestinal mucosal damage and associated barrier dysfunction might trigger chronic GI disease later in life.

C-reactive protein as a tool for monitoring response to treatment in dogs with acute hemorrhagic diarrhea syndrome.

Objectives: C-reactive protein (CRP) is an established marker for systemic inflammation in dogs that is especially elevated in dogs with sepsis. Some dogs with acute hemorrhagic diarrhea syndrome (AHDS) develop bacterial translocation and consequent sepsis during hospitalization. This study aimed to evaluate the course of CRP plasma concentrations during hospitalization and its correlation with clinical and other laboratory variables in dogs with AHDS. Methods: In this prospective, observational study, CRP was evaluated on days 0, 1, 2, and 3 in 27 client-owned dogs who presented with AHDS. Clinical examination data, blood pressure, acute patient physiologic and laboratory evaluation (APPLE) full and APPLE fast scores, and canine hemorrhagic diarrhea severity (CHDS) index were measured on the same days to evaluate the severity of the disease. Results: Twenty-five of the 27 dogs were discharged from hospital. Nineteen dogs received antimicrobial treatment due to sepsis or neutropenia. CRP values were mildly elevated on day 0 (median 27.3 mg/L; 1.0-125.8 mg/L) and markedly elevated on day 1 (median 88.9 mg/L; 1.4-192.7 mg/L). CRP concentrations decreased gradually over the following days. Moreover, CRP concentrations correlated moderately with albumin, leucocyte count, neutrophil count, and APPLE full and fast scores, but not with antimicrobial treatment. Conclusion and relevance: CRP concentrations were significantly elevated in patients with AHDS. In this study population, CRP did not help in detecting the requirement of antimicrobial treatment in dogs with AHDS. Nevertheless, as CRP can monitor the response to treatment, regular analysis can guide treatment.

[Cystoscopic-guided laser ablation for treatment of ectopic ureteroceles in 2 female dogs]. / Zystoskopiegeführte Laserablation zur Korrektur ektopischer Ureterozelen bei 2 Hündinnen.

Two female intact Labrador Retriever dogs (6 and 3 months of age, respectively) presented with a history of urinary incontinence. In both dogs, abdominal ultrasound revealed evidence of a unilateral ectopic ureterocele. Diagnosis of ureteral ectopia was established urethrocystoscopically by visualization of the ureteral orifice in the urethra, and an intramural course was confirmed via retrograde contrast fluoroscopy. Ectopic ureteral orifices were stenotic in both dogs. Cystoscopic- and fluoroscopic-guided laser ablation of the ectopic ureter were performed with a Hol:YAG laser. Following the procedure, both dogs were fully continent without any medical treatment. Cystoscopic- guided laser ablation of ureteroceles was effective and safe in these 2 dogs. Thus, this minimally invasive technique for the treatment of ectopic ureteroceles provides an alternative to surgical intervention.

[Use of antimicrobials in acute canine diarrhea - overview of potential risks, indications and alternatives]. / Antibiotikaeinsatz beim akuten Durchfall des Hundes ­ Übersicht potenzieller Risiken, Indikationen und Alternativen.

In Germany, antibiotics are frequently used in dogs with gastrointestinal disorders such as acute diarrhea. In line with global efforts to limit antibiotic use, this literature review aims to provide a guideline for the rational and judicious use of antibiotics in acute canine diarrhea. Antibiotics can lead to gastrointestinal side effects and may exert a negative influence on the intestinal microbiota in addition to increasing the occurrence of resistant bacteria. There is also evidence that chronic immunological diseases may be triggered by the administration of antibiotics. Therefore, these should not be administered in uncomplicated acute diarrhea without signs of sepsis or systemic inflammatory reaction. In addition, enteropathogenic bacteria usually do not play a role in the etiology of acute diarrhea. For select clinical entities such as acute hemorrhagic diarrhea syndrome, antibiotic therapy should only be recommended in cases displaying signs of bacterial translocation with subsequent sepsis. In the case of parvovirosis, on the other hand, the administration of antibiotics is unavoidable due to the immunological incompetence of the dog caused by the accompanying severe neutropenia.

Diagnostic value of fecal cultures in dogs with chronic diarrhea.

BACKGROUND: Culture-based assessment of the fecal microbiome using fecal culture profiles frequently is performed in dogs with chronic diarrhea, but the diagnostic value of this approach has not been determined. OBJECTIVES: To compare the reported results of fecal culture profiles and the polymerase chain reaction-based dysbiosis index (DI) between dogs with chronic diarrhea and healthy dogs; to assess interlaboratory variability in bacterial and fungal cultures among 3 veterinary diagnostic laboratories (diagnostic laboratory 1 [L1], diagnostic laboratory 2 [L2], diagnostic laboratory 3 [L3]); and to compare the reported interpretation of culture profiles (normobiosis versus dysbiosis) with those of the DI. ANIMALS: Eighteen dogs with chronic diarrhea (CDG) and 18 healthy control dogs (HG). METHODS: In this prospective, case-control study, fecal samples were submitted to 3 commercial laboratories for fecal culture. The microbiota was assessed using PCR assays. Dogs receiving antimicrobials were excluded. RESULTS: Dysbiosis index was significantly increased in CDG (mean, 0.9; SD, 3.8; 95% confidence interval [CI], -1.0; 2.8) compared to HG (mean, -3.0; SD, 2.8; CI, -4.3; -1.6; P = .0002), whereas cultures from all laboratories failed to detect significant differences (P = .66, .18, and .66, respectively). Hemolytic Escherichia coli was the only potential enteropathogen on culture, but no significant difference was found between CDG and HG. For diagnosis of dysbiosis, culture showed no agreement with DI (L1, κ = -0.21; CI, -0.44; -0.02; L2, κ = -0.33; CI, -0.58; -0.08; L3, κ = -0.25; CI, -0.39; -0.11). Furthermore, variability among the 3 laboratories was high (L1/L2, κ = 0.15; CI, -0.05; 0.35; L1/L3, κ = -0.08; CI, -0.01; -0.16; L2/L3, κ = -0.06; CI, -0.33; -0.20). CONCLUSIONS AND CLINICAL IMPORTANCE: Fecal cultures failed to distinguish between diseased and healthy dogs, and a high level of interlaboratory variation for culture was found.

Effect of amoxicillin-clavulanic acid on clinical scores, intestinal microbiome, and amoxicillin-resistant Escherichia coli in dogs with uncomplicated acute diarrhea.

BACKGROUND: Despite limited evidence of efficacy, antibiotic treatment is still frequently prescribed in dogs with uncomplicated acute diarrhea (AD). OBJECTIVE: To assess whether amoxicillin-clavulanic acid has a clinical benefit, an effect on the fecal microbiome, and the proportion of amoxicillin-resistant Escherichia coli in dogs with AD. ANIMALS: Sixteen dogs with AD of <3 days duration. METHODS: Prospective, placebo-controlled, double-blinded study. Clinical scores were compared between client-owned dogs randomly assigned to an antibiotic (AG) or a placebo (PG) group. The intestinal microbiome was analyzed using quantitative PCR assays. Amoxicillin-resistant fecal E. coli were assessed semiquantitatively with microbiological methods. RESULTS: There was no difference in clinical recovery between treated dogs or controls (CADS index day 10: AG group median: 2 (range: 1-3; CI [1.4; 2.6]); PG group median: 1.6 (range: 1-3; CI [1.1; 2.4]); P > .99). All dogs gained normal clinical scores (CADS index ≤3) after 1 to 6 days (median 2 days) after presentation. There was no significant difference in the fecal dysbiosis index (during treatment: AG mean -2.6 (SD 3.0; CI [-5.1; 0.0]); PG mean -0.8 (SD 4.0; CI [-4.2; 2.5]; P > .99) or its bacterial taxa. The proportion of resistant fecal E. coli increased (to median: 100%; range: 35%-100%) during treatment with amoxicillin-clavulanic acid and was still increased (median: 10%; range 2%-67%) 3 weeks after treatment, both of which were significantly higher proportions than in the placebo group for both time points (during treatment AG median 100% versus PG median 0.2% (P < .001); after treatment AG median 10% versus PG median 0.0% (P = .002)). CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that treatment with amoxicillin-clavulanic acid confers no clinical benefit to dogs with AD, but predisposes the development of amoxicillin-resistant E. coli, which persist for as long as 3 weeks after treatment. These findings support international guideline recommendations that dogs with diarrhea should not be treated with antimicrobials unless there are signs of sepsis.

Hepatitis B Virus Particles Activate Toll-Like Receptor 2 Signaling Initially Upon Infection of Primary Human Hepatocytes.

BACKGROUND AND AIMS: To date, conflicting data exist as to whether hepatitis B virus (HBV) has the ability to induce innate immune responses. Here, we investigated cellular changes after the first contact between HBV and primary human hepatocytes (PHH) in vitro and in vivo. APPROACH AND RESULTS: The exposure of PHH to HBV particles resulted in nuclear translocation of NFκB, followed by the expression and secretion of inflammatory cytokines (IL [interleukin] 1B, IL6, and TNF [tumor necrosis factor]). Ultraviolet irradiation of viral particles suppressed HBV infectivity but not the induction of cytokines in PHH, suggesting that the inoculum contains the immune-inducing agent. Purified HBV particles on the whole, which were prepared from HBV DNA-positive and protein-rich fractions after heparin column separation, still had immune-inducing capacity in PHH. The HBV-induced gene expression profile was similar to that induced by toll-like receptor 2 (TLR2) ligand Pam3Cys, but different from those induced by the viral sensors TLR3 or TLR7-9. Treatment of PHH with both HBV particles and Pam3Cys led to phosphorylation of ERK (extracellular signal-regulated kinase), JNK, and p38 mitogen-activated protein kinases as well as NFκB (nuclear factor kappa B). Finally, HBV-induced gene expression could be neutralized by TLR2-specific antibodies. Of note, pretreatment with an HBV entry inhibitor attenuated the TLR2-mediated response to HBV, suggesting a receptor binding-related mechanism. In liver-humanized uPA/severe combined immunodeficient (SCID)/beige mice challenged with HBV in vivo, immune induction could only marginally be seen. CONCLUSIONS: PHHs are able to sense HBV particles through TLR2, leading to an activation of anti-HBV immune responses in vitro. These findings challenge the previously described stealth properties of HBV.

Scale-up of the ex vivo expansion of encapsulated primary human T lymphocytes.

A number of evolving medical therapies call for the controlled expansion of primary human T lymphocytes. After encapsulation in sodium cellulose sulfate-poly(diallyldimethyl) ammonium chloride polyelectrolyte capsules, T lymphocytes can be expanded without persisting activation. Here, the challenge of scaling up this process is addressed. Encapsulated T lymphocytes were cultured in spinner flasks as well as in several types of the bioreactor, including fixed and fluidized beds, a waved cell bag, and a standard stirred tank reactor (STR; 1-L scale). Two proprietary T lymphocyte culture media as well as a standard RPMI-based medium were used. As before, encapsulation coincided with the presence of only a low fraction of activated T lymphocytes (peripheral blood T cells) in the total population. Unexpectedly, growth rates were lower in well-mixed reactors than those in cultivations under static conditions, that is, in T-flasks. Switching the STR to low oxygen conditions (40% air saturation) improved the growth rates to the level of the static cultures and thus forms the potential basis for efficient scale-up of T lymphocyte expansion.

TLR2 Stimulation Strengthens Intrahepatic Myeloid-Derived Cell-Mediated T Cell Tolerance through Inducing Kupffer Cell Expansion and IL-10 Production.

Hepatic APCs play a critical role in promoting immune tolerance in the liver. Recently, we have demonstrated that TLR2 stimulation on liver sinusoidal endothelial cells reverted their suppressive properties to induce T cell immunity. However, there is a paucity of information about how TLR2 stimulation modulates the immunological function of other hepatic APCs. In the current study, we investigated whether TLR2 stimulation influences the function of intrahepatic myeloid-derived cells (iMDCs) and elucidated the mechanisms involved in iMDC-induced T cell immunity. We could show that iMDCs from C57BL/6 mice can potently suppress T cell activation in a cell contact-independent manner. Ag presentation by iMDCs leads to naive CD8 T cell tolerance. To our surprise, instead of inducing cell functional maturation, TLR2 ligand palmitoyl-3-cysteine-serine-lysine-4 (P3C) stimulation further strengthens the suppressive and tolerogenic properties of iMDCs. After P3C administration, the population of Kupffer cells (KCs) of iMDCs dramatically increased. Mechanism analysis shows that KCs are essential for the enhanced inhibition of T cell activation by P3C-stimulated iMDCs. The iMDC-mediated CD8 T cell inhibition was mediated by soluble mediators, one of which was IL-10 secreted by KCs after P3C stimulation. IL-10 blockade could partially abolish iMDC-mediated T cell inhibition. Moreover, hepatitis B virus particle stimulation on iMDCs could also induce IL-10 production by the cells in a TLR2-dependent way. Our results have implications for our understanding of liver-specific tolerance and for the development of strategies to overcome T cell tolerance in situations such as chronic viral liver infections.

Creating a Biomimetic Microenvironment for the Ex Vivo Expansion of Primary Human T Lymphocytes.

The ex vivo expansion of primary human T cells is of considerable interest. Current protocols call for the addition of massive amounts of stimuli. This study presents as alternative the expansion of such cells in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (SCS/PDADMAC) polyelectrolyte microcapsules, which supports at least six cell divisions and results in >40 × 106 cells mLcapsule-1 within less than 10 d. Inside the microcapsules, the T cells are suspended in a viscous SCS-solution. The low molecular weight cut off (<10 000 Da) of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained, while nutrients and metabolites can pass. Expensive additives, such as interleukin-2 (IL-2), can be coencapsulated. Expansion then no longer requires specialized T-cell media. Moreover, these results suggest that an SCS with a low degree of sulfation has biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate.

Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

C-X-C-chemokine ligand 13 (CXCL13), the ligand for C-X-C chemokine receptor type 5 (CXCR5), is a major regulator of B-cell trafficking and plays an integral role in age-dependent clearance of hepatitis B virus (HBV) in the mouse model. However, the expression and function of CXCL13 in patients with chronic hepatitis B (CHB) remain unknown. By use of liver cell subpopulations isolated from CHB patients, we found that CXCL13 mRNA was abundantly expressed in Kupffer cells (KCs), but not in primary hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Interestingly, KC isolated from HBV-positive liver had much higher level of CXCL13 expression than non-HBV-infected controls. And its expression was induced by toll-like receptor 3 ligand poly I:C stimulation. Moreover, intense expression of CXCL13 protein and accumulation of CD4+ T and B cells were evident in follicular-like structures in the liver tissue of CHB patients, which indicated its chemotactic effect on CXCR5+ CD4+ cells and B cells. Consistently, the levels of serum CXCL13 were significantly higher in the CHB patients than in healthy controls. Furthermore, CXCL13 concentration was increased in the complete response (CR) group during weeks 0-12 and did not change significantly during the course of telbivudine treatment, compared with the patients who didn't achieve CR. In conclusion, the HBV-related increase of CXCL13 production in KC and serum CXCL13 level during telbivudine treatment might be associated with immune control of chronic HBV infection.

Nucleic acid-based polymers effective against hepatitis B Virus infection in patients don't harbor immunostimulatory properties in primary isolated liver cells.

Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2'-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.

Advanced Method for Isolation of Mouse Hepatocytes, Liver Sinusoidal Endothelial Cells, and Kupffer Cells.

Separation of pure cell populations from the liver is a prerequisite to study the role of hepatic parenchymal and non-parenchymal cells in liver physiology, pathophysiology, and immunology. Traditional methods for hepatic cell separation usually purify only single cell types from liver specimens. Here, we describe an efficient method that can simultaneously purify populations of hepatocytes (HCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs) from a single mouse liver specimen. A liberase-based perfusion technique in combination with a low-speed centrifugation and magnetic-activated cell sorting (MACS) led to the isolation and purification of HCs, KCs, and LSECs with high yields and purity.