Vaccine responses and hybrid immunity in people living with HIV after SARS-CoV-2 breakthrough infections.

The COVID-19 pandemic posed a challenge for people living with HIV (PLWH), particularly immune non-responders (INR) with compromised CD4 T-cell reconstitution following antiretroviral therapy (CD4 count <350 cells per mm3). Their diminished vaccine responses raised concerns about their vulnerability to SARS-CoV-2 breakthrough infections (BTI). Our in-depth study here revealed chronic inflammation in PLWH and a limited anti-Spike IgG response after vaccination in INR. Nevertheless, the imprinting of Spike-specific B cells by vaccination significantly enhanced the humoral responses after BTI. Notably, the magnitude of cellular CD4 response in all PLWH was comparable to that in healthy donors (HD). However, the polyfunctionality and phenotype of Spike-specific CD8 T cells in INR differed from controls. The findings highlight the need for additional boosters with variant vaccines, and for monitoring ART adherence and the durability of both humoral and cellular anti-SARS-CoV-2 immunity in INR.

Clinical trial: An open-label, randomised trial of different re-start strategies after treatment withdrawal in HBeAg negative chronic hepatitis B.

BACKGROUND: Stopping nucleos(t)ide analogue (NA) therapy in patients with chronic hepatitis B (CHB) may trigger a beneficial immune response leading to HBsAg loss, but clinical trials on re-start strategies are lacking. AIM: To assess whether it is beneficial to undergo a prolonged flare after NA cessation. METHODS: One-hundred-and-twenty-seven patients with HBeAg negative, non-cirrhotic CHB with at least 24 months of viral suppression on NA therapy were included. All study participants stopped antiviral therapy and were randomised to either low-threshold (ALT > 80 U/L and HBV DNA > 2000 IU/mL) or high-threshold (ALT > 100 U/L for >4 months, or ALT > 400 U/L for >2 months) for the re-start of therapy. The primary endpoint was HBsAg loss within 36 months of stopping antiviral treatment. The primary analysis was based on intention-to-treat allocation with last observation carried forward. RESULTS: There was a numerical but not statistically significant difference in HBsAg loss between the low-threshold (3 of 64; 4.7%) and the high-threshold (8 of 63; 12.7%) group (risk difference: 8.0%, 95% CI: -2.3 to 19.6, p = 0.123). None of the patients with end-of-treatment HBsAg > 1000 IU/mL achieved HBsAg loss; among those with end-of-treatment HBsAg < 1000 IU/mL, 8 of 15 (53.3%) achieved HBsAg loss in the high-threshold group compared to 3 of 26 (11.5%) in the low-threshold group. CONCLUSIONS: We could not confirm our hypothesis that a higher threshold for restart of therapy after NA withdrawal improves the likelihood of HBsAg loss within 36 months in patients with HBeAg negative CHB. Further studies including only patients with HBsAg level <1000 IU/mL and/or larger sample size and longer follow-up duration are recommended.

Differences in integrated assessment and management of non-communicable diseases (NCDs) for people with HIV across the WHO European region.

OBJECTIVES: We aimed to assess the extent of integration of non-communicable disease (NCD) assessment and management in HIV clinics across Europe. METHODS: A structured electronic questionnaire with 41 multiple-choice and rating-scale questions assessing NCD assessment and management was sent to 88 HIV clinics across the WHO European Region during March-May 2023. One response per clinic was collected. RESULTS: In all, 51 clinics from 34 countries with >100 000 people with HIV under regular follow-up responded. Thirty-seven clinics (72.6%) reported shared NCD care responsibility with the general practitioner. Systematic assessment for NCDs and integration of NCD management were common overall [median agreement 80%, interquartile range (IQR): 55-95%; and 70%, IQR: 50-88%, respectively] but were lowest in central eastern and eastern Europe. Chronic kidney disease (median agreement 96%, IQR: 85-100%) and metabolic disorders (90%, IQR: 75-100%) were regularly assessed, while mental health (72%, IQR: 63-85%) and pulmonary diseases (52%, IQR: 40-75%) were less systematically assessed. Some essential diagnostic tests such as glycated haemoglobin (HbA1c) for diabetes (n = 38/51, 74.5%), proteinuria for kidney disease (n = 30/51, 58.8%) and spirometry for lung disease (n = 11/51, 21.6%) were only employed by a proportion of clinics. The most frequent barriers for integrating NCD care were the lack of healthcare workers (n = 17/51, 33.3%) and lack of time during outpatient visits (n = 12/51, 23.5%). CONCLUSION: Most HIV clinics in Europe systematically assess and manage NCDs. People with HIV appear to be screened more frequently than the general population at the same age. There are, however, larger gaps among eastern European clinics in general and for clinics in all regions related to mental health, pulmonary diseases and the employment of some essential diagnostic tests.

Treatment cessation in HBeAg-negative chronic hepatitis B: clinical response is associated with increase in specific proinflammatory cytokines.

Patients with HBeAg-negative chronic hepatitis B may experience an immune response after stopping nucleos(t)ide analogue (NA)therapy, which may potentially trigger HBsAg loss or off-therapy sustained viral control. The immunological mechanisms determining clinical response remain poorly understood. To identify inflammatory signatures associated with defined outcomes, we analysed plasma cytokines and chemokines from 57 HBeAg-negative patients enrolled in the Nuc-Stop Study at baseline and 12 weeks after NA cessation. Clinical response at 12 weeks was classified into four groups: immune control, viral relapse, evolving clinical relapse, and resolving clinical relapse. Twelve weeks after treatment cessation 17 patients (30%) experienced immune control, 19 (33%) viral relapse, 6 (11%) evolving clinical relapse, and 15 (26%) resolving clinical relapse. There was a significant increase in interferon-γ-induced protein 10 (IP-10; p = 0.012) and tumor necrosis factor (TNF; p = 0.032) in patients with evolving clinical relapse. Sparse partial least-squares multivariate analyses (sPLS-DA) showed higher first component values for the clinical relapse group compared to the other groups, separation was driven mainly by IP-10, TNF, IL-9, IFN-γ, MIP-1ß, and IL-12. Our results demonstrate that evolving clinical relapse after NA cessation is associated with a systemic increase in the proinflammatory cytokines IP-10 and TNF.Clinical trial registration: ClinicalTrials.gov, Identifier: NCT03681132.

Impact of a TLR9 agonist and broadly neutralizing antibodies on HIV-1 persistence: the randomized phase 2a TITAN trial.

Inducing antiretroviral therapy (ART)-free virological control is a critical step toward a human immunodeficiency virus type 1 (HIV-1) cure. In this phase 2a, placebo-controlled, double-blinded trial, 43 people (85% males) with HIV-1 on ART were randomized to (1) placebo/placebo, (2) lefitolimod (TLR9 agonist)/placebo, (3) placebo/broadly neutralizing anti-HIV-1 antibodies (bNAbs) or (4) lefitolimod/bNAb. ART interruption (ATI) started at week 3. Lefitolimod was administered once weekly for the first 8 weeks, and bNAbs were administered twice, 1 d before and 3 weeks after ATI. The primary endpoint was time to loss of virologic control after ATI. The median delay in time to loss of virologic control compared to the placebo/placebo group was 0.5 weeks (P = 0.49), 12.5 weeks (P = 0.003) and 9.5 weeks (P = 0.004) in the lefitolimod/placebo, placebo/bNAb and lefitolimod/bNAb groups, respectively. Among secondary endpoints, viral doubling time was slower for bNAb groups compared to non-bNAb groups, and the interventions were overall safe. We observed no added benefit of lefitolimod. Despite subtherapeutic plasma bNAb levels, 36% (4/11) in the placebo/bNAb group compared to 0% (0/10) in the placebo/placebo group maintained virologic control after the 25-week ATI. Although immunotherapy with lefitolimod did not lead to ART-free HIV-1 control, bNAbs may be important components in future HIV-1 curative strategies. ClinicalTrials.gov identifier: NCT03837756 .

Use of contraindicated antiretroviral drugs in people with HIV/HCV coinfections receiving HCV treatment with direct-acting antivirals-Results from the EuroSIDA study.

OBJECTIVES: Our objective was to determine whether antiretroviral drugs (ARVs) were used according to the European AIDS Clinical Society (EACS) guidelines for people with HIV/hepatitis C virus (HCV) coinfection treated with direct-acting antivirals (DAAs) between 30 November 2014 and 31 December 2019 in the pan-European EuroSIDA study. METHODS: At each publication date of the EACS guidelines, plus 3 and 6 months, we calculated the number of people receiving DAAs with potential and actual ARV contraindications ('red shading' in the EACS guidelines). We used logistic regression to investigate factors associated with using contraindicated ARVs. RESULTS: Among 1406 people starting DAAs, the median age was 51 years, 75% were male, 57% reported injected drug use as an HIV risk, and 76% were from western Europe. Of 1624 treatment episodes, 609 (37.5%) occurred while the patient was receiving ARVs with potential contraindications; among them, 38 (6.2%; 95% confidence interval [CI] 4.3-8.2) involved a contraindicated ARV (18 non-nucleoside reverse transcriptase inhibitors), 16 involved protease inhibitors, and four involved integrase strand transfer inhibitors. The adjusted odds of receiving a contraindicated ARV were higher (3.25; 95% CI 1.40-7.57) among participants from east/central east Europe (vs. south) and lower (0.22; 95% CI 0.08-0.65) for 2015-2018 guidelines (vs. 2014). In total, 29 of the 32 (90.6%) patients receiving a contraindicated ARV and 441 of the 461 (95.7%) with potential ARV contraindications experienced a sustained virological response ≥12 weeks after stopping treatment (SVR12; p = 0.55). CONCLUSION: In this large heterogenous European cohort, more than one-third of people with HIV/HCV coinfection received DAAs with potential ARV contraindications, but few received a contraindicated ARV. Use of contraindicated ARVs declined over time, corresponding to the increased availability of ARV therapy regimens without interactions with DAA across Europe. Participants who received a contraindicated DAA and ARV combination still had a high rate of SVR12.

A shared mucosal gut microbiota signature in primary sclerosing cholangitis before and after liver transplantation.

BACKGROUND AND AIMS: Several characteristic features of the fecal microbiota have been described in primary sclerosing cholangitis (PSC), whereas data on mucosal microbiota are less consistent. We aimed to use a large colonoscopy cohort to investigate key knowledge gaps, including the role of gut microbiota in PSC with inflammatory bowel disease (IBD), the effect of liver transplantation (LT), and whether recurrent PSC (rPSC) may be used to define consistent microbiota features in PSC irrespective of LT. APPROACH AND RESULTS: We included 84 PSC and 51 liver transplanted PSC patients (PSC-LT) and 40 healthy controls (HCs) and performed sequencing of the 16S ribosomal RNA gene (V3-V4) from ileocolonic biopsies. Intraindividual microbial diversity was reduced in both PSC and PSC-LT versus HCs. An expansion of Proteobacteria was more pronounced in PSC-LT (up to 19% relative abundance) than in PSC (up to 11%) and HCs (up to 8%; Q FDR < 0.05). When investigating PSC before (PSC vs. HC) and after LT (rPSC vs. no-rPSC), increased variability (dispersion) in the PSC group was found. Five genera were associated with PSC before and after LT. A dysbiosis index calculated from the five genera, and the presence of the potential pathobiont, Klebsiella , were associated with reduced LT-free survival. Concomitant IBD was associated with reduced Akkermansia . CONCLUSIONS: Consistent mucosal microbiota features associated with PSC, PSC-IBD, and disease severity, irrespective of LT status, highlight the usefulness of investigating PSC and rPSC in parallel, and suggest that the impact of gut microbiota on posttransplant liver health should be investigated further.

Incidence and risk factors for suicide, death due to substance use, and violent/accidental death in people with HIV.

OBJECTIVE: Deaths due to suicide, substance use and violence/accident may reflect similar risk factors and overlap in their classification. This study aimed to investigate incidence and risk factors of mortality among people with HIV (PWH) due to these three related causes. DESIGN: Prospectively collected data from PWH at least 18 years old and under active follow-up in the EuroSIDA study from 2007 to 2019 were analysed. METHODS: Cause-specific Cox regression analysis was used to assess risk factors. RESULTS: A total of 17 881 participants were included, comprising 149 327 person-years of follow-up (PYFU). Forty participants died by suicide {incidence rate [IR] [95% confidence interval (CI)]: 0.3/1000 PYFU (0.2, 0.4)} 93 from substance use [IR (95% CI): 0.6/1000 PYFU (0.5, 0.8)], and 57 by violence/accident [IR (95% CI): 0.4/1000 PYFU (0.3, 0.5)]. An AIDS diagnosis within the last 12 months was associated with nine-fold increased risk of suicide vs. no history of AIDS [adjusted hazard ratio (aHR): 9.06; 95% CI: 2.07, 39.7]. Male gender was associated with double the risk of violent/accidental death (aHR: 2.28; 95% CI: 1.09, 4.78). PWH in Eastern Europe and those who acquired HIV by injection drug use (IDU) demonstrated a greater risk of death due to substance use or violence/accident. CONCLUSIONS: The association between a recent diagnosis of AIDS and suicide highlights a critical period for intervention. HIV infection acquired through IDU demonstrated an expected relationship with death due to substance use and violent/accidental deaths. Increased risk of death due to substance use and violence/accident in Eastern Europe demands investigation into specific differences that may drive that association.

Probiotics to HIV-Infected Immunological Nonresponders: Altered Mucosal Immunity and Microbial Diversity Restricted to Ileum.

BACKGROUND: HIV-infected immunological nonresponders (INRs) have increased risk of non-AIDS morbidity and compromised gut barrier immunity. Probiotics are widely used to improve health. We assessed the effects of probiotics in INRs with a comprehensive analysis of gut immunity and microbiome in terminal ileum and sigmoid colon. METHODS: The study involved clinical intervention with five-strain probiotic capsules (1.2 × 1010 CFUs/d) for 8 weeks in 20 INRs with CD4+ T-cell counts <400 cells/µL and plasma HIV RNA <50 copies/mL for more than 3.5 years. Colonoscopy with sampling of gut biopsies from terminal ileum and sigmoid colon and fecal and blood sampling were performed before and after the intervention. Flow cytometry (cytokine production, immune activation, and exhaustion), ELISA (inflammation, microbial translocation, and enterocyte damage), and 16S rRNA sequencing analyses were applied. RESULTS: In the terminal ileum, increased alpha diversity, increased abundance of Bifidobacterium sp., and decreased frequencies of IL-22+ CD4+ T cells were observed. The increased abundance of Bifidobacterium sp. in the terminal ileum correlated with increased fraction of CD4+ T cells in the same compartment (r = 0.54, P = 0.05) and increased CD4/CD8 ratio in peripheral blood (r = 0.49, P = 0.05). There were no corresponding changes in the sigmoid colon and no changes in fecal microbiome. Probiotic intervention did not affect peripheral blood CD4 count, viral load, or soluble markers of inflammation and microbial translocation. CONCLUSIONS: Probiotics induced segment-specific changes in the terminal ileum but did not affect systemic CD4 counts in INRs. Further clinical studies are warranted to recommend probiotics to INRs.

Human Immunodeficiency Virus-Infected Immunological Nonresponders Have Colon-Restricted Gut Mucosal Immune Dysfunction.

BACKGROUND: Human immunodeficiency virus (HIV)-infected immunological nonresponders (INRs) fail to reconstitute their CD4+ T-cell pool after initiation of antiretroviral therapy, and their prognosis is inferior to that of immunological responders (IRs). A prevailing hypothesis is that the INR phenotype is caused by a persistently disrupted mucosal barrier, but assessments of gut mucosal immunology in different anatomical compartments are scarce. METHODS: We investigated circulating markers of mucosal dysfunction, immune activation, mucosal Th17 and Th22 cells, and mucosa-adherent microbiota signatures in gut mucosal specimens from sigmoid colon and terminal ileum of 19 INRs and 20 IRs in addition to 20 HIV-negative individuals. RESULTS: INRs had higher blood levels of the enterocyte damage marker intestinal fatty acid-binding protein than IRs. In gut mucosal biopsies, INRs had lower fractions of CD4+ T cells, higher fractions of interleukin 22, and a tendency to higher fractions of interleukin 17-producing CD4+ T cells. These findings were all restricted to the colon and correlated to circulating markers of enterocyte damage. There were no observed differences in gut microbial composition between INRs and IRs. CONCLUSIONS: Restricted to the colon, enterocyte damage and mucosal immune dysfunction play a role for insufficient immune reconstitution in HIV infection independent of the gut microbiota.

A man in his seventies with pleural effusion, knee pain and dysarthria following open heart surgery. / En mann i 70-årene med pleuravæske, knesmerter og dysartri etter åpen hjertekirurgi.

BACKGROUND: Patients receiving immunosuppressive therapy are vulnerable to infections. The wide range of possible causative pathogens, often with unusual manifestations and/or confounding comorbidity, are challenging for diagnosis and treatment. CASE PRESENTATION: An active man in his seventies developed recurrent pleural effusions, peripheral oedemas and fatigue, diagnosed as post-cardiotomy syndrome, within four months of open heart surgery and ablation due to aortic stenosis and atrial fibrillation. Following initial improvement on colchicine and corticosteroids, he deteriorated with respiratory symptoms, dysarthria and knee pain. Investigations revealed abscesses in brain and soft tissue with growth of Nocardia spp. Completion of the long-term broad-spectrum antibiotic treatment was challenging. INTERPRETATION: Systemic nocardiosis that developed in a patient on corticosteroid treatment, initiated to treat post-cardiotomy syndrome, highlights the risk of opportunistic infections by widely used drugs. The case also illustrates the importance of interdisciplinary collaboration for diagnosis and treatment.

Enhanced Gut-Homing Dynamics and Pronounced Exhaustion of Mucosal and Blood CD4+ T Cells in HIV-Infected Immunological Non-Responders.

Immunological non-responders (INR), a subgroup of people living with HIV (PLHIV) who fail to restore CD4+ T cell numbers upon effective antiretroviral treatment, have impaired gut mucosal barrier function and an inferior clinical prognosis compared with immunological responders (IR). The contribution of gut-homing and exhaustion of mucosal T cells to the INR phenotype was previously unknown. Flow cytometry analysis of mononuclear cells from peripheral blood and ileal and colonic lamina propria showed that INR had higher fractions of gut-homing CD4+ T cells in blood compared with IR. In addition, gut-homing cells were more likely to display signs of exhaustion in INR. The increased CD4+ T cell exhaustion in INR was ubiquitous and not restricted to subpopulations defined by activation, differentiation or regulatory T cell markers. In INR, colon CD4+ T cell exhaustion correlated negatively with the fraction of CD4+ T cells in the same compartment, this was not apparent in the ileum. The fraction of exhausted mucosal CD4+ T cells correlated with I-FABP and REG3α, markers of enterocyte damage. We conclude that alterations of gut-homing and exhaustion of T cells may contribute to impaired gut immune and barrier functions associated with immunological non-response in PLHIV.

Probiotics to manage inflammation in HIV infection.

PURPOSE OF REVIEW: To evaluate the current scientific basis for administering probiotics to people living with HIV (PLHIV) to alleviate chronic inflammation and subsequently improve their prognosis. RECENT FINDINGS: The gut microbiome is a potential contributing factor to low-grade inflammation in HIV infection, and there is a scientific rationale for attempting to attenuate inflammation by administering probiotics. Sixteen reports from clinical studies in antiretroviral therapy (ART)-treated PLHIV assessing inflammation after probiotic intervention have been identified; half of them randomized control trials (RCT). Some of the studies report improvement in some parameters of inflammation, but results are inconsistent. No studies report improvement of CD4 counts. None of the RCTs report improvements in any markers of inflammation when analyzed according to protocol. SUMMARY: Current scientific evidence does not support the use of probiotics to alleviate inflammation in HIV infection. The potential effect of probiotic intervention in ART-treated PLHIV with high risk for inflammation remains to be investigated.

Aging and the evolution of comorbidities among HIV-positive individuals in a European cohort.

OBJECTIVES: To describe changes in the prevalence of comorbidities and risk factors among HIV-positive individuals in the EuroSIDA study. DESIGN: Comparison of two cross-sectional cohorts of HIV-positive adults under active follow-up in 2006 and 2014. METHODS: Baseline demographics and prevalence of comorbidities were described. Factors associated with the prevalence of chronic kidney disease (CKD) and cardiovascular disease (CVD) were assessed by logistic regression modelling using generalized estimating equations. RESULTS: Nine thousand, seven hundred and ninety-eight individuals were under active follow-up in EuroSIDA during 2006 and 12 882 during 2014. Compared with study participants in 2006, those in 2014 were older [median age 48.6 years (IQR 40.3-55.1) vs. 43.1 years (37.2-50.0) in 2006] and had higher prevalence of hypertension (59.6 vs. 47% in 2006), diabetes (6.3 vs. 5.4%), CKD (6.9 vs. 4.1%) and CVD (5.0 vs. 3.7%). Individuals in the 2014 cohort had higher odds for CKD (unadjusted OR 2.62, 95% CI 2.30-2.99, P < 0.0001) and CVD (OR 1.88, CI 1.68-2.10, P < 0.0001), but after multivariable adjustment for age group, comorbidities and other factors, year of cohort was no longer significantly associated with the odds of CKD [adjusted OR (aOR) 0.97, CI 0.52-1.82, P = 0.92) or of CVD (aOR 0.94, CI 0.54-1.63, P = 0.82). CONCLUSION: Between 2006 and 2014, the population aged and experienced an overall higher prevalence of non-AIDS comorbidities, including CKD and CVD. The increase in CVD could be explained by the aging population, and the increase in CKD by aging and changes in other factors. Treatment strategies balancing HIV outcomes with long-term management of comorbidities remain a priority.

Neurological melioidosis in Norway presenting with a cerebral abscess.

Neurological melioidosis is a rare condition, as less than 30 cases have been reported in the last 50 years. We present a case of neurological melioidosis, presenting with a cerebral abscess in a returning traveler from an endemic area. While traveling in Cambodia on holiday, the patient was admitted to local hospital for pneumonia. Her condition improved after antimicrobial treatment, and she returned to Norway when discharged. The patient had several contacts with the health care system after returning to Norway, due to recurrent fever and deterioration. Short-term antimicrobial treatment was given with temporary improvement in her condition. Eventually she developed stroke-like symptoms, and a cerebral abscess was found. Cultures from the abscess were positive for Burkholderia pseudomallei and the treatment was adjusted accordingly.

Epithelial-microbial crosstalk in polymeric Ig receptor deficient mice.

Innate and adaptive mucosal defense mechanisms ensure a homeostatic relationship with the large and complex mutualistic gut microbiota. Dimeric IgA and pentameric IgM are transported across the intestinal epithelium via the epithelial polymeric Ig receptor (pIgR) and provide a significant portion of the first line of natural or adaptive antibody-mediated immune defense of the intestinal mucosa. We found that colonic epithelial cells from pIgR KO mice differentially expressed (more than twofold change) more than 200 genes compared with cells from WT mice, and upregulated the expression of antimicrobial peptides in a commensal-dependent manner. Detailed profiling of microbial communities based on 16S rRNA genes revealed differences in the commensal microbiota between pIgR KO and WT mice. Furthermore, we found that pIgR KO mice showed increased susceptibility to dextran sulfate sodium-induced colitis, and that this was driven by their conventional intestinal microbiota. Thus, in the absence of pIgR, the stability of the commensal microbiota is disturbed, gut homeostasis is compromised, and the outcome of colitis is significantly worsened.

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression.

BACKGROUND: Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. METHODOLOGY/PRINCIPAL FINDINGS: Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. CONCLUSION: We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

Increase of regulatory T cells in ileal mucosa of untreated pediatric Crohn's disease patients.

BACKGROUND: Inflammatory bowel disease (IBD) of pediatric and adult onset differs in several aspects although little knowledge exists about pathogenic disparity. Regulatory T cells (Tregs) characterized as CD4+CD25+Foxp3+ are modulators of gut homeostasis, but their role in human IBD remains unclear. OBJECTIVE: To evaluate the mucosal distribution of Foxp3+ and CD25+ cells in untreated pediatric IBD patients at the time of diagnosis. MATERIAL AND METHODS: Untreated pediatric (n = 14) and adult (n = 12) Crohn's disease (CD) patients were prospectively included together with age-matched symptomatic controls. Colonic and ileal mucosal biopsies collected at diagnosis were studied by immunohistochemistry for enumeration of T cells and for mucosal expression of Foxp3 and CD25. Multicolor immunofluorescence staining was performed in situ to phenotype Foxp3+ cells as Tregs and characterize the CD25+ cells. RESULTS: The density of mucosal T cells displayed only small variations, while that of Foxp3+ cells and CD25+ cells was increased in CD patients. Multicolor immunofluorescence showed that most CD25+ cells were macrophages. Interestingly, in the ileum of pediatric CD patients the density of Foxp3+ cells was significantly higher than in adult CD patients. Co-expression of Foxp3 and CD25, as well as Foxp3 and CTLA-4, indicated that the Foxp3+ cells were Tregs. CONCLUSION: Mucosal numbers of Foxp3(+) Tregs and activated (CD25+) macrophages are elevated in both pediatric and adult ileal CD. The greater increase of ileal Foxp3+ Tregs in pediatric CD than in adult CD might contribute to the relatively less frequent phenotype of isolated ileal enteritis in CD children.

Increased number and activation of colonic macrophages in pediatric patients with untreated Crohn's disease.

BACKGROUND: Pediatric inflammatory bowel disease (IBD) may be phenotypically different from adult IBD. In IBD lesions, macrophages are overactivated, suggesting involvement of innate immunity in the pathogenesis. Here, mucosal macrophages were studied in selected untreated pediatric patients compared with adults from a population-based Norwegian cohort of IBD patients. Age-matched non-IBD controls were also included. METHODS: Untreated children (<18 years) and adults (> or =18 years) were included at diagnosis with colonic and ileal biopsies. Controls were symptomatic non-IBD patients with histologically normal gut. Frozen mucosal sections were examined by immunohistochemistry for cellular expression of the pan-macrophage marker CD68 and the costimulatory molecule CD40. Two-color immunofluorescence staining in situ was performed to identify CD40(+) macrophages. RESULTS: Non-IBD adults had significantly higher mucosal density of colonic CD68(+) macrophages than non-IBD children. In pediatric Crohn's disease (CD), macrophages were significantly increased in the colon (but not in the ileum) compared with controls. Their mucosal density in pediatric CD was significantly higher than in pediatric ulcerative colitis. The number of CD40(+) (activated) macrophages was significantly elevated in both histologically inflamed and uninflamed colon and ileum of IBD children. CONCLUSIONS: Histologically normal colon mucosa contains fewer macrophages in children than in adults. However, in colon of children with untreated CD the mucosal macrophage density is increased. Activated mucosal macrophages are increased in untreated pediatric IBD regardless of inflammatory grade. Such upregulated innate mucosal immune activation may contribute to the colonic phenotype of childhood CD.