Ralf Ignatius

Synthetic double-stranded RNAs are adjuvants for the induction of t helper 1 and humoral immune responses to human papillomavirus in rhesus macaques

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C12U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC—but not CpG-C given at the same dose—also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell–activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell–attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates.

Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

ABSTRACT Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.

Calcitonin Gene-Related Peptide Decreases Expression of HLA-DR and CD86 by Human Dendritic Cells and Dampens Dendritic Cell-Driven T Cell-Proliferative Responses Via the Type I Calcitonin Gene-Related Peptide Receptor

These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10−7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10−7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10−7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (∼50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.

High Prevalence of Giardia duodenalis Assemblage B Infection and Association with Underweight in Rwandan Children

Background Giardia duodenalis is highly endemic in East Africa but its effects on child health, particularly of submicroscopic infections, i.e., those below the threshold of microscopy, and of genetic subgroups (assemblages), are not well understood. We aimed at addressing these questions and at examining epidemiological characteristics of G. duodenalis in southern highland Rwanda. Methodology/Principal Findings In 583 children <5 years of age from communities and health facilities, intestinal parasites were assessed by triplicate light microscopy and by PCR assays, and G. duodenalis assemblages were genotyped. Cluster effects of villages were taken into account in statistical analysis. The prevalence of G. duodenalis as detected by microscopy was 19.8% but 60.1% including PCR results. Prevalence differed with residence, increased with age, and was reduced by breastfeeding. In 492 community children without, with submicroscopic and with microscopic infection, underweight (weight-for-age z-score <−2 standard deviations) was observed in 19.7%, 22.1%, and 33.1%, respectively, and clinically assessed severe malnutrition in 4.5%, 9.5%, and 16.7%. Multivariate analysis identified microscopically detectable G. duodenalis infection as an independent predictor of underweight and clinically assessed severe malnutrition. Submicroscopic infection showed respective trends. Overall, G. duodenalis was not associated with gastrointestinal symptoms but assemblages A parasites (proportion, 13%) were increased among children with vomiting and abdominal pain. Conclusions/Significance The prevalence of G. duodenalis in high-endemicity areas may be greatly underestimated by light microscopy, particularly when only single stool samples are analysed. Children with submicroscopic infections show limited overt manifestation, but constitute unrecognized reservoirs of transmission. The predominance of assemblage B in Rwanda may be involved in the seemingly unimposing manifestation of G. duodenalis infection. However, the association with impaired child growth points to its actual relevance. Longitudinal studies considering abundant submicroscopic infections are needed to clarify the actual contribution of G. duodenalis to morbidity in areas of high endemicity.

Acute childhood diarrhoea in northern Ghana: epidemiological, clinical and microbiological characteristics

BackgroundAcute diarrhoea is a major cause of childhood morbidity and mortality in sub-Saharan Africa. Its microbiological causes and clinico-epidemiological aspects were examined during the dry season 2005/6 in Tamale, urban northern Ghana.MethodsStool specimens of 243 children with acute diarrhoea and of 124 control children were collected. Patients were clinically examined, and malaria and anaemia were assessed. Rota-, astro-, noro- and adenoviruses were identified by (RT-) PCR assays. Intestinal parasites were diagnosed by microscopy, stool antigen assays and PCR, and bacteria by culturing methods.ResultsWatery stools, fever, weakness, and sunken eyes were the most common symptoms in patients (mean age, 10 months). Malaria occurred in 15% and anaemia in 91%; underweight (22%) and wasting (19%) were frequent. Intestinal micro-organisms were isolated from 77% of patients and 53% of controls (P < 0.0001). The most common pathogens in patients were rotavirus (55%), adenovirus (28%) and norovirus (10%); intestinal parasites (5%) and bacteria (5%) were rare. Rotavirus was the only pathogen found significantly more frequently in patients than in controls (odds ratio 7.7; 95%CI, 4.2–14.2), and was associated with young age, fever and watery stools. Patients without an identified cause of diarrhoea more frequently had symptomatic malaria (25%) than those with diagnosed intestinal pathogens (12%, P = 0.02).ConclusionRotavirus-infection is the predominant cause of acute childhood diarrhoea in urban northern Ghana. The abundance of putative enteropathogens among controls may indicate prolonged excretion or limited pathogenicity. In this population with a high burden of diarrhoeal and other diseases, sanitation, health education, and rotavirus-vaccination can be expected to have substantial impact on childhood morbidity.

Reduced Peripheral and Mucosal Tropheryma whipplei-Specific Th1 Response in Patients with Whipple’s Disease

Whipple’s disease is a rare infectious disorder caused by Tropheryma whipplei. Major symptoms are arthropathy, weight loss, and diarrhea, but the CNS and other organs may be affected, too. The incidence of Whipple’s disease is very low despite the ubiquitous presence of T. whipplei in the environment. Therefore, it has been suggested that host factors indicated by immune deficiencies are responsible for the development of Whipple’s disease. However, T. whipplei-specific T cell responses could not be studied until now, because cultivation of the bacteria was established only recently. Thus, the availability of T. whipplei Twist-MarseilleT has enabled the first analysis of T. whipplei-specific reactivity of CD4+ T cells. A robust T. whipplei-specific CD4+ Th1 reactivity and activation (expression of CD154) was detected in peripheral and duodenal lymphocytes of all healthy (16 young, 27 age-matched, 11 triathletes) and disease controls (17 patients with tuberculosis) tested. However, 32 Whipple’s disease patients showed reduced or absent T. whipplei-specific Th1 responses, whereas their capacity to react to other common Ags like tetanus toxoid, tuberculin, actinomycetes, Giardia lamblia, or CMV was not reduced compared with controls. Hence, we conclude that an insufficient T. whipplei-specific Th1 response may be responsible for an impaired immunological clearance of T. whipplei in Whipple’s disease patients and may contribute to the fatal natural course of the disease.

Impaired Immune Functions of Monocytes and Macrophages in Whipple's Disease

BACKGROUND & AIMS Whipples disease is a chronic multisystemic infection caused by Tropheryma whipplei. Host factors likely predispose for the establishment of an infection, and macrophages seem to be involved in the pathogenesis of Whipples disease. However, macrophage activation in Whipples disease has not been studied systematically so far. METHODS Samples from 145 Whipples disease patients and 166 control subjects were investigated. We characterized duodenal macrophages and lymphocytes immunohistochemically and peripheral monocytes by flow cytometry and quantified mucosal and systemic cytokines and chemokines indicative for macrophage activation. In addition, we determined duodenal nitrite production and oxidative burst induced by T whipplei and by other bacteria. RESULTS Reduced numbers of duodenal lymphocytes, increased numbers of CD163(+) and stabilin-1(+), reduced numbers of inducible nitric synthase+ duodenal macrophages, and increased percentages of CD163(+) peripheral monocytes indicated a lack of inflammation and a M2/alternatively activated macrophage phenotype in Whipples disease. Incubation with T whipplei in vitro enhanced the expression of CD163 on monocytes from Whipples disease patients but not from control subjects. Chemokines and cytokines associated with M2/alternative macrophage activation were elevated in the duodenum and the peripheral blood from Whipples disease patients. Functionally, Whipples disease patients showed a reduced duodenal nitrite production and reduced oxidative burst upon incubation with T whipplei compared with healthy subjects. CONCLUSIONS The lack of excessive local inflammation and alternative activation of macrophages, triggered in part by the agent T whipplei itself, may explain the hallmark of Whipples disease: invasion of the intestinal mucosa with macrophages incompetent to degrade T whipplei.

Molecular characterization of enteric viral agents from children in northern region of Ghana

Viral gastrointestinal infections are among the most important causes of childhood morbidity and mortality, especially in non‐industrialized countries. The objective of this study was the molecular characterization of rotaviruses, noroviruses, adenoviruses, astroviruses, and enteroviruses obtained from 367 children in the Northern Region of Ghana. One hundred and forty‐two rotavirus‐positive stool samples were examined. The most frequent type identified was G1P[8] occurring in 80% of the cases. Of 27 norovirus positive samples, 5 isolates belonged to genogroup I and 22 to genogroup II. Adenoviruses were detected in 73 samples; 23.3% of these belonged to genogroup F, 31.5% to D, 17.8% to A, 15.1% to C, and 12.3% to B. Astrovirus typing of 12 positive samples displayed a distribution into four different genotypes: five sequences clustered with AstV‐8, four with AstV‐2, two with AstV‐5, and one with AstV‐6. Twenty‐three different enterovirus types were identified in 45 positive samples, coxsackievirus A24 being the most frequent pathogen (18%). This first, comprehensive molecular characterization of enteric viruses in northern Ghana provides baseline data for the molecular epidemiology of these pathogens and immunisation strategies. The available rotavirus vaccines cover the predominant G1P[8] type and would reduce substantially disease burden in that area. J. Med. Virol. 80:1790–1798, 2008.

Aetiology of community-acquired, acute gastroenteritis in hospitalised adults: a prospective cohort study

BackgroundThe aetiology of severe gastroenteritis leading to hospitalisation in adults frequently remains unclear. Our objective was to study the causes and characteristics of community-acquired, acute gastroenteritis in adult hospitalized patients to support the clinical management of these patients.MethodsFrom August 2005 to August 2007, we conducted a prospective cohort study among patients ≥18 y hospitalized with community-acquired gastroenteritis in a university hospital in Berlin, Germany. Stool specimens were examined for 26 gastrointestinal pathogens, supplemented by serologic tests for antibodies to Campylobacter spp., Yersinia spp., and Entamoeba histolytica. Patient data on demographics and clinical presentation were recorded and analyzed. Coexisting medical conditions were assessed using the Charlson Comorbidity Index score.ResultsOf 132 patients presenting with acute community-acquired gastroenteritis, 104 were included in the study. A non-infectious aetiology was diagnosed in 8 patients (8%). In 79 (82%) of the remaining 96 patients at least one microorganism was identified. Campylobacter spp. (35%) was detected most frequently, followed by norovirus (23%), Salmonella spp. (20%), and rotavirus (15%). In 46% of the patients with Campylobacter spp. infection, the diagnosis was made solely by serology. More than one pathogen was found in seventeen (22%) patients. Simultaneous infection was significantly more likely in patients with rotavirus and salmonella infections (RR 3.6; 95% CI: 1.8–7.4; RR 2.5; 95%CI: 1.2–5.5). Length of hospital stay (median: 5.5 days) was independent of the pathogen, but was associated with coexisting medical conditions (OR 4,8; 95%CI:2,0–11,6).ConclusionKnown enteric pathogens were detected in 82% of adult patients who were hospitalized with acute gastroenteritis. We found that currently used culture-based methods may miss a substantial proportion of Campylobacter infections, and additional serological testing for Campylobacter should be considered. Viral infections emerged as an important cause of severe gastroenteritis in adults, and viral-bacterial co-infections in adults are probably underrecognized so far. The presence of coexisting medical conditions – but not the etiological agent – was a predictor for the duration of the hospital stay.

Calreticulin from the intestinal nematode Heligmosomoides polygyrus is a Th2-skewing protein and interacts with murine scavenger receptor-A.

Helminth infections are commonly associated with a Th2 immune response, yet only a few parasite molecules involved in triggering such immune responses have been identified. Here, we describe the Th2-skewing property of calreticulin of Heligmosomoides polygyrus (HpCRT). HpCRT is a secreted protein most abundantly expressed by tissue invasive larvae (L4). Native HpCRT purified from adult worm extract (nHpCRT) stimulated robust IL-4 release from CD4(+) T cells of H. polygyrus infected mice. Interestingly, CD4(+) T cells also produced significant amounts of IL-10 while IFN-gamma was not detectable. Likewise, immunization with recombinant HpCRT (rHpCRT) without extrinsic adjuvant led predominantly to a specific IL-4 production implying the innate ability of HpCRT to drive Th2 responses. The triggering of a Th2-skewed immune response to rHpCRT is corroborated by the induction of HpCRT-specific IgG1 and IgE antibodies. Furthermore, rHpCRT bound to scavenger receptor type A (SR-A) on dendritic cells, and interaction of HpCRT with SR-A led to internalization of HpCRT that could be partially blocked by competition with SR-A ligands as well as with an anti-SR-A monoclonal antibody. Hence, our data imply that nematode calreticulin interacts with a mammalian scavenger receptor and at the same time induces a Th2 response.