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Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-β and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors., Author summary Urinary tract infections affect millions of individuals annually, and many patients suffer from recurring infections several times a year. Antibiotic resistance is increasing rapidly and new strategies are needed to treat even these common bacterial infections. One approach is to use the protective power of asymptomatic bacterial carriage, which has been shown to protect the host against symptomatic urinary tract infection. Instilling “nice” bacteria in the urinary bladder is therefore a promising alternative approach to antibiotic therapy. In an effort to increase the therapeutic use of asymptomatic bacteriuria, we reintroduced bacterial adhesion molecules into the therapeutic Escherichia coli strain 83972 and inoculated patients who are in need of alternative therapy. To our great surprise, the P fimbriated variant caused symptoms, despite lacking other virulence factors commonly thought to be necessary to cause disease. In contrast, type 1 fimbriae, did not provoke symptoms but enhanced the beneficial properties of the wild-type strain. This is explained by a divergent effect of these fimbrial types on host gene expression, where P fimbriae activate the IRF-7 transcription factor that regulates pathology in infected kidneys, suggesting that a single, potent virulence gene may be sufficient to create virulence in human hosts.

The nervous system is engaged by infection, indirectly through inflammatory cascades or directly, by bacterial attack on nerve cells. Here we identify a neuro-epithelial activation loop that participates in the control of mucosal inflammation and pain in acute cystitis. We show that infection activates Neurokinin-1 receptor (NK1R) and Substance P (SP) expression in nerve cells and bladder epithelial cells in vitro and in vivo in the urinary bladder mucosa. Specific innate immune response genes regulated this mucosal response, and single gene deletions resulted either in protection (Tlr4−/− and Il1b−/− mice) or in accentuated bladder pathology (Asc−/− and Nlrp3−/− mice), compared to controls. NK1R/SP expression was lower in Tlr4−/− and Il1b−/− mice than in C56BL/6WT controls but in Asc−/− and Nlrp3−/− mice, NK1R over-activation accompanied the exaggerated disease phenotype, due, in part to transcriptional de-repression of Tacr1. Pharmacologic NK1R inhibitors attenuated acute cystitis in susceptible mice, supporting a role in disease pathogenesis. Clinical relevance was suggested by elevated urine SP levels in patients with acute cystitis, compared to patients with asymptomatic bacteriuria identifying NK1R/SP as potential therapeutic targets. We propose that NK1R and SP influence the severity of acute cystitis through a neuro-epithelial activation loop that controls pain and mucosal inflammation.

Background Most colon cancers start with dysregulated Wnt/β-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. Objective To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc Min/+ mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. Method HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/β-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. Results Peroral HAMLET administration reduced tumour progression and mortality in Apc Min/+ mice. HAMLET accumulated specifically in tumour tissue, reduced β-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered β-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling β-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. Conclusions These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1β)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1β processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1β processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc -/- and Nlrp3 -/- mice. The resulting IL-1β hyper-activation loop included a large number of IL-1β-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc -/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1β and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. Trial Registration The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov)., Author Summary Infections continue to threaten human health as pathogenic organisms outsmart available therapies with remarkable genetic versatility. Fortunately, microbial versatility is matched by the flexibility of the host immune system which provide a rich source of novel therapeutic concepts. Emerging therapeutic solutions include substances that strengthen the immune system rather than killing the bacteria directly. Selectivity is a concern, however, as boosting of the antibacterial immune response may cause collateral tissue damage. This study addresses how the host response to urinary bladder infection causes acute cystitis and how this response can be attenuated in patients who suffer from this very common condition. We identify the cytokine Interleukin-1 beta (IL-1β) as a key immune response determinant in acute cystitis and successfully treat mice with severe acute cystitis by inhibiting IL-1β or the enzyme MMP-7 that processes IL-1β to its active form. Finally, we detect elevated levels of these molecules in urine samples from patients with cystitis, suggesting clinical relevance and a potential role of IL-1β and MMP-7 both as therapeutic targets and as biomarkers of infection. These findings provide a much needed, molecular framework for the pathogenesis and treatment of acute cystitis.

We have established a novel strategy to reduce the risk for recurrent urinary tract infection (UTI), where rapidly increasing antibiotic resistance poses a major threat. Epidemiologic studies have demonstrated that asymptomatic bacteriuria (ABU) protects the host against symptomatic infections with more virulent strains. To mimic this protective effect, we deliberately establish ABU in UTI-prone patients, who are refractory to conventional therapy. The patients are inoculated with Escherichia coli (E. coli) 83972, now widely used as a prototype ABU strain. Therapeutic efficacy has been demonstrated in a placebo-controlled trial, supporting the feasibility of using E. coli 83972 as a tool to prevent recurrent UTI and, potentially, to outcompete antibiotic-resistant strains from the human urinary tract. In addition, the human inoculation protocol offers unique opportunities to study host-parasite interaction in vivo in the human urinary tract. Here, we review the clinical evidence for protection using this approach as well as some molecular insights into the pathogenesis of UTI that have been gained during these studies.

A paradigm shift is needed to improve and personalize the diagnosis of infectious disease and to select appropriate therapies. For many years, only the most severe and complicated bacterial infections received more detailed diagnostic and therapeutic attention as the efficiency of antibiotic therapy has guaranteed efficient treatment of patients suffering from the most common infections. Indeed, treatability almost became a rationale not to analyze bacterial and host parameters in these larger patient groups. Due to the rapid spread of antibiotic resistance, common infections like respiratory tract- or urinary-tract infections (UTIs) now pose new and significant therapeutic challenges. It is fortunate and timely that infectious disease research can offer such a wealth of new molecular information that is ready to use for the identification of susceptible patients and design of new suitable therapies. Paradoxically, the threat of antibiotic resistance may become a window of opportunity, by encouraging the implementation of new diagnostic and therapeutic approaches. The frequency of antibiotic resistance is rising rapidly in uropathogenic organisms and the molecular and genetic understanding of UTI susceptibility is quite advanced. More bold translation of the new molecular diagnostic and therapeutic tools would not just be possible but of great potential benefit in this patient group. This chapter reviews the molecular basis for susceptibility to UTI, including recent advances in genetics, and discusses the consequences for diagnosis and therapy. By dissecting the increasingly well-defined molecular interactions between bacteria and host and the molecular features of excessive bacterial virulence or host-response malfunction, it is becoming possible to isolate the defensive from the damaging aspects of the host response. Distinguishing “good” from “bad” inflammation has been a long-term quest of biomedical science and in UTI, patients need the “good” aspects of the inflammatory response to resist infection while avoiding the “bad” aspects, causing chronicity and tissue damage.

Our knowledge of the molecular mechanisms of urinary tract infection (UTI) pathogenesis has advanced greatly in recent years. In this review, we provide a general background of UTI pathogenesis, followed by an update on the mechanisms of UTI susceptibility, with a particular focus on genetic variation affecting innate immunity. The innate immune response of the host is critically important in the antibacterial defence mechanisms of the urinary tract, and bacterial clearance normally proceeds without sequelae. However, slight dysfunctions in these mechanisms may result in acute disease and tissue destruction. The symptoms of acute pyelonephritis are caused by the innate immune response, and inflammation in the urinary tract decreases renal tubular function and may give rise to renal scarring, especially in paediatric patients. In contrast, in children with asymptomatic bacteriuria (ABU), bacteria persist without causing symptoms or pathology. Pathogenic agents trigger a response determined by their virulence factors, mediating adherence to the urinary tract mucosa, signalling through Toll-like receptors (TLRs) and activating the defence mechanisms. In ABU strains, such virulence factors are mostly not expressed. However, the influence of the host on UTI severity cannot be overestimated, and rapid progress is being made in clarifying host susceptibility mechanisms. For example, genetic alterations that reduce TLR4 function are associated with ABU, while polymorphisms reducing IRF3 or CXCR1 expression are associated with acute pyelonephritis and an increased risk for renal scarring. It should be plausible to "individualize" diagnosis and therapy by combining information on bacterial virulence and the host response.

Boosting innate immunity represents an important therapeutic alternative to antibiotics. However, the molecular selectivity of this approach is a major concern because innate immune responses often cause collateral tissue damage. We identify the transcription factor interferon regulatory factor 7 (IRF-7), a heterodimer partner of IRF-3, as a target for non-antibiotics-based therapy of bacterial infections. We found that the efficient and self-limiting innate immune response to bacterial infection relies on a tight balance between IRF-3 and IRF-7. Deletion of Irf3 resulted in overexpression of Irf7 and led to an IRF-7-driven hyperinflammatory phenotype, which was entirely prevented if Irf7 was deleted. We then identified a network of strongly up-regulated, IRF-7-dependent genes in Irf3(-/-) mice with kidney pathology, which was absent in Irf7(-/-) mice. IRF-3 and IRF-7 from infected kidney cell nuclear extracts were shown to bind OAS1, CCL5, and IFNB1 promoter oligonucleotides. These data are consistent in children with low IRF7 expression in the blood: attenuating IRF7 promoter polymorphisms (rs3758650-T and rs10902179-G) negatively associated with recurrent pyelonephritis. Finally, we identified IRF-7 as a target for immunomodulatory therapy. Administering liposomal Irf7 siRNA to Irf3(-/-) mice suppressed mucosal IRF-7 expression, and the mice were protected against infection and renal tissue damage. These findings offer a response to the classical but unresolved question of "good versus bad inflammation" and identify IRF7 as a therapeutic target for protection against bacterial infection.

The murine chemokine receptor 2 (mCXCR2) controls resistance to urinary tract infection. We have previously shown that mCXCR2 knockout mice develop severe acute pyelonephritis and renal tissue damage with sub-epithelial neutrophil entrapment. In this study we examined the relative importance of neutrophil- and epithelial-specific mCXCR2 expression for bacterial clearance in bone marrow chimeric mice infected with uropathogenic Escherichia coli. Mice expressing mCXCR2 on their neutrophils responded rapidly to experimental urinary tract infection, clearing the infection from the kidneys. Mice lacking epithelial mCXCR2, however, showed delayed exit of neutrophils from the tissues. Mice lacking neutrophil mCXCR2 and mice with no mCXCR2 had no neutrophil recruitment and bacterial clearance. Mice expressing mCXCR2 only in their epithelial cells had a transient epithelial chemokine response; however, neutrophil recruitment was inhibited and bacteria grew without constraint. Our study shows that the expression of mCXCR2 on hematopoietic cells was crucial for bacterial clearance, while its expression on non-bone marrow-derived cells influenced the neutrophil response. These results emphasize the importance of mCXCR2 for the innate defense against urinary tract infection.

Background. Urinary tract infections (UTIs) are important causes of morbidity and death. The present study investigated whether genetic factors influence susceptibility to acute pyelonephritis (APN). CXCR1 expression was investigated as a factor predisposing to APN, because low CXCR1 expression has been associated with disease susceptibility in mice and disease-prone children. Methods. The families of APN-prone children (n=130) and of age-matched control subjects without UTI (n=101) were studied. Three- generation pedigrees of UTI-associated morbidity were established by means of structured interviews of the families. CXCR1 expression was quantified by flow cytometric analysis of peripheral blood neutrophils obtained from family members and control subjects. Results. APN was significantly more common in the family members of the APN-prone children (20 [15%] of 130 family members) than in the relatives of the control subjects (3 [3%] of 101 family members) (P

The defense against mucosal infections relies on chemokines that recruit inflammatory cells to the mucosa. This study examined if the chemokine response to uro-pathogenic Escherichia coli is influenced by fimbrial expression. The CXC (CXCL1, CXCL5, CXCL8, CXCL9, CXCL10) and CC chemokines (CCL2, CCL3, CCL5) were quantified after in vitro infection of uro-epithelial cells with a fimbriated E. coli pyelonephritis isolate, or with P or type 1 fimbriated transformants of an avirulent E. coli K-12 strain. The response profile was shown to vary with the fimbrial type. Type 1 fimbriated E. coli elicited mainly CXCL1 and CXCL8, whereas P fimbriated E. coli stimulated CCL2 and CCL5 and class II were more potent chemokine inducers than class III P fimbriae. Chemokines were also quantified in urine samples from 73 patients with febrile urinary tract infection, and analyzed as a function of disease severity and fimbrial expression by the strain infecting each patient. A complex CXC and CC chemokine response was detected in patient urine, with a significant influence of the fimbrial type. The results show that virulence factors like fimbriae may modify the mucosal chemokine response. This mechanism may allow the host to adjust the inflammatory cell infiltrate to fit the infecting strain.

Urinary tract infections offer an important and highly relevant model to understand innate immune-mediated protection against mucosal infection and innate immune genetics of susceptibility, including promoter polymorphisms reducing Toll-like receptor (TLR) and interferon regulatory factor expression. Whereas virulent Escherichia coli causes severe, potentially life-threatening disease by breaking the inertia of the mucosal barrier, the most common outcome of bacteriuria is an asymptomatic carrier state (ABU) resembling commensalism at other mucosal sites. Pathogens gain a short-term advantage by expressing virulence factors that dysregulate a range of innate signaling pathways and effector functions, including molecules that inactivate TLR signaling. Genome sequencing has revealed that ABU strains, in contrast, have evolved by reductive evolution with a loss of virulence genes and gain of a new class of host RNA polymerase II suppressive activity. These extremes illustrate aspects of “good” versus “bad” inflammation, pathology versus symbiosis. Novel therapeutic approaches based on these molecular interactions are discussed.

This study presents evidence that human platelets bind lipopolysaccharide (LPS) from enterohemorrhagic Escherichia coli (EHEC) through a complex of toll-like receptor 4 (TLR4) and CD62, leading to their activation. TLR4 colocalized with CD62 on the platelet membrane, and the TLR4 specificity of LPS binding to platelets was confirmed using C57BL/10ScN mice lacking Tlr4. Only platelets from TLR4 wild-type mice bound O157LPS in vitro. After in vivo injection, O157LPS bound to platelets from wild-type mice, which had lower platelet counts than did mice lacking TLR4. Mouse experiments confirmed that O157LPS binding to TLR4 is the primary event leading to platelet activation, as shown by CD40L expression, and that CD62 further contributes to this process. Activation of human platelets by EHEC-LPS was demonstrated by expression of the activated GPIIb/IIIa receptor, CD40L, and fibrinogen binding. In perfusion experiments, platelet activation on endothelial cells was TLR4 and CD62 dependent. O157LPS was detected on platelets from 12 of 14 children with EHEC-associated hemolytic uremic syndrome (HUS) and on platelets from 2 children before the development of HUS but not on platelets of EHEC-infected children in whom HUS did not develop (n = 3). These data suggest that O157LPS on platelets might contribute to platelet consumption in HUS. (Blood. 2006;108:167-176)

Mucosal pathogens recognize glycoconjugate receptors at the site of infection, and attachment is an essential first step in disease pathogenesis. Inhibition of attachment may prevent disease, and several approaches have been explored. This review discusses the prevention of bacterial attachment and disease by agents that modify the glycosylation of cell surface glycoconjugates. Glycosylation inhibitors were tested in the urinary tract infection model, where P-fimbriated Escherichia coli rely on glycosphingolipid receptors for attachment and tissue attack. N-butyldeoxynojirimycin blocked the expression of glucosylceramide-derived glycosphingolipids and attachment was reduced. Bacterial persistence in the kidneys was impaired and the inflammatory response was abrogated. N-butyldeoxynojirimycin was inactive against strains which failed to engage these receptors, including type 1 fimbriated or nonadhesive strains. In vivo attachment has been successfully prevented by soluble receptor analogues, but there is little clinical experience of such inhibitors. Large-scale synthesis of complex carbohydrates, which could be used as attachment inhibitors, remains a technical challenge. Antibodies to bacterial lectins involved in attachment may be efficient inhibitors, and fimbrial vaccines have been developed. Glycosylation inhibitors have been shown to be safe and efficient in patients with lipid storage disease and might therefore be tested in urinary tract infection. This approach differs from current therapies, including antibiotics, in that it targets the pathogens which recognize these receptors.

Purpose of review Urinary tract infections (UTIs) are common, dangerous and interesting. Susceptible individuals experience multiple, often clustered episodes, and in a subset of patients, infections progress to acute pyelonephritis (APN), sometimes accompanied by uro-sepsis. Others develop asymptomatic bacteriuria (ABU). Here, we review the molecular basis for these differences, with the intention to distinguish exaggerated host responses that drive disease from attenuated responses that favour protection and to highlight the genetic basis for these extremes, based on knock-out mice and clinical studies. Recent findings The susceptibility to UTI is controlled by specific innate immune signalling and by promoter polymorphisms and transcription factors that modulate the expression of genes controlling these pathways. Gene deletions that disturb innate immune activation either favour asymptomatic bacteriuria or create acute morbidity and disease. Promoter polymorphisms and transcription factor variants affecting those genes are associated with susceptibility in UTI-prone patients. Summary It is time to start using genetics in UTI-prone patients, to improve diagnosis and to assess the risk for chronic sequels such as renal malfunction, hypertension, spontaneous abortions, dialysis and transplantation. Furthermore, the majority of UTI patients do not need follow-up, but for lack of molecular markers, they are unnecessarily investigated.

Human renal epithelial cells express iNOS in response to cytokines but not bacteria.BackgroundEpithelial cells form the mucosal barriers that prevent the entry of mucosal pathogens, and respond to bacterial infections by producing various host defense molecules. In this study, we examined the inducible nitric oxide synthase (iNOS) response of primary human renal tubular epithelial cells (HRTEC) following infection with uropathogenic Escherichia coli Hu734, or stimulation with lipopolysaccharide (LPS) or cytokines.MethodsInduction of iNOS was examined by RT-PCR, Western blot, immunohistochemistry and nitrite measurements. The effects of endogenously produced nitric oxide (NO), and exogenously applied DETA/NO, SIN-1 and H2O2 on cell viability were analyzed using a respiration assay.ResultsHRTEC did not produce NO following infection with E. coli Hu734, LPS alone, or in combination with interferon-γ (IFN-γ), even though these agents caused a marked increase in iNOS expression by RAW 264.7, a macrophage cell line. In contrast, iNOS protein and mRNA expression by HRTEC increased after exposure to a cytokine mixture consisting of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α) and IFN-γ. This was due to the combination of IL-1β and IFN-γ, but the individual cytokines had no effect. Inducible NOS-expressing cell cultures showed reduced viability, and this effect was inhibited with the NOS inhibitor L-NMMA in RAW 264.7 cells, but not in HRTEC. HRTEC were more sensitive to oxidative stress induced by H2O2 than to nitrogen stress induced by DETA/NO.ConclusionsWe conclude that uropathogenic E. coli that attach to HRTEC fail to directly activate iNOS expression, and that iNOS expression during bacterial infection is more likely to result from stimulation by local cytokines such as IL-1β and IFN-γ.

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.

Microbial determinants of acute-disease severity and tissue damage have been extensively studied, but less is known about genetic variation influencing host susceptibility. This chapter discusses two candidate genes with strong effects on the innate immune response and the antibacterial defense in the urinary tract and with major but opposite effects on urinary tract infection (UTI) susceptibility. The chapter explains that defects in TLR4 expression are protective and associated with asymptomatic bacteriuria (ABU) while defects in CXCR1 expression promote acute pyelonephritis (APN) and renal scarring. C3H/HeJ mice, then known as lipopolysaccharide (LPS)-nonresponder mice, had an increased susceptibility to UTI, as shown by delayed bacterial clearance. It also had an impaired innate immune response, suggesting that defects in innate immunity are of great importance for the antibacterial defense of the urinary tract. Studies of the murine model showed that the antibacterial defense of the urinary tract mucosa relies on innate immunity and that TLR4 plays a central role in the early host defense against infection. The results suggest that genetic variation of the TLR4 promoter is an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility. It was found that the protein expression was reduced and additionally the level of CXCR1 transcript and protein expression was lower in this new subset of pediatric patients. There is a great clinical need to identify genetic variants that improve resistance or increase susceptibility to infectious pathogens.

This chapter describes the signaling pathways in the urinary tract and their relevance to asymptomatic carriage, acute symptomatic disease, and chronic infection with tissue damage. There is evidence that P fimbriae enhance bacterial virulence by promoting both intestinal colonization and spread to the urinary tract, by promoting the establishment of bacteriuria, by facilitating the establishment of bacteremia, by activating the innate host response, and by resisting neutrophil killing. Escherichia coli P fimbriae use glycosphingolipid receptors (GSLs) as primary receptors to adhere to the host cells and use TLR4 as coreceptors in transmembrane signaling and cell activation. The nonfimbriated E. coli strain did not induce a host response in either Tlr4+/+ or Tlr4-/- mice, demonstrating that P fimbriae and Tlr4 both were needed to trigger the innate host response. The fimbriae have been identified as virulence factors in the murine experimental model of urinary tract infection (UTI) and as colonization factors of the large intestine, but a role in virulence is potentially difficult to reconcile with the occurrence of type 1 fimbriae in both virulent and commensal strains. The GSL recognition receptors are essential for P fimbriae to adhere and to recruit TLR4 for signaling. The expression of receptors for P fimbriae reflects the P blood group, since the receptor structures also act as the P blood group of the host.

Most studies on innate immunity have focused on macrophages, as these cells are crucial defenders against infection of the systemic compartments. This chapter talks about (i) the molecular mechanisms used by bacteria to trigger the innate host response, (ii) neutrophils as effectors of the antimicrobial defense of the urinary tract, and (iii) genetic defects in innate host defense pathways that explain the susceptibility to urinary tract infections (UTIs). P-fimbriated Escherichia coli is used to examine the role of recognition receptors and toll-like receptor-4 (TLR4) coreceptors in epithelial cell activation. Cell activation can proceed in two steps, involving a primary ligand-binding receptor and a second receptor responsible for transmembrane signaling, which in this model is TLR4. The authors have speculated that epithelial unresponsiveness to lipopolysaccharide (LPS) may be essential to maintain mucosal integrity, and have used the murine IL-8 receptor homologue (mIL-8Rh-/-) mouse to study chemokines and chemokine receptors in the defense against UTI. Inactivation of a single gene encoding mIL-8Rh is sufficient to convert the mice from a resistant to a susceptible phenotype, as defined both by acute disease susceptibility and by chronic disease development. The chemokine receptors must be functional to avoid the trapping of neutrophils that results in tissue destruction.

International audience; Immunity in the urinary tract has distinct and poorly understood pathophysiological characteristics and urinary tract infections (UTIs) are important causes of morbidity and mortality. We investigated the role of the soluble pattern recognition molecule pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, in UTIs. PTX3-deficient mice showed defective control of UTIs and exacerbated inflammation. Expression of PTX3 was induced in uroepithelial cells by uropathogenic Escherichia coli (UPEC) in a Toll-like receptor 4 (TLR4)- and MyD88-dependent manner. PTX3 enhanced UPEC phagocytosis and phagosome maturation by neutrophils. PTX3 was detected in urine of UTI patients and amounts correlated with disease severity. In cohorts of UTI-prone patients, PTX3 gene polymorphisms correlated with susceptibility to acute pyelonephritis and cystitis. These results suggest that PTX3 is an essential component of innate resistance against UTIs. Thus, the cellular and humoral arms of innate immunity exert complementary functions in mediating resistance against UTIs.

Uropathogenic Escherichia coli elicit a host response that determines the severity of urinary tract infection (UTI). Specific adherence mechanisms allow the bacteria to initiate this process by targeting epithelial cells in the urinary tract mucosa. Epidemiological studies show a strong association of P-fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo. The present study examined the ability of P-fimbriae to induce inflammation in the human urinary tract. Patients were subjected to intravesical inoculation with a non-fimbriated E. coli strain or transformants of this strain expressing P-fimbriae. The inflammatory response was analysed as a function of P-fimbrial expression. The P-fimbriated transformants invariably caused higher interleukin (IL)-8, IL-6 and neutrophil responses in the urinary tract than the ABU strain. Furthermore, loss of P-fimbrial expression in vivo was accompanied by a return to background levels of neutrophils, IL-6 and IL-8 in individual patients. The results demonstrate that the pap sequences confer on a non-fimbriated, avirulent strain the ability to induce a host response in the human urinary tract. P-fimbriae thus fulfil the 'molecular Koch-Henle postulates' linking a single virulence factor to host response induction.

Urinary tract infections (UTIs) vary in pathogenesis and severity. After their ascent into the urinary tract, bacteria may establish asymptomatic bacteriuria (ABU), cause acute cystitis, or cause acute pyelonephritis. Research during the last few decades has established that the site of infection and the disease severity are influenced by bacterial virulence. In the 1940s, hemolysin was shown to identify Escherichia coli that cause extraintestinal infections [1]. “Uropathogenic” E. coli strains were later shown to belong to a restricted set of serotypes or clones [2], and acute pyelonephritis and ABU strains were shown to differ in surface antigen repertoire [3]. Studies in the 1970s started to involve host cell interactions with attachment to the urinary tract mucosa [4]. We proposed that the disease severity was a direct result of bacterial virulence and that tissue attachment is a first critical step. The special virulence of the uropathogenic clones has subsequently been shown to include numerous virulence factors encoded on the pathogenicity islands (see Middendorf et al., this issue). The variation in urinary tract virulence reflects the ability of bacteria to trigger mucosal and systemic host responses. Through different molecular interactions, bacteria may trigger epithelial cell responses, cause cell detachment, and invade or kill cells by apoptosis (for review see [5]). Inflammation has received special attention because it determines the severity of UTI and the clearance of infection [6] (also see Agace et al. in [5]). We have studied how the inflammatory response is initiated and how it determines the resistance to UTI. Herein we argue that individuals differ in the ability to respond to UTI. We propose that pyelonephritis occurs more readily in “high responders” and that their abnormalities exaggerate the damaging rather than the protective aspects of inflammation. The “low responders,” on the other hand, have suppressed inflammatory signals, allowing bacteriuria to establish without harm

Bacterial attachment is thought to enhance virulence by promoting colonization of the urinary tract and by attacking tissue [1]. Uropathogenic Escherichia coli express several classes of fimbriae-associated adhesins that mediate attachment through specific binding to different glycoconjugate receptors [2] (for review see Mirelman [3]). P fimbriae, encoded by the pap gene cluster, show the most clear-cut association with virulence as defined by acute disease severity [4]. It has been proposed that P fimbriae augment the virulence of uropathogenic E. coli at different stages during the pathogenesis of urinary tract infection (UTI). pap+ strains remain longer in the intestinal flora and spread more efficiently to the urinary tract than do papstrains [5]. Once in the urinary tract, P-fimbriated strains establish bacteriuria and may cross the epithelial barrier into the bloodstream [6]. P fimbriae enhance epithelial cytokine responses in vitro [7] and in the mouse UTI model [8]. The role of P fimbriae per se for colonization and host response induction is not clear and has been the subject of some discussion. While fimbriae-mediated attachment enhances bac

Urinary tract infections (UTIs) provide an excellent model to identify mucosal resistance mechanisms. The urinary tract is open to microbial attack, but most individuals are resistant to UTIs, demonstrating the presence of a highly efficient host defense along the mucosa. The clustering of infections in certain individuals indicates, on the other hand, that specific defense defects render certain individuals susceptible to infection. The mechanisms of this susceptibility to infection can be studied in the murine UTI model, in which injected human disease isolates first establish in the lower urinary tract and then ascend into the renal pelvis. Mice resistant to injected isolates eliminate 108 cfu/mL within a few days. The largest reduction in bacterial numbers occurs during the first 24 h, suggesting that critical defense mechanisms are activated within this short time frame.

Neutrophil migration across infected mucosal surfaces is chemokine dependent, but the role of chemokine receptors has not been investigated. In this study, chemokine receptors were shown to be expressed by epithelial cells lining the urinary tract, and to play an essential role for neutrophil migration across the mucosal barrier. Uroepithelial CXCR1 and CXCR2 expression was detected in human urinary tract biopsies, and in vitro infection of human uroepithelial cell lines caused a dramatic increase in both receptors. As a consequence, there was higher binding of IL-8 to the cells and the IL-8-dependent neutrophil migration across the infected epithelial cell layers was enhanced. Abs to IL-8 or to the CXCR1 receptor inhibited this increase by 60% (p < 0.004), but anti-CXCR2 Abs had no effect, suggesting that CXCR1 was the more essential receptor in this process. Similar observations were made in the mouse urinary tract, where experimental infection stimulated epithelial expression of the murine IL-8 receptor, followed by a rapid flux of neutrophils into the lumen. IL-8 receptor knockout mice, in contrast, failed to express the receptor, their neutrophils were unable to cross the epithelial barrier, and accumulated in massive numbers in the tissues. These results demonstrate that epithelial cells express CXC receptors and that infection increases receptor expression. Furthermore, we show that CXCR1 is required for neutrophil migration across infected epithelial cell layers in vitro, and that the murine IL-8 receptor is needed for neutrophils to cross the infected mucosa of the urinary tract in vivo.

This study tested the protective activity of antibodies to the LPS core of Haemophilus influenzae (Borrelli et al., Infect. Immun. 1995;63: 3683–92) in a hematogenous meningitis model. Meningitis was established by intraperitoneal inoculation of infant rats with H. influenzae type b (Hib). The severity of infection was determined by daily assessment of mortality, symptoms of disease and weight changes. Mortality occurred rapidly after infection with 105cfu/rat and most animals died within 24 h. At a lower infection dose (104cfu/rat) the rats survived, but developed symptoms of disease such as tremor, hypothermia, lethargy and anorexia within 12–72 h post challenge. Surviving animals showed decreased weight gain. Bacteremia was detected by daily blood-cultures in 10/10 rats and cleared 6 days after inoculation. The monoclonal anti-LPS antibody MAHI 3 was used in passive protection studies. MAHI 3 increased the survival in the high inoculum group (105cfu/rat) from 10–17% in control animals to 60–90%. At the lower inoculum concentration (104cfu/rat) MAHI 3 treatment reduced the symptoms and blood counts. Intraperitoneal injection of MAHI 3 was more effective than intranasal injection as shown by the effect on bacteremia. We conclude that anti-LPS antibodies can protect against mortality caused by hematogenous Hib infections in infant rats.

This study examined the role of neutrophil leukocytes for the antibacterial defense at mucosal infection sites. Urinary tract infection (UTI) was established by injection into the bladder lumen of Escherichia coli 1177, a fully virulent clinical isolate. Infection of C3H/HeN (lpsn, lpsn) mice recruited neutrophils into the urinary tract, and bacteria were cleared from kidneys and bladders. The neutrophil response was absent in C3H/HeJ (lpsd, lpsd) mice, and bacteria persisted in the tissues. Peripheral neutrophil depletion of C3H/HeN mice was subsequently achieved by pretreatment with the granulocyte-specific antibody RB6-8C5. The E. coli-induced neutrophil recruitment was inhibited, as shown by immunohistochemistry and tissue myeloperoxidase quantitation. As a consequence, bacterial clearance from kidneys and bladders was drastically impaired. Antibody treatment of C3H/HeJ mice had only a marginal effect. The results show that neutrophils are essential for bacterial clearance from the urinary tract and that the neutrophil recruitment deficiency in C3H/HeJ mice explains their susceptibility to gram-negative UTI.

Mucosal pathogens use diverse and highly specific molecular mechanisms to activate mucosal inflammation. It may even be argued that their virulence depends on the inflammatory response that they induce. Some bacteria target epithelial cells and trigger them to produce inflammatory mediators but others cross the mucosa and activate macrophages or dendritic cells. Although systemic release of inflammatory mediators causes many symptoms and signs of infection, local chemokine production leads to the recruitment of inflammatory cells and lymphocytes that participate directly in the clearance of bacteria from mucosal sites. In this way, mucosal inflammation is a two-edged sword responsible for disease associated tissue destruction and crucial for the antimicrobial defence. Understanding of these pathways should create tools to enhance the defence and interfere with disease.

An interleukin-6 (IL-6) response was detected in 81 patients with febrile urinary tract infections (UTIs). Bacteremic patients (n=24) had higher serum IL-6 at inclusion and throughout the first 24 h (P

The severity of an infection is a function of the resistance of the host and the virulence of the infecting strain. Infections of the urinary tract can be caused by a wide range of gram-negative and gram-positive bacteria, which also are constituents of the normal flora. Indeed, most uropathogens originate from the intestinal tract. Still, the virulence concept implies that bacteria associated with urinary tract infections differ from members of the indigenous flora not causing infections. This review attempts to summarize the mechanisms known to contribute to bacterial virulence in the urinary tract and their relevance in different patient groups.

Escherichia coli stimulates neutrophil migration across human uroepithelial cell layers. This study investigated the role of the neutrophil chemokine interleukin-8 (IL-8) in this process. E. coli and IL-1alpha stimulated urinary tract epithelial layers to secrete IL-8 and induced transepithelial neutrophil migration. Anti-IL-8 antibody reduced neutrophil migration across epithelial cell layers, indicating a central role for this chemokine in the migration process. Furthermore, addition of recombinant IL-8 to unstimulated cell layers was sufficient to induce migration. The IL-8 dependence of neutrophil migration was maintained after removal of soluble IL-8 by washing of the cell layers. Flow cytometry analysis with fluorescein isothiocyanate-labelled IL-8 confirmed IL-8's ability to bind to the epithelial cell surface. Indirect immunofluorescence with confocal laser scanning microscopy showed that IL-8 associated with the epithelial cell layers. Prior incubation of neutrophils with antibodies to IL-8 receptor A (IL-8RA) reduced neutrophil migration. Anti-IL-8 RB antibody had no effect on neutrophil migration. These results demonstrate that IL-8 plays a key role in E. coli- or IL-1alpha-induced transuroepithelial migration and suggest that epithelial cell-produced IL-8 interacts with IL-8RA on the neutrophil surface.

The frequency of humoral immunodeficiencies was analysed in 39 patients with a history of recurrent (> or = 3) episodes of community-acquired pneumonia. Total immunoglobulin levels and/or IgG subclass levels were low in 14 patients (36%), including eight patients with IgG or IgG2 deficiency. The specific antibody activity to pneumococcal capsular polysaccharides (serotypes 3, 6A, 19F, and 23F) and to phosphorylcholine was low in the IgG/IgG2-deficient patients compared to 36 healthy controls, and they also responded poorly to vaccination with a 23-valent pneumococcal capsular polysaccharide vaccine. The remaining 25 patients, with normal immunoglobulin and IgG subclass levels, had specific anti-pneumococcal antibody levels comparable to the healthy controls, and all but 3 responded to vaccination. We conclude that immunoglobulin deficiencies and the inability to respond to polysaccharide antigens are common risk factors for recurrent pneumonia in adult patients. Immunoglobulin levels (including IgG subclasses) and antibody response to polysaccharide antigens should be investigated in these patients.

Asymptomatic bacteriuria established by intravesical inoculation of Escherichia coli 83972 is protective in patients with recurrent urinary tract infections. In this randomized, controlled crossover study a total of 3 symptomatic urinary tract infection episodes developed in 2 patients while they carried E. coli 83972. We examined whether virulence reacquisition by symptom isolates may account for the switch from asymptomatic bacteriuria to symptomatic urinary tract infection.We used E. coli 83972 re-isolates from 2 patients in a prospective study and from another 2 in whom symptoms developed after study completion. We phylogenetically classified the re-isolates, and identified the genomic restriction patterns and gene expression profiles as well as virulence gene structure and phenotypes. In vivo virulence was examined in the murine urinary tract infection model.The fim, pap, foc, hlyA, fyuA, iuc, iroN, kpsMT K5 and malX genotypes of the symptomatic re-isolates remained unchanged. Bacterial gene expression profiles of flagellated symptomatic re-isolates were unique to each host, providing no evidence of common deregulation. Symptomatic isolates did not differ in virulence from the wild-type strain, as defined in the murine urinary tract infection model by persistence, symptoms or innate immune activation.The switch from asymptomatic E. coli 83972 carriage to symptomatic urinary tract infection was not explained by reversion to a functional virulence gene repertoire.

Haemophilus influenzae and Streptococcus pneumoniae are common causes of respiratory tract infections. H. influenzae attach to receptor epitopes in mucins and in epithelial cell membranes. Attachment is followed by an epithelial cell cytokine response. Secreted cytokines then initiate inflammation, upset the integrity of the mucosal barrier, and lead to disease. S. pneumoniae do not bind to mucins but attach to respiratory tract epithelial cells. Attachment is increased by viral infection of the epithelial cells. Unlike H. Influenzae, S. pneumoniae induce apoptosis in epithelial cells, thus disrupting the mucosal barrier. Attachment and persistence is counterbalanced by antiadhesive as well as bactericidal molecules in secretions such as human milk. These examples illustrate the balance between host defenses and microbial virulence as it has coevolved to maintain the health of the respiratory mucosa.

This study analysed the effects of immunoregulatory cytokines on uroepithelial cell cytokine responses. The A-498 human kidney cell line was treated with the interleukins IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, interferon gamma (IFN-alpha) and transforming growth factor beta (TGF-beta 1). Secreted IL-6 and IL-8 were quantitated by enzyme-linked immunoabsorbent assay (ELISA) and bioassay; IL-6 and IL-8 mRNA species were quantitated by reverse transcriptase polymerase chain reaction (RT-PCR). IL-4, IL-13, IFN-gamma, and TGF-beta 1, but not IL-2, IL-5, IL-10 or IL-12, stimulated IL-6 secretion. At high concentrations, IL-4 and IL-13 stimulated low levels of IL-8 secretion. Immunoregulatory cytokines were analysed for their ability to modify the A-498 cells' IL-6 and IL-8 secretion in response to Escherichia coli. IL-5, IL-12, IL-13 and TGF-beta 1 additively enhanced the bacterially induced IL-6 secretion, but they did not affect IL-8 secretion. The strongest affects on uroepithelial cell IL-6 and IL-8 responses in the presence of bacteria were observed in conjunction with IL-4 and IFN-alpha. IL-4 induced IL-6 production in synergy with E. coli. IFN-alpha both enhanced and inhibited IL-6 and IL-8 responses in combination with E. coli, depending on the order of stimulant addition. The results demonstrate that immunoregulatory cytokines can modify the uroepithelial cell responses to bacteria in vitro. In this way, T cells may regulate the cytokine responses of uroepithelial and possibly other mucosal epithelial cells in vivo.

BACKGROUND: This study analyzed antibodies to pneumococcal polysaccharides in human milk and their effect on nasopharyngeal colonization and acute otitis media in breast-fed infants.METHODS: A total of 503 milk samples were collected from 310 mothers. Nasopharyngeal cultures were obtained from their children at 2, 6 and 10 months postpartum, and the capsular groups/types of the Streptococcus pneumoniae isolates were determined.RESULTS: Types 6A, 6B, 19A, 19F and 23F accounted for 54% of the pneumococcal isolates, but type 3 isolates were uncommon. Milk samples were analyzed for antibody activity to the common capsular polysaccharide types 6A, 19F and 23F; to the type 3 polysaccharide; to C-polysaccharide; and to phosphorylcholine (PC), a major component of the pneumococcal cell wall polysaccharide (CWPS). Anti-capsular antibody activity was low or absent in > 90% of the milk samples. In contrast anti-PC antibody activity was detected in 88% and anti-CWPS in 84% of the samples. The frequency of acute otitis media did not vary with the milk anti-capsular, anti-PC or anti-CWPS antibody activity.CONCLUSIONS: There was no reduction in nasopharyngeal carriage of S. pneumoniae among children fed milk with anti-capsular or anti-PC antibody activity, but carriage was increased in those children who received milk with anti-CWPS antibody activity. A protective role of antipolysaccharide or anti-CWPS antibodies in milk was not detected under the study conditions. (Less)

Urinary tract infections (UTI) are common, dangerous and interesting. This review includes a general background on UTIs and molecular mechanisms of pathogenesis. In addition, we discuss UTI susceptibility and especially the effect of genetic variation on innate immunity. The symptoms of acute pyelonephritis are caused by the innate immune response and inflammation in the urinary tract decreases renal tubular function and may give rise to renal scarring, especially in childhood. The disease severity is explained by pathogens and their virulence factors triggering signaling through Toll-like receptors (TLRs), interferon regulatory factors (IRFs) and type 1 interferons, and the activation of a host response mediating disease or pathology or clearance of infection. In children with asymptomatic bacteriuria (ABU), in contrast, bacteria persist without causing symptoms or pathology. ABU strains mostly lack virulence factors, and the lack of symptoms has largely been attributed to their lack of virulence. Recently, rapid progress has been made in the understanding of host susceptibility mechanisms. For example, genetic alterations that reduce TLR4 function are associated with ABU while polymorphisms reducing IRF3 or CXCR1 expression are associated with acute pyelonephritis and an increased risk for renal scarring. Understanding bacterial virulence and host resistance promises new tools to improve the diagnostic accuracy in children with UTI. By combining information on bacterial virulence and the host response, it should be possible to start individualizing diagnosis and therapy. Finally, we propose that the prediction of future disease risk and decisions on prophylaxis and invasive diagnostic procedures might be improved by genetic analysis.

Serum and urine cytokines were analyzed in children with hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Interleukin-6 (IL-6) was elevated in the serum of 33 of 35 children with HUS (94%) and in 2 of 2 children with recurrent TTP. Serum IL-6 was higher in children with HUS who developed anuria, extrarenal manifestations during the acute phase of illness and/or chronic renal sequelae. Tumor necrosis factor-alpha (TNF-alpha) was detected in the serum of 7 patients with HUS (20%) and 1 patient with TTP. IL-6 and TNF-alpha were elevated in the urine of 4 of 4 children with HUS and 2 of 2 children with TTP. Urinary levels were higher than serum levels, suggesting local production of cytokines in the urinary tract. Sequential serum and urine samples showed that IL-6 levels varied with disease activity. IL-6 and TNF-alpha were not detected in the serum (n = 25) and urine (n = 15) of healthy children. We conclude that IL-6 in urine may be used to monitor disease activity in HUS and TTP.

Localized at the border between the external environment and the internal tissue, epithelial cells are exposed to stimulants from two directions. Microorganisms in the lumen can activate the transcription of cytokine mRNA and cytokine secretion, and cytokines in the mucosal environment can modify endogenous and microbially induced epithelial cytokine responses. Epithelial cells thus actively participate in mucosal immunity and inflammation.

Viruses are thought to facilitate bacterial infections of the respiratory tract, but the mechanisms are poorly understood. The present study analyzed the effect of adenovirus on bacterial adherence to human respiratory tract epithelial cells. The human lung carcinoma cell line A549 was infected with adenovirus of types 1, 2, 3, 4, 5, and 9. At a multiplicity of infection of 75 particles per cell, cytopathic effects occurred in 75 to 100% of the cells within 48 h. The virus-infected cells were harvested at various times after infection and analyzed for the ability to bind strains of Haemophilus influenzae and Streptococcus pneumoniae. Adenovirus (types 1, 2, 3, and 5) commonly causing respiratory tract infections increased the binding of adherent S. pneumoniae strains to the cells. This effect was not seen for other adenovirus types. Adenovirus infection did not change the adherence of cells of poorly adhering strains of S. pneumoniae or H. influenzae. The increase in adherence of S. pneumoniae could be inhibited by the DNA synthesis inhibitor cytosine arabinofuranoside, which is known to block the late phase of the adenovirus infection. When electron microscopy was used, there was no evidence that virus particles bound directly to bacteria. Adherence was not affected by pretreatment of the cells with virus particles or viral proteins. This suggested that adenovirus infection upregulated receptors for S. pneumoniae. The increased attachment may be one mechanism by which viruses precondition the respiratory mucosa for bacterial infection.

It has been shown previously that secretory IgA interacts with the mannose-specific lectin of Escherichia coli. The purpose of the study described here was to evaluate whether the N-linked oligosaccharide chains of the human IgA isotypes IgA1 and IgA2 differ in lectin receptor activity. A range of plant lectins specific for N-linked oligosaccharide chains were tested for their ability to precipitate IgA1 and IgA2 myeloma proteins, secretory IgA and free secretory component. IgA2 myeloma proteins reacted more strongly than IgA1 with the mannose-specific lectin ConA, whereas IgA1 myeloma proteins reacted more strongly than IgA2 with two galactose-specific lectins, Ricinus communis agglutinin I and Abrus precatorius agglutinin. This suggests that IgA2 possesses a larger proportion of short truncated complex type oligosaccharide chains and/or oligomannose type chains than IgA1. Further, IgA2 reacted more strongly than IgA1 myeloma proteins with Lens culinaris (lentil) lectin, and Pisum sativum (pea) lectin, suggesting that IgA2 exposes more of short, complex type chains fucosylated on the core than IgA1. The differences demonstrated in receptor activity between IgA1 and IgA2 may be important in their interaction with the microbial flora, as well with endogenous lectins, such as phagocyte receptors.

A functional and well-balanced immune response is required to resist most infections. Slight dysfunctions in innate immunity can turn the 'friendly' host defense into an unpleasant foe and give rise to disease. Beneficial and destructive forces of innate immunity have been discovered in the urinary tract and mechanisms by which they influence the severity of urinary tract infections (UTIs) have been elucidated. By modifying specific aspects of the innate immune response to UTI, genetic variation either exaggerates the severity of acute pyelonephritis to include urosepsis and renal scarring or protects against symptomatic disease by suppressing innate immune signaling, as in asymptomatic bacteriuria (ABU). Different genes are polymorphic in patients prone to acute pyelonephritis or ABU, respectively, and yet discussions of UTI susceptibility in clinical practice still focus mainly on social and behavioral factors or dysfunctional voiding. Is it not time for UTIs to enter the era of molecular medicine? Defining why certain individuals are protected from UTI while others have severe, recurrent infections has long been difficult, but progress is now being made, encouraging new approaches to risk assessment and therapy in this large and important patient group, as well as revealing promising facets of 'good' versus 'bad' inflammation.

Bacterial virulence factors influence the site and severity of urinary tract infections. While pyelonephritis-associated molecular traits have been defined, virulence factors specific for acute cystitis strains have not been identified. This study examined the virulence factor repertoire of 247 Escherichia coli strains, prospectively isolated from women with community-acquired acute cystitis. Fim sequences were present in 96% of the isolates, which also expressed Type 1 fimbriae. Curli were detected in 75%, 13% of which formed cellulose. Pap sequences were present in 47%, 27% were papG+, 23% were prsG+ and 42% expressed P fimbriae. TcpC was expressed by 33% of the strains, 32% in a subgroup of patients who only had symptoms of cystitis and 42% in patients with signs of upper urinary tract involvement; most frequently by the papG+/prsG+ subgroup. Strains with the full fim, pap and TcpC and curli virulence profile were more common in cystitis patients with than in patients without upper tract involvement (p

The severity of urinary tract infection (UTI) reflects the quality and magnitude of the host response. While strong local and systemic innate immune activation occurs in patients with acute pyelonephritis, the response to asymptomatic bacteriuria (ABU) is low. The immune response repertoire in ABU has not been characterized, due to the inherent problem to distinguish bacterial differences from host-determined variation. In this study, we investigated the host response to ABU and genetic variants affecting innate immune signaling and UTI susceptibility. Patients were subjected to therapeutic urinary tract inoculation with E. coli 83972 to ensure that they were exposed to the same E. coli strain. The innate immune response repertoire was characterized in urine samples, collected from each patient before and after inoculation with bacteria or PBS, if during the placebo arm of the study. Long-term E. coli 83972 ABU was established in 23 participants, who were followed for up to twelve months and the innate immune response was quantified in 233 urine samples. Neutrophil numbers increased in all but two patients and in an extended urine cytokine/chemokine analysis (31 proteins), the chemoattractants IL-8 and GRO-α, RANTES, Eotaxin-1 and MCP-1, the T cell chemoattractant and antibacterial peptide IP-10, inflammatory regulators IL-1-α and sIL-1RA and the T lymphocyte/dendritic cell product sIL-2Rα were detected and variably increased, compared to sterile samples. IL-6, which is associated with symptomatic UTI, remained low and numerous specific immune mediators were not detected. The patients were also genotyped for UTI-associated IRF3 and TLR4 promoter polymorphisms. Patients with ABU associated TLR4 polymorphisms had low neutrophil numbers, IL-6, IP-10, MCP-1 and sIL-2Rα concentrations. Patients with the ABU-associated IRF3 genotype had lower neutrophils, IL-6 and MCP-1 responses than the remaining group. The results suggest that the host-specific, low immune response to ABU mainly includes innate immune mediators and that host genetics directly influence the magnitude of this response.

Urinary tract infections activate a mucosal inflammatory response, which includes cytokine secretion and neutrophil influx. The mechanisms involved in the neutrophil influx have not been identified. Interleukin-8, a potent chemoattractant for neutrophils, is produced by urinary tract epithelial cell lines in vitro. This study analyzed the human IL-8 response to deliberate Escherichia coli infection of the urinary tract. Urine and serum samples were obtained before and after intravesical instillation of E. coli. Neutrophil numbers were determined on uncentrifuged urine, and IL-8 levels were measured by ELISA. A urinary IL-8 response was found in all patients after bacterial instillation, but no serum IL-8 was detected. There was a strong correlation between urinary IL-8 levels and urinary neutrophil numbers. The same E. coli strains used to colonize the patients stimulated IL-8 production in urinary tract epithelial cells. The level of IL-8 secreted by epithelial cell lines was influenced by the fimbrial properties of the E. coli. These results demonstrated that E. coli elicit a mucosal IL-8 response in humans, and suggested that IL-8 is involved in the onset of pyuria. Epithelial cells may be an important source of IL-8 during urinary tract infection.

Hemolysins are cytolytic proteins which have been extensively characterized at the molecular level, however, their in vivo functions remain unclear. This study analyzed the association of hemolysin production with the inflammatory response in patients with urinary tract infection (UTI). Infants and children with their first episode of UTI (n = 644) were followed prospectively. The body temperature, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), urinary leucocyte count and renal concentrating capacity were used as measures of the inflammatory response. The hemolytic genotype (hly) of the Escherichia coli strain from each UTI episode was defined by DNA-DNA hybridization, and the phenotype by hemolysis in blood agar. There was no significant increase in the level of fever, CRP, ESR, or decrease in renal concentrating capacity during UTI episodes caused by hly positive compared to hly negative E. coli. Multiple regression analysis did not demonstrate significant associations of hly with elevated fever, CRP, ESR or reduced renal concentrating capacity. In contrast, patients infected with P fimbriated E. coli strains had higher fever, CRP, ESR and lower renal concentrating capacity than those infected with other strains. This association was not influenced by the hly genotype of the P fimbriated strains. The frequency of hly+ strains was not significantly higher in the subset of patients assigned a diagnosis of acute pyelonephritis compared to asymptomatic bacteriuria. This was in contrast to P fimbriae, which were accumulated in acute pyelonephritis. The results suggested that the acute inflammatory response to E. coli UTI is independent of hemolysin production. The inflammatogenic potential of uropathogenic E. coli clones was better described by the presence or absence of P-fimbriae than by hemolysin.