Impaired cortical energy metabolism but not major antioxidant defenses in experimental bacterial meningitis.

The loss of soluble brain antioxidants and protective effects of radical scavengers implicate reactive oxygen species in cortical neuronal injury caused by bacterial meningitis. However, the lack of significant oxidative damage in cortex [J. Neuropathol. Exp. Neurol. 61 (2002) 605-613] suggests that cortical neuronal injury may not be due to excessive parenchymal oxidant production. To see whether this tissue region exhibits a prooxidant state in bacterial meningitis, we examined the state of the major cortical antioxidant defenses in infant rats infected with Streptococcus pneumoniae. Adenine nucleotides were co-determined to assess possible changes in energy metabolism. Arguing against heightened parenchymal oxidant production, the high NADPH/NADP(+) ratio ( approximately 3:1) and activities of the major antioxidant defense and pentose phosphate pathway enzymes remained unchanged at the time of fulminant meningitis. In contrast, cortical ATP, ADP and total adenine nucleotides were on average decreased by approximately 25%. However, energy depletion did not lead to a significant decrease in adenylate energy charge (AEC). ATP depletion was likely a consequence of metabolic degradation, since it correlated with both the loss of total adenine nucleotides and accumulation of purine degradation products. Furthermore, the loss of ATP and decrease in AEC correlated significantly with the extent of neuronal injury. These results strongly suggest that energy depletion rather than parenchymal oxidative damage is involved in the observed cortical neuronal injury.

Bacterial meningitis causes two distinct forms of cellular damage in the hippocampal dentate gyrus in infant rats.

Bacterial meningitis causes neurological sequelae in up to 50% of survivors. Two pathogens known for their propensity to cause severe neurological damage are Streptococcus pneumoniae and group B streptococci. Some forms of neuronal sequelae, such as learning and memory deficits, have been associated with neuronal injury in the hippocampus. To learn more about hippocampal injury in meningitis, we performed a comparative study in bacterial meningitis due to S. pneumoniae and group B streptococcus, in which 11-day-old infant rats were infected intracisternally with either of the two pathogens. Histopathological examination of the neuronal injury in the dentate gyrus of the hippocampus showed that S. pneumoniae caused predominantly classical apoptotic cell death. Cells undergoing apoptosis were located only in the subgranular zone and stained positive for activated caspase-3 and TUNEL. Furthermore, dividing progenitor cells seemed particularly sensitive to this form of cell death. Group B streptococcus was mainly responsible for a caspase-3-independent (and TUNEL-negative) form of cell death. Compared with the morphological features found in apoptosis (e.g., apoptotic bodies), this form of neuronal death was characterized by clusters of uniformly shrunken cells. It affected the dentate gyrus throughout the blade, showing no preferences for immature or mature neurons. Thus, depending on the infecting agent, bacterial meningitis causes two distinct forms of cell injury in the dentate gyrus.

The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality.

Ca2+-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

The mood-stabilizer lithium prevents hippocampal apoptosis and improves spatial memory in experimental meningitis.

Pneumococcal meningitis is associated with high morbidity and mortality rates. Brain damage caused by this disease is characterized by apoptosis in the hippocampal dentate gyrus, a morphological correlate of learning deficits in experimental paradigms. The mood stabilizer lithium has previously been found to attenuate brain damage in ischemic and inflammatory diseases of the brain. An infant rat model of pneumococcal meningitis was used to investigate the neuroprotective and neuroregenerative potential of lithium. To assess an effect on the acute disease, LiCl was administered starting five days prior to intracisternal infection with live Streptococcus pneumoniae. Clinical parameters were recorded, cerebrospinal fluid (CSF) was sampled, and the animals were sacrificed 42 hours after infection to harvest the brain and serum. Cryosections of the brains were stained for Nissl substance to quantify brain injury. Hippocampal gene expression of Bcl-2, Bax, p53, and BDNF was analyzed. Lithium concentrations were measured in serum and CSF. The effect of chronic lithium treatment on spatial memory function and cell survival in the dentate gyrus was evaluated in a Morris water maze and by quantification of BrdU incorporation after LiCl treatment during 3 weeks following infection. In the hippocampus, LiCl significantly reduced apoptosis and gene expression of Bax and p53 while it increased expression of Bcl-2. IL-10, MCP-1, and TNF were significantly increased in animals treated with LiCl compared to NaCl. Chronic LiCl treatment improved spatial memory in infected animals. The mood stabilizer lithium may thus be a therapeutic alternative to attenuate neurofunctional deficits as a result of pneumococcal meningitis.

The antidepressant fluoxetine protects the hippocampus from brain damage in experimental pneumococcal meningitis.

BACKGROUND High mortality and morbidity rates are observed in patients with bacterial meningitis (BM) and urge for new adjuvant treatments in addition to standard antibiotic therapies. In BM the hippocampal dentate gyrus is injured by apoptosis while in cortical areas ischemic necrosis occurs. Experimental therapies aimed at reducing the inflammatory response and brain damage have successfully been evaluated in animal models of BM. Fluoxetine (FLX) is an anti-depressant of the selective serotonin reuptake inhibitors (SSRI) and was previously shown to be neuroprotective in vitro and in vivo. We therefore assessed the neuroprotective effect of FLX in experimental pneumococcal meningitis. METHODS Infant rats were infected intracisternally with live Streptococcus pneumoniae. Intraperitoneal treatment with FLX (10mgkg(-1)d(-1)) or an equal volume of NaCl was initiated 15min later. 18, 27, and 42h after infection, the animals were clinically (weight, clinical score, mortality) evaluated and subject to a cisternal puncture and inflammatory parameters (i.e., cyto-/chemokines, myeloperoxidase activity, matrix metalloproteinase concentrations) were measured in cerebrospinal fluid (CSF) samples. At 42h after infection, animals were sacrificed and the brains collected for histomorphometrical analysis of brain damage. RESULTS A significant lower number of animals treated with FLX showed relevant hippocampal apoptosis when compared to littermates (9/19 animals vs 18/23, P=0.038). A trend for less damage in cortical areas was observed in FLX-treated animals compared to controls (13/19 vs 13/23, P=ns). Clinical and inflammatory parameters were not affected by FLX treatment. CONCLUSION A significant neuroprotective effect of FLX on the hippocampus was observed in acute pneumococcal meningitis in infant rats.

The matrix metalloproteinase inhibitor RS-130830 attenuates brain injury in experimental pneumococcal meningitis.

BACKGROUND Pneumococcal meningitis (PM) is characterized by high mortality and morbidity including long-term neurofunctional deficits. Neuropathological correlates of these sequelae are apoptosis in the hippocampal dentate gyrus and necrosis in the cortex. Matrix metalloproteinases (MMPs) play a critical role in the pathophysiology of PM. RS-130830 (Ro-1130830, CTS-1027) is a potent partially selective inhibitor of MMPs of a second generation and has been evaluated in clinical trials as an anti-arthritis drug. It inhibits MMPs involved in acute inflammation but has low activity against MMP-1 (interstitial collagenase), MMP-7 (matrilysin) and tumour necrosis factor α converting enzyme (TACE). METHODS A well-established infant rat model of PM was used where live Streptococcus pneumoniae were injected intracisternally and antibiotic treatment with ceftriaxone was initiated 18 h post infection (hpi). Treatment with RS-130830 (75 mg/kg bis in die (bid) i.p., n = 40) was started at 3 hpi while control littermates received the vehicle (succinylated gelatine, n = 42). RESULTS Cortical necrosis was significantly attenuated in animals treated with RS-130830, while the extent of hippocampal apoptosis was not influenced. At 18 hpi, concentrations of interleukin (IL)-1β and IL-10 were significantly lower in the cerebrospinal fluid of treated animals compared to controls. RS-130830 significantly reduced weight loss and leukocyte counts in the cerebrospinal fluid of survivors of PM. CONCLUSION This study identifies MMP inhibition, specifically with RS-130830, as an efficient strategy to attenuate disease severity and cortical brain injury in PM.

Patients with community acquired pneumonia admitted to European intensive care units: an epidemiological survey of the GenOSept cohort.

INTRODUCTION Community acquired pneumonia (CAP) is the most common infectious reason for admission to the Intensive Care Unit (ICU). The GenOSept study was designed to determine genetic influences on sepsis outcome. Phenotypic data was recorded using a robust clinical database allowing a contemporary analysis of the clinical characteristics, microbiology, outcomes and independent risk factors in patients with severe CAP admitted to ICUs across Europe. METHODS Kaplan-Meier analysis was used to determine mortality rates. A Cox Proportional Hazards (PH) model was used to identify variables independently associated with 28-day and six-month mortality. RESULTS Data from 1166 patients admitted to 102 centres across 17 countries was extracted. Median age was 64 years, 62% were male. Mortality rate at 28 days was 17%, rising to 27% at six months. Streptococcus pneumoniae was the commonest organism isolated (28% of cases) with no organism identified in 36%. Independent risk factors associated with an increased risk of death at six months included APACHE II score (hazard ratio, HR, 1.03; confidence interval, CI, 1.01-1.05), bilateral pulmonary infiltrates (HR1.44; CI 1.11-1.87) and ventilator support (HR 3.04; CI 1.64-5.62). Haematocrit, pH and urine volume on day one were all associated with a worse outcome. CONCLUSIONS The mortality rate in patients with severe CAP admitted to European ICUs was 27% at six months. Streptococcus pneumoniae was the commonest organism isolated. In many cases the infecting organism was not identified. Ventilator support, the presence of diffuse pulmonary infiltrates, lower haematocrit, urine volume and pH on admission were independent predictors of a worse outcome.

Cigarette Smoke Exposure Increases Myeloid Cell Production and Lung Bacterial Clearance in Mice

Pneumonia is a leading cause of hospitalization in patients with chronic obstructive pulmonary disease (COPD). Although most COPD patients are smokers, the effects of cigarette smoke exposure on clearance of lung bacterial pathogens and on immune and inflammatory responses are incompletely defined. Here, clearance of Streptococcus pneumoniae and Pseudomonas aeruginosa and associated immune responses were examined in mice exposed to cigarette smoke or following smoking cessation. Mice exposed to cigarette smoke for 6 weeks or 4 months demonstrated decreased lung bacterial burden compared to air-exposed mice when infected 16-24 hours post-exposure. When infection was performed after smoke cessation, bacterial clearance kinetics of mice previously exposed to smoke reversed to comparable levels as those of control mice suggesting that the observed defects were not dependent on adaptive immunological memory to bacterial determinants found in smoke. Comparing cytokine levels and myeloid cell production prior to infection in mice exposed to cigarette smoke relative to mice never exposed or following smoke cessation revealed that reduced bacterial burden was most strongly associated with higher levels of IL-1β and GM-CSF in the lungs and with increased neutrophil reserve and monocyte turnover in the bone marrow. Using serpinb1a-deficient mice with reduced neutrophil numbers and treatment with G-CSF showed that increased neutrophil numbers contribute only in part to the effect of smoke on infection. Our findings indicate that cigarette smoke induces a temporary and reversible increase in clearance of lung pathogens, which correlates with local inflammation and increased myeloid cell output from the bone marrow.

Cetoacidosis inducida por corticoides y gatifloxacina en una mujer joven no diabética

Objetivo: Comunicar un caso de cetoacidosis inducida por corticoides y gatifloxacina y discutir los mecanismos de esta inusual y seria complicación. Caso clínico: Mujer de 32 años, ingresa por neumonía adquirida en la comunidad de 5 días de evolución. Antecedentes: AR probable diagnosticada 4 meses antes tratada con metotrexate y corticoides intermitente. Examen físico: regular estado general, IMC 21, Tº 38ºC, FR 32/min, derrame pleural derecho, FC 96/min, PA 110/70, artralgias sin artritis. Exámenes complementarios: Hto 23%, GB 16300/mm3, VSG 96mm/1ºh, glucemia 0.90mg/dl, función hepática y amilasa normales, uremia 1.19g/l, creatinina 19mg/l. Hemocultivos (2) y esputo positivos para Neumococo penicilina-sensible. La neumonía responde a gatifloxacina. Deteriora la función renal hasta la anuria con acidosis metabólica. Se interpreta como glomerulonefritis lúpica rápidamente progresiva por proteinuria de 2g/24hs, FR (+) 1/1280, FAN (+) 1/320 homogéneo, Anti ADN (+) , complemento bajo: C3 29.4mg/dl y C4 10mg/dl, Ac anti Ro, La, Scl70, RNP y anticardiolipinas positivos. Se indica metilprednisolona EV (3 bolos 1g), complicándose con hiperglucemias de >6 g/l y cetoacidosis con cetonuria (+); Ac anti ICA y antiGAD negativos con HbA1C 5.2%. Es tratada en UTI (insulina y hemodiálisis). La paciente mejora, se desciende la dosis de corticoides, con normalización de la glucemia sin tratamiento hipoglucemiante. Comentarios 1) La presencia de HbA1C nomal, Ac anti ICA y GAD negativos permite descartar con razonable grado de certeza una diabetes tipo1 asociada al lupus. 2) El desarrollo de la cetoacidosis durante el tratamiento con corticoides y gatifloxacina y su resolución posterior avalan el rol etiológico de los mismos. 3) La cetoacidosis puede explicarse por estimulación de la gluconeogénesis y la insulinoresistencia a nivel de receptor y post-receptor generada por los fármacos potenciado por el estado inflamatorio relacionado con el lupus y la sepsis.

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

Identification of a structural determinant for resistance to β-lactam antibiotics in Gram-positive bacteria

Streptococcus pneumoniae is the main causal agent of pathologies that are increasingly resistant to antibiotic treatment. Clinical resistance of S. pneumoniae to β-lactam antibiotics is linked to multiple mutations of high molecular mass penicillin-binding proteins (H-PBPs), essential enzymes involved in the final steps of bacterial cell wall synthesis. H-PBPs from resistant bacteria have a reduced affinity for β-lactam and a decreased hydrolytic activity on substrate analogues. In S. pneumoniae, the gene coding for one of these H-PBPs, PBP2x, is located in the cell division cluster (DCW). We present here structural evidence linking multiple β-lactam resistance to amino acid substitutions in PBP2x within a buried cavity near the catalytic site that contains a structural water molecule. Site-directed mutation of amino acids in contact with this water molecule in the “sensitive” form of PBP2x produces mutants similar, in terms of β-lactam affinity and substrate hydrolysis, to altered PBP2x produced in resistant clinical isolates. A reverse mutation in a PBP2x variant from a clinically important resistant clone increases the acylation efficiency for β-lactams and substrate analogues. Furthermore, amino acid residues in contact with the structural water molecule are conserved in the equivalent H-PBPs of pathogenic Gram-positive cocci. We suggest that, probably via a local structural modification, the partial or complete loss of this water molecule reduces the acylation efficiency of PBP2x substrates to a point at which cell wall synthesis still occurs, but the sensitivity to therapeutic concentrations of β-lactam antibiotics is lost.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.