Di-mannosylation enhances the adjuvant properties of adamantane-containing desmuramyl peptides in vivo.

Muramyl dipeptide (MDP) is the smallest essential peptidoglycan substructure capable of promoting both innate and adaptive immune responses. Herein, we report on the design, synthesis, and in vivo study of the adjuvant properties of two novel MDP analogs containing an achiral adamantyl moiety attached to the desmuramyl dipeptide (DMP) pharmacophore and additionally modified by one mannosyl subunit (derivative 7) or two mannosyl subunits (derivative 11). Mannose substructures were introduced in order to assess how the degree of mannosylation affects the immune response and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) binding affinity, compared to the reference compound ManAdDMP. Both mannosylated MDP analogs showed improved immunomodulating properties, while the di-mannosylated derivative 11 displayed the highest, statistically significant increase in anti-OVA IgG production. In this study, for the first time, the di-mannosylated DMP derivative was synthesized and immunologically evaluated. Derivative 11 stimulates a Th-2-polarized type of immune reaction, similar to the reference compound ManAdDMP and MDP. Molecular dynamics (MD) simulations demonstrate that 11 has a higher NOD2 binding affinity than 7, indicating that introducing the second mannose significantly contributes to the binding affinity. Mannose interacts with key amino acid residues from the LRR hydrophobic pocket of the NOD2 receptor and loop 2.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Synthesis and Immunological Evaluation of Mannosylated Desmuramyl Dipeptides Modified by Lipophilic Triazole Substituents.

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the smallest peptidoglycan fragment able to trigger an immune response by activating the NOD2 receptor. Structural modification of MDP can lead to analogues with improved immunostimulating properties. The aim of this work was to prepare mannosylated desmuramyl peptides (ManDMP) containing lipophilic triazole substituents to study their immunomodulating activities in vivo. The adjuvant activity of the prepared compounds was evaluated in the mouse model using ovalbumin as an antigen and compared to the MDP and referent adjuvant ManDMPTAd. The obtained results confirm that the α-position of D-isoGln is the best position for the attachment of lipophilic substituents, especially adamantylethyl triazole. Compound 6c exhibited the strongest adjuvant activity, comparable to the MDP and better than referent ManDMPTAd.

Mannosylated adamantane-containing desmuramyl peptide recognition by the NOD2 receptor: a molecular dynamics study.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular receptor that recognizes the bacterial peptidoglycan fragment muramyl dipeptide (MDP). Our group has synthesized and biologically evaluated desmuramyl peptides containing adamantane and its mannose derivatives. The most active mannosylated derivative, ManAdDMP (Man-OCH2-d-(1-Ad)Gly-l-Ala-d-isoGln), is further characterized in silico in this study. We built intact model structures of the rabbit NOD2 protein, whose crystal structure lacks seven loops, and explored the binding of ManAdDMP. Two main binding sites for ManAdDMP are located within the nucleotide-binding oligomerization domain (NOD) and C-terminal leucine-rich repeat (LRR) domains. Our analysis shows that the dipeptide isoGln moiety of ManAdDMP significantly contributes to the binding, whereas the mannose moiety interacts with modelled loop 7, which is a part of the NOD helical domain 2. The presented results point to the importance of loops 2 and 7 in ligand recognition that could be useful for further investigation of NOD2 activation/inhibition.

Design, Synthesis, and Biological Evaluation of Desmuramyl Dipeptides Modified by Adamantyl-1,2,3-triazole.

Muramyl dipeptide (MDP) is the smallest peptidoglycan fragment able to trigger the immune response. Structural modification of MDP can lead to the preparation of analogs with improved immunostimulant properties, including desmuramyl peptides (DMPs). The aim of this work was to prepare the desmuramyl peptide (L-Ala-D-Glu)-containing adamantyl-triazole moiety and its mannosylated derivative in order to study their immunomodulatory activities in vivo. The adjuvant activity of the prepared compounds was evaluated in a murine model using ovalbumin as an antigen, and compared to the reference adjuvant ManAdDMP. The results showed that the introduction of the lipophilic adamantyl-triazole moiety at the C-terminus of L-Ala-D-Glu contributes to the immunostimulant activity of DMP, and that mannosylation of DMP modified with adamantyl-triazole causes the amplification of its immunostimulant activity.

Solvophobically Driven Complexation of Adamantyl Mannoside with ß-Cyclodextrin in Water and Structured Organic Solvents.

Invited for the cover of this issue is Josip Pozar with collaborators from the University of Zagreb. The image depicts the differences in high- and low-temperature water effect on the complexation thermodynamics of adamantyl mannoside with ß-cyclodextrin. Read the full text of the article at 10.1002/chem.202000282.

Solvophobically Driven Complexation of Adamantyl Mannoside with ß-Cyclodextrin in Water and Structured Organic Solvents.

The effects of solvent and temperature on the complexation of adamantyl mannoside with ß-cyclodextrin and 6-O-monotosyl-6-deoxy-ß-cyclodextrin were explored experimentally and by means of molecular dynamics simulations. Efficient binding was observed only in hydrogen-bonded solvents, which indicated solvophobically driven complexation. The stability of the inclusion complex was considerably higher in aqueous media. A pronounced temperature dependence of Δr H○ and Δr S○ , resulting in perfect enthalpy-entropy compensation, was observed in water. The complexation thermodynamics was in line with classical rationale for the hydrophobic effect at lower temperatures and the nonclassical explanation at higher temperatures. This finding linked cyclodextrin complexation thermodynamics with insights regarding the effect of temperature on the hydration water structure. The complexation enthalpies and entropies were weakly dependent on temperature in organic media. The signs of Δr H○ and Δr S○ were in accordance with the nonclassical hydrophobic (solvophobic) effect. The structures of the optimized product corresponded to those deduced spectroscopically, and the calculated and experimentally obtained values of Δr G○ were in very good agreement. This investigation clearly demonstrated that solvophobically driven formation of cyclodextrin complexes could be anticipated in structured solvents in general. However, unlike in water, adamantane and the host cavity behaved solely as structure breakers in the organic media explored so far.

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages.

We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Contrasting Vantage Points between Caregivers and Residents on the Perception of Elder Abuse and Neglect During Long-Term Care.

BACKGROUND: Elder abuse and neglect can be defined as refusing or failing to fulfil a caregiver's obligation to meet the needs of elderly individuals in order to punish or hurt them. We aimed to explore perceptions of elder mistreatment of both caregivers and residents during long-term care, and highlight significant differences in the overall mistreatment perception regarding socio-demographic variables, as well as the type of care facility. SUBJECTS AND METHODS: The study involved 171 caregivers and 245 elderly individuals in stationary facilities. Two structured questionnaires were used - one for caregivers and the other for institutionalized elderly residents, whose initial validation concerning question and factor selection has been based upon exploratory factor analysis and discriminant validity. Parametric and nonparametric tests were employed in the statistical analysis, and statistical significance was set at p<0.05 (two-sided). RESULTS: We found significant differences in the perception of elder abuse and neglect between caregivers and elderly residents. More specifically, caregivers tend to recognize unnecessary or inappropriate medical/care procedures as indicators of elder mistreatment, while the elderly residents emphasize the removal of their personal belongings and inappropriate physical contact. According to the care facility, residents reported abuse/neglect more frequently in extended care units (21.4%), compared to the county-owned nursing home (11.4%) and private nursing home (12.1%) (p=0.001). Similarly, caregivers reported abuse/neglect more frequently in extended care units (75.4%), in comparison to county-owned nursing home (24.6%) and private nursing home (0%) (p=0.039). Shift work was also a significant predictor, as the morning nursing staff perceived abuse/neglect more frequently (p=0.011). CONCLUSIONS: This study has shown that residents and caregivers have contrasting vantage points in relation to elder abuse/neglect perception, which underlines the need for evidence-based standardization of procedures to prevent any type of elder mistreatment.

Design, synthesis and biological evaluation of immunostimulating mannosylated desmuramyl peptides.

Muramyl dipeptide is the minimal structure of peptidoglycan with adjuvant properties. Replacement of the N-acetylmuramyl moiety and increase of lipophilicity are important approaches in the preparation of muramyl dipeptide analogues with improved pharmacological properties. Mannose receptors present on immunocompetent cells are pattern-recognition receptors and by mannose ligands binding they affect the immune system. Here we present the design, synthesis and biological evaluation of novel mannosylated desmuramyl peptide derivatives. Mannose was coupled to dipeptides containing a lipophilic adamantane on N- or C-terminus through a glycolyl or hydroxyisobutyryl linker. Adjuvant activities of synthesized compounds were investigated in the mouse model using ovalbumin as an antigen. Their activities were compared to the previously described mannosylated adamantane-containing desmuramyl peptide and peptidoglycan monomer. Tested compounds exhibited adjuvant activity and the strongest enhancement of IgG production was stimulated by compound 21 (Man-OCH2-ᴅ-(1-Ad)Gly-ʟ-Ala-ᴅ-isoGln).

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer.

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,ß-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.

Changes in hair cortisol levels and cognitive function among older patients in palliative care.

This monocentric pilot study aimed to assess whether there are significant changes in hair cortisol levels and cognitive functioning at baseline and after 3 weeks of hospitalisation in older palliative care patients, and to ascertain whether hair cortisol can be used as a pertinent stress biomarker in this population. Hair cortisol concentration levels were measured with the use of an enzyme-linked immunosorbent assay (ELISA), while cognitive functions were assessed by using 16-point Mini-Mental State Examination 2 (MMSE-2): brief version. A statistically significant difference in mean hair cortisol levels at the start of the hospitalisation was found when compared to the 3-week follow-up (p=0.007). No statistically significant differences were observed regarding MMSE-2 scores using the same time points. These results have direct clinical implications and set the stage for further research endeavours in the field.

Prevalence of Primary Dysmenorrhoea and Its Impact on Academic Performance among Croatian Students during the COVID-19 Pandemic.

Background: Dysmenorrhoea is one of the most common gynaecological problems. Therefore, it is important to investigate its impact during the COVID-19 pandemic which has a great impact on the lives of menstruating people all over the world. Aim: To determine the prevalence and impact of primary dysmenorrhoea on academic performance among students during the pandemic. Materials and Methods: This cross-sectional study was conducted in April 2021. All data were collected by an anonymous self-assessed web-based questionnaire. Due to voluntary participation in the study, 1210 responses were received, but 956 were left for analysis after exclusion criteria were applied. Descriptive quantitative analysis was performed and Kendall rank correlation coefficient was used. Results: The prevalence of primary dysmenorrhoea was 90.1%. Menstrual pain was mild in 7.4% of cases, moderate in 28.8%, and severe in 63.8%. The study found that primary dysmenorrhoea has a great perceived impact on all included aspects of academic performance. Most affected were concentration in class in 810 (94.1%) and doing homework and learning in 809 (94.0%) female students. There is also a correlation between menstrual pain intensity and its impact on academic performance (p < 0.001). Conclusions: Our study found that the prevalence of primary dysmenorrhoea among students at the University of Zagreb is high. Painful menstruation greatly impacts academic performance and therefore it is important to do more research on this topic.

Synthesis and immunostimulating properties of novel adamant-1-yl tripeptides.

The aim of this work was to prepare L- and D-(adamant-1-yl)-Gly-L-Ala-D-isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant-1-yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; ß-D-GlcNAc-(1→4)-D-MurNAc-L-Ala-D-isoGln-mesoDAP(εNH(2) )-D-Ala-D-Ala) and structurally related adamant-2-yl tripeptides.

Influence of mannosylation on immunostimulating activity of adamant-1-yl tripeptide.

The mannosylated derivative of adamant-1-yl tripeptide (D-(Ad-1-yl)Gly-L-Ala-D-isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant-1-yl tripeptide (Man-OCH(2) CH(Me)CO-D-(Ad-1-yl)Gly-L-Ala-D-isoGln) is a non-pyrogenic, H(2) O-soluble, and non-toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, ß-D-GlcNAc-(1→4)-D-MurNAc-L-Ala-D-isoGln-mesoDAP(εNH(2) )-D-Ala-D-Ala), a well-known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.

Knowledge, Attitudes, Psychosocial Perspectives and Applied Epidemiology in the Control of Head Lice (pediculosis capitis) in Croatian Preschool Children: A Qualitative Study on Childcare Professionals and Health Coordinators.

This study aimed to concurrently determine the perceived knowledge, attitudes and perspectives of childcare professionals working in kindergartens towards pediculosis capitis, a common ectoparasitic disease also known as head lice, as well as gain insights into procedures and control measures that are implemented in an outbreak setting. We used a qualitative approach with a problem-centered, semi-structured and three-part interview technique conducted in selected kindergartens of Varazdin and Medimurje counties of the Republic of Croatia. Based on a purposive (deliberate) sampling method, the study included both childcare professionals and on-site health coordinators aged between 21 and 56 years of age. Five main themes were put forth as a result of the conducted thematic analysis: prevention and control measures for managing head lice, information and knowledge, social issues, psychological issues and disease perception. Each of these themes also had specific emerging categories based on participants' responses. Even though all respondents confirmed that the disease is continuously monitored only a few of them mentioned that a scalp examination was an inherent part of disease surveillance within the kindergarten community. Moreover, we found that information on pediculosis capitis is available to both parents and childcare professionals, but with a questionable uptake. Additionally, the majority of the respondents reported that parents tend to hide the infestation in their children due to shame and/or to avoid social stigma, and thus often fail to inform kindergarten teachers and health coordinators about the problem. In conclusion, our findings have implications for further practice and the introduction of tailored public health measures for the most vulnerable populations, most notably kindergarten children.

Targeted Delivery of Adamantylated Peptidoglycan Immunomodulators in Lipid Nanocarriers: NMR Shows That Cargo Fragments Are Available on the Surface.

We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles.

Novel mannosyl derivatives of peptidoglycan monomer: Synthesis and biological evaluation of immunomodulatory properties.

The aim of this work was to prepare mannosyl derivatives of peptidoglycan monomer (PGM, beta-d-GlcNAc-(1-->4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(epsilonNH(2))-d-Ala-d-Ala) in order to study the effects of mannosylation on adjuvant (immunostimulating) activity. Novel Man-OCH(2)CH(CH(3))CO-PGM isomers were substrates for N-acetylmuramyl-l-alanine amidase, like the parent PGM molecule. Adjuvant activity of Man-OCH(2)CH(CH(3))CO-PGM was tested in the mouse model using ovalbumin as an antigen.

Proposed dual antagonist approach for the prevention and treatment of urinary tract infections caused by uropathogenic Escherichia coli.

Urinary tract infections are among the most common infectious diseases worldwide, primarily caused by uropathogenic Escherichia coli (UPEC) strains that harbor type I pili and P pili on the surface. Standard E. coli therapy still entails antibiotic consumption, but urinary tract infections tend to recur at a very high rate. Due to the emergence of antibiotic resistant strains of UPEC, as well as high infection recurrence rates, there is a need for new approaches to efficiently treat and prevent urinary tract infections. Since aforementioned adhesive organelles are the principal virulence factors in UPEC, anti-adhesion strategy seems to be the most promising (and hitherto unexplored) treatment option. Here we propose an antiadhesive dual targeting approach towards FimH and PapG adhesive proteins placed on two key virulence factors for UPEC - type I fimbriae and P pili. Such dual antagonists will contain appropriate pharmacophores (mannose and natural cranberry-containing polyphenol) joined together and will more efficiently block the infection and prevent the progression of the disease in comparison to FimH and PapG as isolated targets. More specifically, polyphenol mannosides (due to the structural similarities with the most potent biaryl inhibitors) can act as high-affinity FimH ligands, while cranberry-associated polyphenol moiety can additionally inhibit the PapG-mediated adhesion. Proposed compound may also contribute to the antioxidant capacity of the human organism. In conclusion, this dual-target hypothesis for the prevention and treatment of UPEC infections represents an important foundation for further research on this topic.

A 'pathogenic needle' in a 'commensal haystack': Genetic virulence signatures of Corynebacterium glucuronolyticum that may drive its infectious propensity for the male urogenital system.

The predominance of the genus Corynebacterium in the healthy male urogenital system contributes to the resident microbiome of not only the distal urethra, but potentially the proximal urethra and urinary bladder as well. However, for certain species in this genus, pathogenic potential was described, and the salient representative is Corynebacterium glucuronolyticum (C. glucuronolyticum) implicated in cases of urethritis and prostatitis in men. Nonetheless, some still question whether C. glucuronolyticum can actually be considered pathogenic or rather just a commensal species fortuitously isolated in patients with urogenital symptoms and/or syndromes. Although pathogen/commensal dichotomy is not always clear-cut, we hypothesize that specific genetic markers may expose C. glucuronolyticum as a convincingly pathogenic Corynebacterium. More specifically, characteristic pathogenic gene constellation inherent to this species (most notably the presence of specific sortase/SpaA-type pili gene clusters, but also the augmentative role of type VII secretion system) may significantly facilitate host tissue adhesion, with subsequent suppression/evasion of the immune response and acquisition of vitally important nutrients. Consequently, these genetic markers differentiate C. glucuronolyticum from its commensal counterparts, and give this species a pathogenic facet, which can be even further influenced by the Allee effect. In this paper we also propose a specific methodological approach on how to analyze C. glucuronolyticum epithelial colonization capacity and explore inceptive host cell-pathogen interactions that manipulate host environment and immune responses. This entails moving from approaches based primarily on overall homology of primary sequences towards specific structure-function studies to precisely evaluate all stakeholders involved in pili assemblage, cell adhesion and the expression of other virulence traits. In the era of high precision medicine, the hypothesized roles of C. glucuronolyticum adhesion systems in both virulence and nutrient acquisition may also reveal promising targets for future drug developments.