Comparison of polysaccharide glycoconjugates as candidate vaccines to combat Clostridiodes (Clostridium) difficile.

Two known Clostridiodes (Clostridium) difficile surface antigens, a lipoteichoic acid (LTA) and a polysaccharide (PS-II) were isolated and purified in order to prepare glycoconjugate vaccines to the carrier protein human serum albumin utilising a reductive amination strategy. Mice and rabbits were immunized with a prime and two boost strategy and the resulting sera were examined for their ability to recognise the purified homologous antigens and subsequently killed whole cells of C. difficile strains and other Clostridia species. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, with generally similar titers from animals that received the LTA or the PS-II conjugates. Sera raised to the LTA conjugates were able to recognise other Clostridia species C. butyricum, C. bifermentans and C. subterminale whereas sera raised to the PS-II conjugates were not. These LTA and PS-II sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has confirmed that the LTA and PS-II polysaccharides are both highly conserved surface polymers of C. difficile that are easily accessible to the immune system and as such may have potential as vaccine antigens or as targets for therapeutics to combat C. difficile infection.

Proceedings of a workshop to discuss the epidemiology of invasive Haemophilus influenzae disease with emphasis on serotype a and b in the Americas, 2019.

On March 9, 2019, a one-day workshop titled "The current epidemiology of invasive Haemophilus influenzae disease in the Americas", jointly organized by the Public Health Agency of Canada (PHAC), the Canadian Institute of Health Research (CIHR), and the National Research Council Canada (NRC), brought together experts in the epidemiology and surveillance of invasive Haemophilus influenzae (Hi) disease from the Pan American Health Organization (PAHO) and its five regional reference laboratories in South America, USA, and Canada in Ottawa, Ontario, Canada. This workshop built upon recommendations of previous related workshops and incorporated updated data.

First characterization of immunogenic conjugates of Vi negative Salmonella Typhi O-specific polysaccharides with rEPA protein for vaccine development.

Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the first time, we report the synthesis and evaluation of polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Four different conjugates were synthesized using recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA) as the carrier proteins, using either direct reductive amination or an intermediate linker molecule, adipic acid dihydrazide (ADH). Upon injection into mice, a significantly higher antibody titer was observed in mice administrated with conjugate-1 (OSP-HSA) (P=0.0001) and conjugate 2 (OSP-rEPA) (P≤0.0001) as compared to OSP alone. In contrast, the antibody titer elicited by conjugate 3 (OSPADH-HSA) and conjugate 4 (OSPADH-rEPA) were insignificant (P=0.1684 and P=0.3794, respectively). We conclude that reductive amination is the superior method to prepare the S. Typhi OSP glycoconjugate. Moreover, rEPA was a better carrier protein than HSA. Thus OSP-rEPA conjugate seems to be efficacious typhoid vaccines candidate, it may be evaluated further and recommended for the clinical trials.

INO80 is required for oncogenic transcription and tumor growth in non-small cell lung cancer.

Epigenetic regulators are attractive targets for the development of new cancer therapies. Among them, the ATP-dependent chromatin remodeling complexes control the chromatin architecture and have important roles in gene regulation. They are often found to be mutated and de-regulated in cancers, but how they influence the cancer gene expression program during cancer initiation and progression is not fully understood. Here we show that the INO80 chromatin remodeling complex is required for oncogenic transcription and tumor growth in non-small-cell lung cancer (NSCLC). Ino80, the SWI/SNF ATPase in the complex, is highly expressed in NSCLC cells compared with normal lung epithelia cells. Further, its expression, as well as that of another subunit Ino80B, negatively correlates with disease prognosis in lung cancer patients. Functionally, INO80 silencing inhibits NSCLC cell proliferation and anchorage-independent growth in vitro and tumor formation in mouse xenografts. It occupies enhancer regions near lung cancer-associated genes, and its occupancy correlates with increased genome accessibility and enhanced expression of downstream genes. Together, our study defines a critical role of INO80 in promoting oncogenic transcription and NSCLC tumorigenesis, and reveals a potential treatment strategy for inhibiting the cancer transcription network by targeting the INO80 chromatin remodeling complex.

The emerging Haemophilus influenzae serotype a infection and a potential vaccine: Implementation science in action.

Haemophilus influenzae serotype b (Hib) was a major cause of meningitis in children until Hib conjugate vaccine was introduced into the routine infant immunization program and Hib disease in children was almost eliminated. In Alaska, northern Canada and other countries with Indigenous peoples, H. influenzae serotype a (Hia) has emerged as a significant cause of pneumonia, meningitis and septic arthritis especially in children under 24 months of age. A joint government initiative between the Public Health Agency of Canada (PHAC) and the National Research Council of Canada (NRC) was carried out to assess whether an Hia vaccine could be developed for the common good. The initiative included strategic partnerships with clinician researchers in Thunder Bay, Ontario who provide health services to Indigenous people and the Artic Investigations Program (AIP) of the United States Centers for Disease Control and Prevention (CDC) in Alaska. This government initiated and funded research identified that the development of an Hia vaccine is possible and ongoing surveillance that includes strain characterization is essential to understand the potential spread of Hia in North America and around the world.

Developing a vaccine for Haemophilus influenzae serotype a: Proceedings of a workshop.

Since the late 1990s there has been an emergence of Haemophilus influenzae serotype a (Hia) infections, especially in Indigenous communities in the northern regions of Canada and Alaska associated with significant morbidity and approximately a 10% mortality. A Hia vaccine could potentially prevent this disease and save the health care system millions of dollars in both acute and long-term care. On March 23-24, 2016, the National Research Council (NRC), the Public Health Agency of Canada (PHAC) and the Canadian Institutes of Health Research (CIHR) co-organized a meeting on H. influenzae serotype a (Hia) to examine the current state of disease epidemiology and a potential vaccine solution path. The meeting included representatives from academia, federal and territorial public health units, hospital laboratories, federal departments involved in Aboriginal health, advocacy organizations for Indigenous peoples and industry. Representatives from industry confirmed having the capacity and the interest to support preparation of clinical trial batches. Canadian regulatory authorities have expressed a willingness to help ensure appropriate measures are in place for licensure purposes. Furthermore, there is the capacity and interest in performing some clinical trials in Indigenous communities in both Canada and Alaska. Recommendations for next steps included: complete pre-clinical studies, improve epidemiological surveillance to better understand the extent of the disease in the rest of North America and globally, establish engagement mechanisms with national Indigenous organizations to ensure their peoples are fully involved in the process and explore funding opportunities to prepare clinical lots and undertake clinical trials.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: conjugates based on core oligosaccharides.

In this study we have prepared glycoconjugates with core oligosaccharides (OS) from the lipopolysaccharide (LPS) of Neisseria meningitidis, thus avoiding the neo-epitopes of the deacylated lipid A region of the derived LPS molecule identified in our previous studies. A comprehensive investigation was performed with glycoconjugates prepared from the most extended to the most truncated core OS still maintaining the conserved inner core epitope. As previously, we have established reproducible bactericidal killing of the homologous antigen elaborating strain, but a failure to kill wild-type strains. In these studies it was evident that the linker molecules used in the conjugation methodologies were dominating the immune response. However, when galE core OS based conjugates were prepared without utilizing linkers, via direct reductive amination, we failed to generate an immune response to even the homologous antigen. We also identified that immunisation with the galE antigen via linker methodologies provoked an immune response that was dependent upon key residues of the conserved inner core OS structure, whereas the immune responses to lgtB and lgtA antigens did not involve the inner core OS. This comprehensive study has, despite our best efforts, cast significant doubt as to the utility of the conserved inner core region of the meningococcal LPS as a potential vaccine antigen.

Rasosomes originate from the Golgi to dispense Ras signals.

Ras proteins undergo an incompletely understood trafficking process in the cell. Rasosomes are protein nanoparticles of 80-100 nm diameter that carry lipidated Ras isoforms (H-Ras and N-Ras) as well as their effectors through the cytoplasm and near the plasma membrane (PM). In this study, we identified the subcellular origin of rasosomes and how they spread Ras proteins through the cell. We found no dependency of rasosome formation on galectins, or on the GDP-/GTP-bound state of Ras. We found that significantly more rasosomes are associated with forms of Ras that are localized to the Golgi, namely N-Ras or the singly palmitoylated H-Ras mutant (C181S). To explore the possibility that rasosome originate from the Golgi, we used photoactivatable (PA)-GFP-H-Ras mutants and showed that rasosomes bud from the Golgi in a two-step mechanism. Newly released rasosomes first move in an energy-dependent directed fashion and then convert to randomly diffusing rasosomes. Dual fluorescence time-lapse imaging revealed the appearance of dually labeled rasosomes, indicating a dynamic exchange of cytoplasmic and PM-associated Ras with rasosome-associated Ras. Finally, higher levels of rasosomes correlate with higher levels of ERK phosphorylation, a key marker of Ras downstream signaling. We suggest that H-Ras and N-Ras proteins exchange with rasosomes that can function as carriers of palmitoylated Ras and its signals.

Differential requirement of CAAX-mediated posttranslational processing for Rheb localization and signaling.

The Rheb1 and Rheb2 small GTPases and their effector mTOR are aberrantly activated in human cancer and are attractive targets for anti-cancer drug discovery. Rheb is targeted to endomembranes via its C-terminal CAAX (C=cysteine, A=aliphatic, X=terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl isoprenoid. After farnesylation, Rheb undergoes two additional CAAX-signaled processing steps, Ras converting enzyme 1 (Rce1)-catalyzed cleavage of the AAX residues and isoprenylcysteine carboxyl methyltransferase (Icmt)-mediated carboxylmethylation of the farnesylated cysteine. However, whether these postprenylation processing steps are required for Rheb signaling through mTOR is not known. We found that Rheb1 and Rheb2 localize primarily to the endoplasmic reticulum and Golgi apparatus. We determined that Icmt and Rce1 processing is required for Rheb localization, but is dispensable for Rheb-induced activation of the mTOR substrate p70 S6 kinase (S6K). Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks Rheb localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of Rheb. We conclude that inhibitors of Icmt and Rce1 will not block Rheb function, but FTS could be a promising treatment for Rheb- and mTOR-dependent cancers.

Candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: developmental chemistry and investigation of immunological responses following immunization of mice and rabbits.

Glycoconjugates were prepared by covalently linking the immunogenic protein carrier CRM(197) to O-deacylated lipopolysaccharide (LPS) derived from Neisseria meningitidis (strain H44/76), immunotype L3 galE LPS. This mutant strain elaborates a truncated LPS structure that displays immunological epitopes characteristic of 76% of Group B meningococcal (NmB) strains. CRM(197) was covalently linked either to the reducing glucosamine residue of the lipid A region of the O-deacylated LPS or to a 2-keto-3-deoxy-octulosonic acid (Kdo) residue in the inner core region of the O-deacylated LPS. In both rabbits and mice a much stronger IgG response to the immunising antigen was generated in those animals that received conjugates linked via the lipid A region. Sera from mice that were immunized with these conjugates were assayed for their reactivity with LPS, both mutant and wild-type, of several homologous and heterologous NmB strains. Sera obtained from mice immunized with conjugates in which the carrier protein was linked via the Kdo moiety were only able to react with O-deacylated, but not fully acylated (native), LPS from the homologous strain. However, sera obtained from mice that were immunized with conjugates, in which the carrier protein was coupled to the lipid A region, reacted predominately with inner core epitopes that contained phosphoethanolamine at the same 3-position of the distal heptose residue (HepII) of the inner core LPS as was present on the immunising antigen. Additionally it was observed that sera from rabbits immunised with lipid A linked conjugates, unlike the mice responses, were generally not as specific for LPS antigens that contained phosphoethanolamine at the same 3-position as was present on the immunising antigen, but showed a broader inner core recognition, whereas those rabbits that received the Kdo-linked conjugates gave only a very weak non-specific response to all immunotypes. Finally, the sera from two out of six mice that had received lipid A linked conjugates had bactericidal activity against L3 wild-type NmB strain 8047 and one of these was able to passively protect against meningococcal infection in an infant rat model. This study demonstrates evidence towards the proof-in-principle that by using Nm inner core LPS conjugates coupled via the lipid A region with an intact phosphoethanolamine at the O-3 position of the HepII of the inner core LPS, it is possible to elicit functional and protective antibodies against meningococcal infection.

Mapping bacterial glycolipid complexity using capillary electrophoresis and electrospray mass spectrometry.

This chapter presents the application of capillary electrophoresis coupled to electrospray mass spectrometry (CE-ES-MS) for the analysis of complex bacterial lipopolysaccharides (LPS) from pathogenic strains of Haemophilus influenzae and Neisseria meningitidis. A discussion is included of the development of electrophoretic conditions conducive to trace-level enrichment and separation of closely related glycoforms and isoforms, which provided sensitive detection of glycolipids from as little as five bacterial colonies. The chapter also describes the use of mixed MS scanning functions to aid the identification of specific functionalities and immunodeterminants of LPS, such as pyrophosphoethanolamine, phosphocholine, and N-acetyl neuraminic acid (Neu5Ac), which represent less than 2% of the overall LPS population. The combination of high-resolution capillary electrophoresis with sensitive tandem mass spectrometry (MS/MS) provides a unique analytical tool to probe the subtle structural changes resulting from oligosaccharide branching and location of substituted LPS isoforms. The ability to detect a diverse LPS population over a wide dynamic range of expression using CE-MS enables the correlation of structural changes between bacterial strains and isogenic mutants to assign functional gene relationship.

Intergenerational transmission of somatization behaviour: 2. Observations of joint attention and bids for attention.

BACKGROUND: Somatoform disorders may have their roots in childhood through processes that involve an enhanced parental focus on health. The aim of this study was to test the hypothesis that somatizing mothers will show less joint involvement than other mothers during play but greater responsiveness when this play involves a 'medical' theme. METHOD: Cross-sectional observational study of 42 chronic somatizers, 44 organically ill and 50 healthy mothers and their 4-8 year-old children during structured play and a meal. Tasks comprised boxes containing tea-set items, 'medical' items and a light snack. RESULTS: Somatizing mothers were emotionally flatter and showed lower rates of joint attention than other mothers during both play tasks. While the three groups had similar rate of bids for attention, somatizing mothers were more responsive to their child's bids during play with the medical box than at other times. In contrast, the children of somatizing mothers ignored a greater proportion of their mother's bids during play with the medical box than did children of other mothers or during play with a non-medical theme. CONCLUSION: The study has demonstrated tentative evidence in support of the hypothesis.

Intergenerational transmission of somatization behaviour: a study of chronic somatizers and their children.

BACKGROUND: Exposure to an ill parent in childhood may be a risk factor for adult somatization. This study examines the hypothesis that somatizing adults are more likely to have been exposed to illness as a child and that in turn, their children are more likely to report ill health and to have more contact with medical services than children of other mothers. METHOD: A cross-sectional comparative investigation of three groups of mothers and their children of 4-8 years of age: (i) 48 mothers suffering from chronic somatization; (ii) 51 mothers with chronic 'organic' illness; and (iii) 52 healthy mothers was carried out. RESULTS: Somatizing mothers were more likely than other women to report exposure to childhood neglect and to physical illness in a parent (OR 2.9; 95% CI 1.4-6.1). The children of these somatizing mothers were more likely to have health problems than were the children of organically ill or healthy women and had more consultations with family doctors (average annual rates: somatizers 4.9 (S.D. 3.8), organic 3.0 (S.D. 3.5) and healthy 2.8 (S.D. 2.6)). Multivariate modelling of consultation rates among children found significant main effects for maternal somatization, maternal childhood adversity, the child's tendency to worry about health and a two-way interaction of maternal childhood adversity and her somatization status. CONCLUSIONS: The hypotheses are broadly supported. However, it is important to emphasize the extent to which these findings are based on maternal reports.

Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H. influenzae strain Rd (RM118).

A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)-]-L-alpha-D-Hepp-(1-->5)-alpha-Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D (1)H-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes involved in the addition of each glycose residue.

Structural analysis of the lipopolysaccharide from the nontypable Haemophilus influenzae strain SB 33.

The structure of the core region of the lipopolysaccharide (LPS) from the nontypable Haemophilus influenzae strain SB 33 was elucidated. The LPS was subjected to a variety of degradative procedures. The structures of the derived oligosaccharide products were established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. These analyses revealed a series of related phosphocholine (PCho) containing structures differing in the number of hexose residues. The results pointed to each species containing a conserved phosphoethanolamine (PEtn) substituted heptose-containing trisaccharide inner-core moiety. The major LPS glycoforms were identified as 2-Hex, 3-Hex and 4-Hex species according to the number of hexose residues present.

Farnesyltransferase inhibitors: potential role in the treatment of cancer.

New targets for drug discovery have been identified rapidly as a result of the many recent and rapid advances in the understanding of signal transduction pathways that contribute to oncogenesis. In particular, oncogenic Ras proteins have been seen as an important target for novel anti-cancer drugs. Since the decade-old identification and cloning of farnesyltransferase (FTase), a critical enzyme that post-translationally modifies Ras and other farnesylated proteins, FTase inhibitors (FTIs) have been under intense investigation designed to bring them to clinical practice for cancer therapy. FTIs can inhibit the growth of tumour cells in culture and in animal models, and are now in clinical trials. Interestingly, their mechanism of action is not as simple as originally envisioned, and Ras is probably not the most important farnesylated protein whose modification is inhibited as a result of FTI treatment. Although K-Ras can escape inhibition of processing by FTIs, tumours with oncogenically mutated K-Ras proteins can still be inhibited by FTI treatment. Indeed, Ras mutation status does not correlate with FTI sensitivity or resistance. Instead, it now appears likely that inhibition of the processing of other farnesylated proteins such as RhoB and the centromere-binding proteins CENP-E and CENP-F can explain the ability of FTIs to cause cell cycle arrest and apoptosis in preclinical studies, and even to cause regression in animal tumour models. Preclinical studies suggest the likelihood that FTIs will be useful in combination therapies with conventional treatment modalities including cytotoxics (especially paclitaxel) and radiation. Phase I combination trials are underway, and early phase II/III trials using FTIs as monotherapy are open for patients with a wide variety of cancers. Early preclinical results also suggest the possibility of using FTIs as chemopreventive agents. Studies to be completed over the next 2 or 3 years should define the appropriate patient populations, administration and scheduling necessary to optimise the use of these novel anticancer agents.

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.

We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.