Stimulation of Potent Humoral and Cellular Immunity via Synthetic Dual-Antigen MVA-Based COVID-19 Vaccine COH04S1 in Cancer Patients Post Hematopoietic Cell Transplantation and Cellular Therapy.

Hematopoietic cell transplantation (HCT) and chimeric antigen receptor (CAR)-T cell patients are immunocompromised, remain at high risk following SARS-CoV-2 infection, and are less likely than immunocompetent individuals to respond to vaccination. As part of the safety lead-in portion of a phase 2 clinical trial in patients post HCT/CAR-T for hematological malignancies (HM), we tested the immunogenicity of the synthetic modified vaccinia Ankara-based COVID-19 vaccine COH04S1 co-expressing spike (S) and nucleocapsid (N) antigens. Thirteen patients were vaccinated 3-12 months post HCT/CAR-T with two to four doses of COH04S1. SARS-CoV-2 antigen-specific humoral and cellular immune responses, including neutralizing antibodies to ancestral virus and variants of concern (VOC), were measured up to six months post vaccination and compared to immune responses in historical cohorts of naïve healthy volunteers (HV) vaccinated with COH04S1 and naïve healthcare workers (HCW) vaccinated with the FDA-approved mRNA vaccine Comirnaty® (Pfizer, New York, NY, USA). After one or two COH04S1 vaccine doses, HCT/CAR-T recipients showed a significant increase in S- and N-specific binding antibody titers and neutralizing antibodies with potent activity against SARS-CoV-2 ancestral virus and VOC, including the highly immune evasive Omicron XBB.1.5 variant. Furthermore, vaccination with COH04S1 resulted in a significant increase in S- and N-specific T cells, predominantly CD4+ T lymphocytes. Elevated S- and N-specific immune responses continued to persist at six months post vaccination. Furthermore, both humoral and cellular immune responses in COH04S1-vaccinated HCT/CAR-T patients were superior or comparable to those measured in COH04S1-vaccinated HV or Comirnaty®-vaccinated HCW. These results demonstrate robust stimulation of SARS-CoV-2 S- and N-specific immune responses including cross-reactive neutralizing antibodies by COH04S1 in HM patients post HCT/CAR-T, supporting further testing of COH04S1 in immunocompromised populations.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Aerobic bacterial methane synthesis.

Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.

The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen-deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the 'DFD patch'), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

BACKGROUND AND AIMS: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined. APPROACH AND RESULTS: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver. CONCLUSIONS: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages.

ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection.

Hematopoietic stem cells (HSCs) undergo rapid expansion in response to stress stimuli. Here we investigate the bioenergetic processes which facilitate the HSC expansion in response to infection. We find that infection by Gram-negative bacteria drives an increase in mitochondrial mass in mammalian HSCs, which results in a metabolic transition from glycolysis toward oxidative phosphorylation. The initial increase in mitochondrial mass occurs as a result of mitochondrial transfer from the bone marrow stromal cells (BMSCs) to HSCs through a reactive oxygen species (ROS)-dependent mechanism. Mechanistically, ROS-induced oxidative stress regulates the opening of connexin channels in a system mediated by phosphoinositide 3-kinase (PI3K) activation, which allows the mitochondria to transfer from BMSCs into HSCs. Moreover, mitochondria transfer from BMSCs into HSCs, in the response to bacterial infection, occurs before the HSCs activate their own transcriptional program for mitochondrial biogenesis. Our discovery demonstrates that mitochondrial transfer from the bone marrow microenvironment to HSCs is an early physiologic event in the mammalian response to acute bacterial infection and results in bioenergetic changes which underpin emergency granulopoiesis.

Nurse-Driven mHealth Implementation Using the Technology Inpatient Program for Smokers (TIPS): Mixed Methods Study.

BACKGROUND: Smoking is the leading cause of preventable death and disease, yet implementation of smoking cessation in inpatient settings is inconsistent. The Technology Inpatient Program for Smokers (TIPS) is an implementation program designed to reach smokers with a mobile health (mHealth) intervention using stakeholder-supported strategies. OBJECTIVE: The purpose of this study was to determine the impact of the TIPS implementation strategies on smoker-level engagement of the mHealth intervention during care transition. METHODS: We examined varying intensities (passive motivational posters only and posters + active nurse-led facilitation) of TIPS strategies on four hospital units located in two sites. Unit-level and smoker-level adoption was monitored during active implementation (30 weeks) and sustainability follow-up (30 weeks). Process measures reflecting the reach, effectiveness, adoption, implementation, maintenance (RE-AIM) framework, stakeholder reported adaptations of strategies, and formative evaluation data were collected and analyzed. RESULTS: For our smoker-level reach, 103 smokers signed up for the mHealth intervention in-hospital, with minimal decline during sustainability follow-up. While posters + nurse facilitation did not lead to higher reach than posters alone during active implementation (27 vs 30 signed up), it did lead to higher engagement of smokers (85.2% vs 73.3% completion of the full 2-week intervention). TIPS strategy adoption and fidelity varied by unit, including adoption of motivational posters (range: weeks 1 and 5), fidelity of posters (0.4% to 16.2% of posters missing per unit weekly) and internal facilitation of nurse training sessions (average of 2 vs 7.5 by site). Variable maintenance costs of the program totaled US $6.63 (US $683.28/103) per smoker reached. Reported family-member facilitation of mHealth sign-up was an observation of unintended behavior. CONCLUSIONS: TIPS is a feasible and low-cost implementation program that successfully engages smokers in an mHealth intervention and sustains engagement after discharge. Further testing of nurse facilitation and expanding reach to patient family and friends as an implementation strategy is needed.

Elevated apoptosis impairs epithelial cell turnover and shortens villi in TNF-driven intestinal inflammation.

The intestinal epithelial monolayer, at the boundary between microbes and the host immune system, plays an important role in the development of inflammatory bowel disease (IBD), particularly as a target and producer of pro-inflammatory TNF. Chronic overexpression of TNF leads to IBD-like pathology over time, but the mechanisms driving early pathogenesis events are not clear. We studied the epithelial response to inflammation by combining mathematical models with in vivo experimental models resembling acute and chronic TNF-mediated injury. We found significant villus atrophy with increased epithelial cell death along the crypt-villus axis, most dramatically at the villus tips, in both acute and chronic inflammation. In the acute model, we observed overexpression of TNF receptor I in the villus tip rapidly after TNF injection and concurrent with elevated levels of intracellular TNF and rapid shedding at the tip. In the chronic model, sustained villus atrophy was accompanied by a reduction in absolute epithelial cell turnover. Mathematical modelling demonstrated that increased cell apoptosis on the villus body explains the reduction in epithelial cell turnover along the crypt-villus axis observed in chronic inflammation. Cell destruction in the villus was not accompanied by changes in proliferative cell number or division rate within the crypt. Epithelial morphology and immunological changes in the chronic setting suggest a repair response to cell damage although the villus length is not recovered. A better understanding of how this state is further destabilised and results in clinical pathology resembling IBD will help identify suitable pathways for therapeutic intervention.

Deciphering the Complex Signaling Systems That Regulate Intestinal Epithelial Cell Death Processes and Shedding.

Intestinal epithelial cells play a fundamental role in maintaining homeostasis. Shedding of intestinal cells in a controlled manner is critical to maintenance of barrier function. Barrier function is maintained during this shedding process by a redistribution of tight junctional proteins to facilitate closure of the gap left by the shedding cell. However, despite the obvious importance of epithelial cell shedding to gut health, a central question is how the extrusion of epithelial cells is achieved, enabling barrier integrity to be maintained in the healthy gut and restored during inflammation remains largely unanswered. Recent studies have provided evidence that excessive epithelial cell shedding and loss of epithelial barrier integrity is triggered by exposure to lipopolysaccharide or tumor necrosis factor alpha. Subsequent studies have provided evidence of the involvement of specific cellular components and signaling mechanisms as well as the functionality of microbiota that can be either detrimental or beneficial for intestinal barrier integrity. This review will focus on the evidence and decipher how the signaling systems through which the mucosal immune system and microbiota can regulate epithelial cell shedding and how these mechanisms interact to preserve the viability of the epithelium.

The state of cancer survivorship programming in Commission on Cancer-accredited hospitals in Georgia.

PURPOSE: In Georgia, there are more than 356,000 cancer survivors. Although many encounter challenges as a result of treatment, there is limited data on the availability of survivorship programming. This paper highlights findings from two surveys assessing survivorship care in Commission on Cancer (CoC)-accredited hospitals in Georgia. METHODS: In 2010, 38 CoC-accredited hospitals were approached to complete a 36-item survey exploring knowledge of national standards and use of survivorship care plans (SCPs), treatment summaries (TSs), and psychosocial assessment tools. In 2012, 37 CoC-accredited hospitals were asked to complete a similar 21-item survey. RESULTS: Seventy-nine percent (n = 30) of cancer centers completed the 2010 survey. Sixty percent (n = 18) reported having a cancer survivorship program in place or in development. Forty-three percent (n = 13) provided survivors with a SCP and 40% (n = 12) a TS. Sixty percent (n = 18) reported either never or rarely using a psychosocial assessment tool. Sixty-two percent (n = 23) completed the 2012 survey. Ninety-six percent (n = 22) were aware of the new CoC guideline 3.3. Thirty-nine percent (n = 9) provided a SCP and/or TS. Eighty-seven percent (n = 20) stated they were very confident or somewhat confident their organization could implement a SCP and/or TS by 2015. CONCLUSIONS: The data indicated the importance of collaboration and shared responsibility for survivorship care. Broad implementation of SCPs and TSs can help address the late and long-term effects of treatment. IMPLICATIONS FOR CANCER SURVIVORS: Increasing knowledge on survivorship care is imperative as the Georgia oncology community engages oncologists and primary care providers to achieve higher quality of life for all survivors.

Syndecan-3 is selectively pro-inflammatory in the joint and contributes to antigen-induced arthritis in mice.

INTRODUCTION: Syndecans are heparan sulphate proteoglycans expressed by endothelial cells. Syndecan-3 is expressed by synovial endothelial cells of rheumatoid arthritis (RA) patients where it binds chemokines, suggesting a role in leukocyte trafficking. The objective of the current study was to examine the function of syndecan-3 in joint inflammation by genetic deletion in mice and compare with other tissues. METHODS: Chemokine C-X-C ligand 1 (CXCL1) was injected in the joints of syndecan-3-/-and wild-type mice and antigen-induced arthritis performed. For comparison chemokine was administered in the skin and cremaster muscle. Intravital microscopy was performed in the cremaster muscle. RESULTS: Administration of CXCL1 in knee joints of syndecan-3-/-mice resulted in reduced neutrophil accumulation compared to wild type. This was associated with diminished presence of CXCL1 at the luminal surface of synovial endothelial cells where this chemokine clustered and bound to heparan sulphate. Furthermore, in the arthritis model syndecan-3 deletion led to reduced joint swelling, leukocyte accumulation, cartilage degradation and overall disease severity. Conversely, CXCL1 administration in the skin of syndecan-3 null mice provoked increased neutrophil recruitment and was associated with elevated luminal expression of E-selectin by dermal endothelial cells. Similarly in the cremaster, intravital microscopy showed increased numbers of leukocytes adhering and rolling in venules in syndecan-3-/-mice in response to CXCL1 or tumour necrosis factor alpha. CONCLUSIONS: This study shows a novel role for syndecan-3 in inflammation. In the joint it is selectively pro-inflammatory, functioning in endothelial chemokine presentation and leukocyte recruitment and cartilage damage in an RA model. Conversely, in skin and cremaster it is anti-inflammatory.

A chemokine self-presentation mechanism involving formation of endothelial surface microstructures.

Endothelial surface microstructures have been described previously under inflammatory conditions; however, they remain ill-characterized. In this study, CXCL8, an inflammatory chemokine, was shown to induce the formation of filopodia-like protrusions on endothelial cells; the same effects were observed with CXCL10 and CCL5. Chemokines stimulated filopodia formation by both microvascular (from bone marrow and skin) and macrovascular (from human umbilical vein) endothelial cells. Use of blocking Abs and degradative enzymes demonstrated that CXCL8-stimulated filopodia formation was mediated by CXCR1 and CXCR2, Duffy Ag/receptor for chemokines, heparan sulfate (HS), and syndecans. HS was present on filopodial protrusions appearing as a meshwork on the cell surface, which colocalized with CXCL8, and this glycosaminoglycan was 2,6-O- and 3-O-sulfated. Transmission electron microscopy revealed that CXCL8-stimulated filopodial and microvilli-like protrusions that interacted with leukocytes before transendothelial migration and removal of HS reduced this migration. iTRAQ mass spectrometry showed that changes in the levels of cytoskeletal, signaling, and extracellular matrix proteins were associated with CXCL8-stimulated filopodia/microvilli formation; these included tropomyosin, fascin, and Rab7. This study suggests that chemokines stimulate endothelial filopodia and microvilli formation, leading to their presentation to leukocytes and leukocyte transendothelial migration.

Behaviour change to treat overactive bladder syndrome.

Overactive bladder syndrome has a negative impact on quality of life. This article discusses a definition of the condition and its the prevalence and aetiology, as well as some of the more common behavioural therapies. Although there is a good evidence base for bladder retraining, there is often conflicting research to support other interventions including caffeine reduction, the amount and types of fluid recommended, smoking cessation and weight reduction. A standardised approach from a national level is needed; nurses also need to demonstrate more robustly the effectiveness of their interventions.

Survivorship navigation outcome measures: a report from the ACS patient navigation working group on survivorship navigation.

Survivorship navigation is a relatively new concept in the field of patient navigation but an important one. This article highlights the essential functions of the survivorship navigator and defines core outcomes and measures for navigation in the survivorship period. Barriers to access to care experienced by patients during active cancer treatment can continue into the post-treatment period, affecting quality follow-up care for survivors. These barriers to care can be particularly acute for non-English speakers, immigrants, the uninsured, the underinsured, and other vulnerable populations. The survivorship navigator can help reduce barriers and facilitate access to survivorship care and services through communication and information exchange for patients. Survivorship navigation may improve appropriate health care utilization through education and care coordination, potentially improving health outcomes and quality of life of survivors while reducing cost to the health care system. Survivorship navigators can also educate survivors on how to improve their overall wellness, thereby directly impacting the health of a growing population of cancer survivors.

Improving information for men with postoperative urinary incontinence.

Despite urinary incontinence being a recognised complication of prostate surgery, the physical and psychological needs of men with postoperative urinary incontinence are not well met and there is little written information specifically designed for this group of patients (Moore and Estey, 1999; Harris, 1997). Men rarely seek help for urinary incontinence and I used to feel frustrated at finding that some put up with a continence problem in secret for many years. I decided to explore the reasons for their failing to ask for help and what sort of information they would require if they were going to have prostate surgery. The information I elicited helped me to formulate a strategy to tackle this difficult issue.

Differential binding of chemokines to macrophages and neutrophils in the human inflamed synovium.

In chronic inflammatory foci, such as the rheumatoid joint, there is enhanced recruitment of phagocytes from the blood into the tissues. Chemokines are strongly implicated in directing the migration of these cells, although little is known regarding the chemokine receptors that could mediate their chemotaxis into the joint tissue. Therefore the objective of the study was to identify chemokine binding sites on macrophages and neutrophils within the rheumatoid synovium using radiolabeled ligand binding and in situ autoradiography. Specific binding sites for CCL3 (macrophage inflammatory protein-1alpha), CCL5 (RANTES), CCL2 (monocyte chemoattractant protein-1) and CXCL8 (IL-8) were demonstrated on CD68+ macrophages in the subintimal and intimal layers. The number and percentage of intimal cells that bound chemokines were greater in inflamed regions compared to noninflamed regions. The intensity of intimal binding varied between chemokines with the rank order, CCL3 > CCL5 > CCL2 > CXCL8. Neutrophils throughout the synovium bound CXCL8 but did not show any signal for binding CCL2, CCL3 or CCL5. Immunohistochemistry showed that both CXCR1 and CXCR2 are expressed by macrophages and neutrophils in the rheumatoid and nonrheumatoid synovia, suggesting that both of these receptors are responsible for the CXCL8 binding. The chemokine binding sites described on phagocytes may be involved in the migration of these cells into the inflamed joint.