Mixed-method evaluation study of a targeted mass drug administration of long-acting anti-malarials among children aged 3 months to 15 years in the Bossangoa sub-prefecture, Ouham, Central African Republic, during the COVID-19 pandemic.

BACKGROUND: In 2020, during the COVID-19 pandemic, Médecins Sans Frontières (MSF) initiated three cycles of dihydroartemisin-piperaquine (DHA-PQ) mass drug administration (MDA) for children aged three months to 15 years within Bossangoa sub-prefecture, Central African Republic. Coverage, clinical impact, and community members perspectives were evaluated to inform the use of MDAs in humanitarian emergencies. METHODS: A household survey was undertaken after the MDA focusing on participation, recent illness among eligible children, and household satisfaction. Using routine surveillance data, the reduction during the MDA period compared to the same period of preceding two years in consultations, malaria diagnoses, malaria rapid diagnostic test (RDT) positivity in three MSF community healthcare facilities (HFs), and the reduction in severe malaria admissions at the regional hospital were estimated. Twenty-seven focus groups discussions (FGDs) with community members were conducted. RESULTS: Overall coverage based on the MDA card or verbal report was 94.3% (95% confidence interval (CI): 86.3-97.8%). Among participants of the household survey, 2.6% (95% CI 1.6-40.3%) of round 3 MDA participants experienced illness in the preceding four weeks compared to 30.6% (95% CI 22.1-40.8%) of MDA non-participants. One community HF experienced a 54.5% (95% CI 50.8-57.9) reduction in consultations, a 73.7% (95% CI 70.5-76.5) reduction in malaria diagnoses, and 42.9% (95% CI 36.0-49.0) reduction in the proportion of positive RDTs among children under five. A second community HF experienced an increase in consultations (+ 15.1% (- 23.3 to 7.5)) and stable malaria diagnoses (4.2% (3.9-11.6)). A third community HF experienced an increase in consultations (+ 41.1% (95% CI 51.2-31.8) and malaria diagnoses (+ 37.3% (95% CI 47.4-27.9)). There were a 25.2% (95% CI 2.0-42.8) reduction in hospital admissions with severe malaria among children under five from the MDA area. FGDs revealed community members perceived less illness among children because of the MDA, as well as fewer hospitalizations. Other indirect benefits such as reduced household expenditure on healthcare were also described. CONCLUSION: The MDA achieved high coverage and community acceptance. While some positive health impact was observed, it was resource intensive, particularly in this rural context. The priority for malaria control in humanitarian contexts should remain diagnosis and treatment. MDA may be additional tool where the context supports its implementation.

Multiple introductions of monkeypox virus to Ireland during the international mpox outbreak, May 2022 to October 2023.

BackgroundMpox, caused by monkeypox virus (MPXV), was considered a rare zoonotic disease before May 2022, when a global epidemic of cases in non-endemic countries led to the declaration of a Public Health Emergency of International Concern. Cases of mpox in Ireland, a country without previous mpox reports, could reflect extended local transmission or multiple epidemiological introductions.AimTo elucidate the origins and molecular characteristics of MPXV circulating in Ireland between May 2022 and October 2023.MethodsWhole genome sequencing of MPXV from 75% of all Irish mpox cases (182/242) was performed and compared to sequences retrieved from public databases (n = 3,362). Bayesian approaches were used to infer divergence time between sequences from different subclades and evaluate putative importation events from other countries.ResultsOf 242 detected mpox cases, 99% were males (median age: 35 years; range: 15-60). All 182 analysed genomes were assigned to Clade IIb and, presence of 12 distinguishable subclades suggests multiple introductions into Ireland. Estimation of time to divergence of subclades further supports the hypothesis for multiple importation events from numerous countries, indicative of extended and sustained international spread of mpox. Further analysis of sequences revealed that 92% of nucleotide mutations were from cytosine to thymine (or from guanine to adenine), leading to a high number of non-synonymous mutations across subclades; mutations associated with tecovirimat resistance were not observed.ConclusionWe provide insights into the international transmission dynamics supporting multiple introductions of MPXV into Ireland. Such information supported the implementation of evidence-informed public health control measures.

Estimated incubation period distributions of mpox using cases from two international European festivals and outbreaks in a club in Berlin, May to June 2022.

BackgroundSince May 2022, an mpox outbreak affecting primarily men who have sex with men (MSM) has occurred in numerous non-endemic countries worldwide. As MSM frequently reported multiple sexual encounters in this outbreak, reliably determining the time of infection is difficult; consequently, estimation of the incubation period is challenging.AimWe aimed to provide valid and precise estimates of the incubation period distribution of mpox by using cases associated with early outbreak settings where infection likely occurred.MethodsColleagues in European countries were invited to provide information on exposure intervals and date of symptom onset for mpox cases who attended a fetish festival in Antwerp, Belgium, a gay pride festival in Gran Canaria, Spain or a particular club in Berlin, Germany, where early mpox outbreaks occurred. Cases of these outbreaks were pooled; doubly censored models using the log-normal, Weibull and Gamma distributions were fitted to estimate the incubation period distribution.ResultsWe included data on 122 laboratory-confirmed cases from 10 European countries. Depending on the distribution used, the median incubation period ranged between 8 and 9 days, with 5th and 95th percentiles ranging from 2 to 3 and from 20 to 23 days, respectively. The shortest interval that included 50% of incubation periods spanned 8 days (4-11 days).ConclusionCurrent public health management of close contacts should consider that in approximately 5% of cases, the incubation period exceeds the commonly used monitoring period of 21 days.

Adeno-Associated Virus 2 and Human Adenovirus F41 in Wastewater during Outbreak of Severe Acute Hepatitis in Children, Ireland.

During April-July 2022, outbreaks of severe acute hepatitis of unknown etiology (SAHUE) were reported in 35 countries. Five percent of cases required liver transplantation, and 22 patients died. Viral metagenomic studies of clinical samples from SAHUE cases showed a correlation with human adenovirus F type 41 (HAdV-F41) and adeno-associated virus type 2 (AAV2). To explore the association between those DNA viruses and SAHUE in children in Ireland, we quantified HAdV-F41 and AAV2 in samples collected from a wastewater treatment plant serving 40% of Ireland's population. We noted a high correlation between HAdV-F41 and AAV2 circulation in the community and SAHUE clinical cases. Next-generation sequencing of the adenovirus hexon in wastewater demonstrated HAdV-F41 was the predominant HAdV type circulating. Our environmental analysis showed increased HAdV-F41 and AAV2 prevalence in the community during the SAHUE outbreak. Our findings highlight how wastewater sampling could aid in surveillance for respiratory adenovirus species.

SARS-CoV-2 variant trends in Ireland: Wastewater-based epidemiology and clinical surveillance.

SARS-CoV-2 RNA quantification in wastewater is an important tool for monitoring the prevalence of COVID-19 disease on a community scale which complements case-based surveillance systems. As novel variants of concern (VOCs) emerge there is also a need to identify the primary circulating variants in a community, accomplished to date by sequencing clinical samples. Quantifying variants in wastewater offers a cost-effective means to augment these sequencing efforts. In this study, SARS-CoV-2 N1 RNA concentrations and daily loadings were determined and compared to case-based data collected as part of a national surveillance programme to determine the validity of wastewater surveillance to monitor infection spread in the greater Dublin area. Further, sequencing of clinical samples was conducted to determine the primary SARS-CoV-2 lineages circulating in Dublin. Finally, digital PCR was employed to determine whether SARS-CoV-2 VOCs, Alpha and Delta, were quantifiable from wastewater. No lead or lag time was observed between SARS-CoV-2 wastewater and case-based data and SARS-CoV-2 trends in Dublin wastewater significantly correlated with the notification of confirmed cases through case-based surveillance preceding collection with a 5-day average. This demonstrates that viral RNA in Dublin's wastewater mirrors the spread of infection in the community. Clinical sequence data demonstrated that increased COVID-19 cases during Ireland's third wave coincided with the introduction of the Alpha variant, while the fourth wave coincided with increased prevalence of the Delta variant. Interestingly, the Alpha variant was detected in Dublin wastewater prior to the first genome being sequenced from clinical samples, while the Delta variant was identified at the same time in clinical and wastewater samples. This work demonstrates the validity of wastewater surveillance for monitoring SARS-CoV-2 infections and also highlights its effectiveness in identifying circulating variants which may prove useful when sequencing capacity is limited.

Mortality beyond emergency threshold in a silent crisis- results from a population-based mortality survey in Ouaka prefecture, Central African Republic, 2020.

BACKGROUND: The Central African Republic (CAR) suffers a protracted conflict and has the second lowest human development index in the world. Available mortality estimates vary and differ in methodology. We undertook a retrospective mortality study in the Ouaka prefecture to obtain reliable mortality data. METHODS: We conducted a population-based two-stage cluster survey from 9 March to 9 April, 2020 in Ouaka prefecture. We aimed to include 64 clusters of 12 households for a required sample size of 3636 persons. We assigned clusters to communes proportional to population size and then used systematic random sampling to identify cluster starting points from a dataset of buildings in each commune. In addition to the mortality survey questions, we included an open question on challenges faced by the household. RESULTS: We completed 50 clusters with 591 participating households including 4000 household members on the interview day. The median household size was 7 (interquartile range (IQR): 4-9). The median age was 12 (IQR: 5-27). The birth rate was 59.0/1000 population (95% confidence interval (95%-CI): 51.7-67.4). The crude and under-five mortality rates (CMR & U5MR) were 1.33 (95%-CI: 1.09-1.61) and 1.87 (95%-CI: 1.37-2.54) deaths/10,000 persons/day, respectively. The most common specified causes of death were malaria/fever (16.0%; 95%-CI: 11.0-22.7), violence (13.2%; 95%-CI: 6.3-25.5), diarrhoea/vomiting (10.6%; 95%-CI: 6.2-17.5), and respiratory infections (8.4%; 95%-CI: 4.6-14.8). The maternal mortality ratio (MMR) was 2525/100,000 live births (95%-CI: 825-5794). Challenges reported by households included health problems and access to healthcare, high number of deaths, lack of potable water, insufficient means of subsistence, food insecurity and violence. CONCLUSIONS: The CMR, U5MR and MMR exceed previous estimates, and the CMR exceeds the humanitarian emergency threshold. Violence is a major threat to life, and to physical and mental wellbeing. Other causes of death speak to poor living conditions and poor access to healthcare and preventive measures, corroborated by the challenges reported by households. Many areas of CAR face similar challenges to Ouaka. If these results were generalisable across CAR, the country would suffer one of the highest mortality rates in the world, a reminder that the longstanding "silent crisis" continues.

Supplemental Fiber Affects Body Temperature and Fecal Metabolites but Not Respiratory Rate or Body Composition in Mid-Distance Training Sled Dogs.

Dietary fiber affects canine physiology in many ways, such as increasing colonic absorption of water and improving gut health, both of which may positively impact exercise performance. The objectives of this study were to investigate the effects of increased dietary soluble fiber and incremental training on respiratory rate (RR), internal body temperature (BT), body composition, and fecal metabolites in mid-distance training sled dogs. Fourteen dogs (12 Siberian and 2 Alaskan Huskies) were blocked by age, sex, and body weight (BW) and then randomly allocated into one of two diet groups. Seven dogs were fed a dry extruded control diet (Ctl) with an insoluble:soluble fiber ratio of 4:1 (0.74% soluble fiber on a dry-matter basis), and seven dogs were fed a dry extruded treatment diet (Trt) with an insoluble:soluble fiber ratio of 3:1 (2.12% soluble fiber on a dry-matter basis). Fecal samples were taken once a week. All dogs underwent 9 weeks of incremental exercise conditioning where the running distance was designed to increase each week. Every 3 weeks, external telemetry equipment was used to non-invasively measure and record RR and internal BT at resting, working, and post-exercise recovery states. Body composition was measured on weeks -1 and 9 using quantitative magnetic resonance. Body composition, RR, BT, and fecal metabolites were analyzed using a mixed model with dog as a random effect and week and diet group as fixed effects. Dogs on Trt had lower working and post-exercise BT than Ctl (P < 0.05). In addition, Trt dogs had lower recovery BT at weeks 2 and 5 than Ctl dogs (P < 0.05). Treatment dogs had greater fecal short-chain fatty acid concentrations than Ctl (P < 0.05). Diet had no effect on RR or body composition (P > 0.10), but exercise resulted in an overall 7% increase in lean and 3.5% decrease in fat mass (P < 0.05). These data suggest that increasing dietary soluble fiber may positively influence BT and gut health; however, it has no effect on RR or body composition. Soluble fiber did not negatively impact any measures of overall health and performance and should be considered for use in performance dogs.

Multimodal imaging of drug and excipients in rat lungs following an inhaled administration of controlled-release drug laden PLGA microparticles.

Controlled-release formulations, in the form of micro- or nanoparticles, are increasingly attractive to the pharmaceutical industry for drug delivery. For respiratory illnesses, controlled-release microparticle formulations provide an opportunity to deliver a higher percentage of an inhaled medicament dose to the lung, thus potentially reducing the therapeutic dose, frequency of dosing, and minimising side-effects. We describe the use of a multimodal approach consisting of MALDI MS imaging, 3D depth profiling TOF-SIMS analysis, and histopathology to monitor the distribution of drug and excipients in sections taken from excised rat lungs following an inhaled administration of drug-laden microparticles. Following a single dose, the administered drug was detected in the lung via both MALDI MS and TOF-SIMS over a range of time points. Both imaging techniques enabled the characterisation of the distribution and retention of drug particles and identified differences in the capabilities of both imaging modalities. Histochemical staining of consecutive sections was used to provide biological context to the findings and will also be discussed in this presentation. We demonstrate how this multimodal approach could be used to help increase our understanding of the use of controlled release microparticles.

National perinatal survey demonstrates a decreasing seroprevalence of Toxoplasma gondii infection among pregnant women in France, 1995 to 2016: impact for screening policy.

BackgroundToxoplasmosis during pregnancy can result in congenital anomalies or fetal death. Universal antenatal screening is recommended in France, a strategy in place since the 1970s.AimWe determined the seroprevalence of toxoplasmosis among pregnant women participating in the 2016 national perinatal survey (ENP), compared results with previous ENPs, and investigated factors associated with Toxoplasma gondii infection.MethodsUsing the 2016 ENP data, which contain sociodemographic and clinical information from all women giving birth during a one week period, we calculated adjusted prevalence ratios (aPR) by sociodemographic factors. Using available data from prior ENPs (1995, 2003 and 2010), we calculated age-standardised seroprevalences and aPRs for French women.ResultsIn 2016, seroprevalence was 31.3% overall. Among French women, associations with increasing age (aPR: 1.54; 95% CI: 1.39-1.70), residence in Paris (aPR: 1.19; 95% CI: 1.08-1.31) or south-western regions (aPR: 1.19; 95% CI: 1.08-1.31), and higher professional status (aPR: 1.12; 95%CI 1.04-1.21) were observed. An association with increasing age was also evident among women from North Africa and sub-Saharan Africa. Age-standardised seroprevalence decreased from 55.0% in 1995 to 33.7% in 2016. Among French women, significant associations with age, Paris and south-west regions persisted across all ENPs.ConclusionHigher prevalences in older women may reflect a higher past risk of exposure while persistent geographical differences may reflect dietary or environmental differences. Toxoplasma seroprevalence among pregnant women continues to fall and will impact screening effectiveness. This warrants a comprehensive review to determine the appropriate future of prevention in France.

Changes in Behaviour and Voluntary Physical Activity Exhibited by Sled Dogs throughout Incremental Exercise Conditioning and Intermittent Rest Days.

Participation in repetitive endurance training decreases sled dogs' voluntary activity and locomotive behaviours; however, the changes in their voluntary physical activity over consecutive rest days has not been examined to assess exercise-recovery. The objective of this study was to examine the changes in behaviour and voluntary activity of sled dogs throughout repetitive incremental conditioning and intermittent rest days. Fourteen dogs (6 males, 8 females; age 3.7 ± 2.7 years; BW 21.5 ± 2.8 kg) underwent 10 weeks of conditioning. Once a week, 5-min video recordings were taken pre- and post-exercise to measure the time spent performing agonistic behaviours, chewing on the gangline, digging, jumping, lunging, posture changing, sitting, standing and lying. Additionally, voluntary physical activity was measured on a day with an exercise bout during baseline, week 4, 5 and 7 and two consecutive rest days during baseline, week 1, 4, 5 and 7. A repeated-measures mixed model was used to analyse data in SAS (v 9.4.). As dogs progressed through their conditioning, the time spent changing posture prior to an exercise bout decreased (p < 0.05), suggesting that dogs may reduce their voluntary locomotive behaviours with increasing exercise. Additionally, dogs were more active during the second consecutive rest day than the first (p < 0.05), suggesting that rest days may provide a short-term recovery period.

Investigating the effects of incremental conditioning and supplemental dietary tryptophan on the voluntary activity and behaviour of mid-distance training sled dogs.

Serotonin is a neurotransmitter synthesized by the amino acid tryptophan, that has the potential to impact the behaviour and activity of dogs. The objective of this study was to assess the effects of supplemental tryptophan and a 12-week incremental training regimen on the voluntary activity and behaviour of client-owned Siberian Huskies. Sixteen dogs were blocked for age, BW and sex and then randomly allocated to either the control or treatment group. Both groups were fed the same dry extruded diet; however, the treatment group were supplemented with tryptophan to achieve a tryptophan: large neutral amino acid ratio of 0.075:1. Once a week, a 5-minute video recording was taken immediately pre- and post- exercise to evaluate dogs' behaviours. Activity monitors were used to record voluntary activity on both training and rest days. Linear regression analysis was used to assess the relationship between training week and time spent performing each behaviour. Additionally, a repeated measure mixed model was used to test differences between diet groups and training week for both behavioural and activity count data. The time spent performing agonistic behaviours prior to exercise was negatively associated with week for treatment dogs (ß = -0.32, 95% CI [-0.55, -0.10], P < 0.05) and no change was observed for control dogs (ß = -0.13, 95% CI [-0.41, 0.15], P > 0.10). Treatment did not have any effect on activity levels (P > 0.10). For all dogs, locomotive behaviours decreased prior to exercise as weeks progressed (P < 0.05), while run day voluntary activity depended on the distance run that day (P < 0.05). These data suggest that sled dogs experience an exercise-induced reduction in voluntary locomotion in response to both single bouts and repetitive bouts of exercise. Additionally, tryptophan supplementation may decrease agonistic behaviours, without having any effect on voluntary activity.

Transimmunization restores immune surveillance and prevents recurrence in a syngeneic mouse model of ovarian cancer.

Ovarian cancer accounts for most deaths from gynecologic malignancies. Although more than 80% of patients respond to first-line standard of care, most of these responders present with recurrence and eventually succumb to carcinomatosis and chemotherapy-resistant disease. To improve patient survival, new modalities must, therefore, target or prevent recurrent disease. Here we describe for the first time a novel syngeneic mouse model of recurrent high-grade serous ovarian cancer (HGSOC), which allows immunotherapeutic interventions in a time course relevant to human carcinomatosis and disease course. Using this model, we demonstrate the efficacy of Transimmunization (TI), a dendritic cell (DC) vaccination strategy that uses autologous and physiologically derived DC loaded with autologous whole tumor antigens. TI has been proven successful in the treatment of human cutaneous T cell lymphoma and we report for the first time its in vivo efficacy against an intra-peritoneal solid tumor. Given as a single therapy, TI is able to elicit an effective anti-tumor immune response and inhibit immune-suppressive crosstalks with sufficient power to curtail tumor progression and establishment of carcinomatosis and recurrent disease. Specifically, TI is able to inhibit the expansion of tumor-associated macrophages as well as myeloid-derived suppressive cells consequently restoring T cell immune-surveillance. These results demonstrate the possible value of TI in the management of ovarian cancer and other intra-peritoneal tumors.

Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes.

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.

Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy.

Generation of large numbers of dendritic cells (DC) for research or immunotherapeutic purposes typically involves in vitro conversion of murine bone marrow precursors or human blood monocytes to DC via cultivation with supraphysiologic concentrations of cytokines such as GM-CSF and IL-4 for up to 7 days. Alternatively, our group has recently established a new approach, based on the underlying mechanism of action of a widely used cancer immunotherapy termed Extracorporeal Photochemotherapy (ECP). Our method of rapid and cytokine-free production of therapeutically relevant DC populations, leveraging the innate physiologic programs likely responsible for DC differentiation from blood monocytes in vivo, potentially offers a novel, inexpensive, and easily accessible source of DC for clinical and research uses. This approach involves ex vivo physiologic reprogramming of blood monocytes to immunologically tunable dendritic antigen-presenting cells, which we term "phDC," for physiological DC. To facilitate access and utilization of these new DC populations by the research community, in this chapter, we describe the use of a scaled-down version of the clinical ECP leukocyte-treatment device termed the Transimmunization (TI) chamber or plate, suitable for processing both mouse and human samples. We highlight the methodological sequences necessary to isolate mouse or human peripheral blood mononuclear cell (PBMC) from whole blood, and to expose those PBMC to the TI chamber for facilitating monocyte activation and conversion to physiological DC (phDC) through interaction with blood proteins and activated platelets under controlled flow conditions. We then provide sample protocols for potential applications of the generated DC, including their use as vaccinating antigen-presenting cells (APC) in murine in vivo antitumor models, and in human ex vivo T-cell stimulation and antigen cross-presentation assays which mimic clinical vaccination. We additionally highlight the technical aspects of loading mouse or human phDC with tumor-associated antigens (TAA) in the form of peptides or apoptotic tumor cells. We provide a simple and clinically relevant means to reprogram blood monocytes into functional APC, potentially replacing the comparatively expensive and clinically disappointing cytokine-derived DC which have previously dominated the dendritic cell landscape.

Mortality and health survey, Walikale, Democratic Republic of the Congo, 2017: an example of the use of survey data for humanitarian program planning.

BACKGROUND: During humanitarian crises, health information systems are often lacking and surveys are a valuable tool to assess the health needs of affected populations. In 2013, a mortality and health survey undertaken by Médecins Sans Frontières (MSF) in the conflict affected Walikale territory of North Kivu, Democratic Republic of the Congo (DRC), indicated mortality rates exceeding humanitarian crisis thresholds and a high burden of mortality and morbidity due to malaria. In late 2017, after a period of relative stability, MSF reassessed the health status of the population through a second survey to guide ongoing operations. METHODS: A two-stage cluster survey, selecting villages using probability proportional to size and households using random walk procedures, was conducted. Household members were interviewed on morbidity and mortality, healthcare use, vaccination status, and bednet availability. RESULTS: The sample included 5711 persons in 794 households. The crude mortality rate (CMR) and under-five mortality rate (U5MR) were 0.98 per 10,000 persons/day (95% confidence interval (CI) 0.78-1.2) and 1.3 per 10,000 persons/day (95% CI): 0.82-2.0), respectively. The most frequently reported causes of death were fever/malaria (31%), diarrhoea (15%) and respiratory infections (8%). In 89% of households at least one person was reported as falling ill in the previous 2 weeks, and 58% sought healthcare. Cost was the main barrier amongst 58% of those who did not seek healthcare. Coverage of measles-containing-vaccine was 62% in under-fives. Sufficient bednet coverage (1 bednet/2 people) was reported from 17% of households. CONCLUSION: The second survey illustrates that although mortality is now just below crisis thresholds, the area still experiences excess mortality and has substantial health needs. The study results have supported the further expansion of integrated community case management to improve access to care for malaria, diarrhoea and respiratory infections. Such surveys are important to orient operations to the health needs of the population being served and also highlight the ongoing vulnerability of populations after humanitarian crises.

Extracorporeal photochemotherapy induces bona fide immunogenic cell death.

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8+ T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8+ T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells.

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP's clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity.

Extracorporeal photochemotherapy (ECP) is a cancer immunotherapy for cutaneous T-cell lymphoma (CTCL) operative in more than 350 centers worldwide. Although its efficacy and favorable safety profile have driven its widespread use, elucidation of its underlying mechanism has been difficult. In this study, we identify the principal contributors to the anticancer immunotherapeutic effects of ECP, with the goal of enhancing potency and broadening applicability to additional malignancies. First, we scaled down the clinical ECP leukocyte-processing device to mouse size. Second, we used that miniaturized device to produce a cellular vaccine that regularly initiated therapeutic antimelanoma immunity. Third, we individually subtracted key factors from either the immunizing inoculum or the treated animal to ascertain their contribution to the in vivo antimelanoma response. Platelet-signaled monocyte-to-dendritic cell (DC) differentiation followed by sorting/processing/presentation of tumor antigens derived from internalized apoptotic tumor cells were absolute requirements. As in clinical ECP, immunogenic cell death of tumor cells was finely titrated by DNA cross-linkage mediated by photoactivated 8-methoxypsoralen (8-MOPA). ECP-induced tumor-loaded DC were effective immunotherapeutic agents only if they were spared exposure to 8-MOPA, indicating that healthy DC are required for ECP. Infusion of responder T cells into naïve tumor-challenged mice established the protective role of stimulated T-cell antitumor immunity. Collectively, these results reveal that selective antitumor effects of ECP are initiated by tumor antigen-loaded, ECP-induced DC, which promote potent collaboration between CD4 and CD8 tumor-specific T cells. These mechanistic insights suggest potential therapeutic applicability of ECP to solid tumors in addition to CTCL.Significance: These findings identify principal cellular contributors to the anticancer immunotherapeutic impact of ECP and suggest this treatment may be applicable to a broad spectrum of immunogenic malignancies. Cancer Res; 78(14); 4045-58. ©2018 AACR.

Ongoing nationwide outbreak of Salmonella Agona associated with internationally distributed infant milk products, France, December 2017.

On 1 December 2017, an outbreak of Salmonella Agona infections among infants was identified in France. To date, 37 cases (median age: 4 months) and two further international cases have been confirmed. Five different infant milk products manufactured at one facility were implicated. On 2 and 10 December, the company recalled the implicated products; on 22 December, all products processed at the facility since February 2017. Trace-forward investigations indicated product distribution to 66 countries.

Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen.

Targeting antigen to dendritic cells (DCs) is a powerful and novel strategy for vaccination. Priming or loading DCs with antigen controls whether subsequent immunity will develop and hence whether effective vaccination can be achieved. The goal of our present work was to increase the potency of DC-based antitumor vaccines by overcoming inherent limitations associated with antigen stability and cross-presentation. Nanoparticles prepared from the biodegradable polymer poly(lactic-co-glycolic acid) have been extensively used in clinical settings for drug delivery and are currently the subject of intensive investigation as antigen delivery vehicles for vaccine applications. Here we describe a nanoparticulate delivery system with the ability to simultaneously carry a high density of protein-based antigen while displaying a DC targeting ligand on its surface. Utilizing a targeting motif specific for the DC-associated surface ligand DEC-205, we show that targeted nanoparticles encapsulating a MART-127-35 peptide are both internalized and cross-presented with significantly higher efficiency than isotype control-coated nanoparticles in human cells. In addition, the DEC-205-labeled nanoparticles rapidly escape from the DC endosomal compartment and do not colocalize with markers of early (EEA-1) or late endosome/lysosome (LAMP-1). This indicates that encapsulated antigens delivered by nanoparticles may have direct access to the class I cytoplasmic major histocompatibility complex loading machinery, overcoming the need for "classical" cross-presentation and facilitating heightened DC stimulation of anti-tumor CD8(+) T-cells. These results indicate that this delivery system provides a flexible and versatile methodology to deliver melanoma-associated antigen to DCs, with both high efficiency and heightened potency.