Enhanced potency of immunotherapy against B-cell precursor acute lymphoblastic leukemia by combination of an Fc-engineered CD19 antibody and CD47 blockade.

CD19-directed immunotherapy has become a cornerstone in the therapy of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). CD19-directed cellular and antibody-based therapeutics have entered therapy of primary and relapsed disease and contributed to improved outcomes in relapsed disease and lower therapy toxicity. However, efficacy remains limited in many cases due to a lack of therapy response, short remission phases, or antigen escape. Here, BCP-ALL cell lines, patient-derived xenograft (PDX) samples, human macrophages, and an in vivo transplantation model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were used to examine the therapeutic potency of a CD19 antibody Fc-engineered for improved effector cell recruitment (CD19-DE) and antibody-dependent cellular phagocytosis (ADCP), in combination with a novel modified CD47 antibody (Hu5F9-IgG2σ). For the in vivo model, only samples refractory to CD19-DE monotherapy were chosen. Hu5F9-IgG2σ enhanced ADCP by CD19-DE in various BCP-ALL cell line models with varying CD19 surface expression and cytogenetic backgrounds, two of which contained the KMT2A-AFF1 fusion. Also, the antibody combination was efficient in inducing ADCP by human macrophages in pediatric PDX samples with and adult samples with and without KMT2A-rearrangement in vitro. In a randomized phase 2-like PDX trial using seven KMT2A-rearranged BCP-ALL samples in NSG mice, the CD19/CD47 antibody combination proved highly efficient. Our findings support that the efficacy of Fc-engineered CD19 antibodies may be substantially enhanced by a combination with CD47 blockade. This suggests that the combination may be a promising therapy option for BCP-ALL, especially in relapsed patients and/or patients refractory to CD19-directed therapy.

Effectiveness of Neuropediatric Inpatient Rehabilitation.

AIM: Inpatient rehabilitation plays an important role in treating neurological diseases in children and adolescents. However, there is a lack of current research concerning this matter. This retrospective study aims to analyze the effectiveness of neuropediatric inpatient rehabilitation, to identify influencing factors, and to examine the importance of inpatient rehabilitation programs. METHODS: We reviewed medical records of patients, diagnosed with cerebral palsy, traumatic brain injury (TBI), or stroke who had an inpatient rehabilitation at the Department of Neuropediatrics of St. Mauritius Therapieklinik in Meerbusch from 2012 to 2019. The patients received several units of different therapies such as motor and cognitive rehabilitation or speech therapy per day, depending on their individual needs and aims. Rehabilitation outcome was assessed by comparing Gross Motor Function Measure-88 and Pediatric Evaluation of Disability Inventory admission and discharge scores. Influences of sex, age, length of stay (LOS), and admission score were analyzed. RESULTS: A total of 738 patients with a mean age of 9.2 (± 5.1) years and a mean LOS of 53.8 (± 33.7) days were included; 38.5% were female. Patients, regardless of their diagnosis, sex, or age, demonstrated highly significant and meaningful improvements of self-care, mobility, and social function during inpatient rehabilitation. Especially, the group of patients with TBI and stroke could approximate their skills substantially to the ones of healthy peers. A longer LOS correlated significantly with greater improvement of skills. INTERPRETATION: This is a current study, supporting the effectiveness of neuropediatric inpatient rehabilitation and affirming its value in treating neurological diseases in children and adolescents.

Virtual reality-based attention training in patients with neurological damage: A pilot study.

Virtual Reality has been shown to be a valid tool to assess cognitive functions in an ecologically valid way. However, evidence regarding its effectiveness as a treatment option for cognitive rehabilitation has been limited. Furthermore, its potential to facilitate the transfer of training effects to patients' everyday life is still poorly studied. This study aimed to evaluate the efficacy of a VR-based attention training and its transfer to attentional functioning in everyday life. Nineteen inpatients with neurological disorders and attentional deficits underwent daily 30-min VR training sessions over a two-week period. Attentional functions were assessed before and after the training period using several computerized tests, two self-constructed behavioral tasks, and a questionnaire assessing patients' subjective attentional functioning. Pre-Post-analyses show significant decreases in reaction times in the computerized alertness and selective attention tests. Transfer to the behavioral tasks and self-report data could not be observed. Despite the specificity of the changes, confounding effects cannot entirely be ruled out due to the lack of a control group. Results suggest that training was effective in improving attentional functioning along neuropsychological measures, but did not elicit transfer to an ecologically valid or subjective level. Implications for the future development of VR interventions are discussed.

Practicability of a Time-Limited Welfare Assessment Protocol for Pasture-Based Dairy Farms, and a Preliminary Assessment of Welfare Outcome Thresholds.

This study assessed a new time-limited protocol developed for pasture-based cows across 23 dairy farms. The process started prior to milking with a questionnaire, followed by an assessment of resources (16 farms only) and behavioural observation of cows at pasture. Remaining animal-based measures were assessed during milking, usually by two assessors (one parlour based and one outside). The protocol proved to be practical and feasible with limited changes needed, except for the assessment of water availability and behaviour. As most cows could access only one water trough, distance between troughs was not a measure of water availability, while the observation of a large numbers of cows at pasture for 30 min resulted in few observations and an uncertain denominator (effective number of observed cows). Further research is needed to determine the best way of assessing water availability and cow behaviour in a time-limited assessment of pasture-based cows. Three animal-based measures (broken tails, dirtiness, and coughing) had mean values higher than the author-determined acceptable thresholds, while <50% of farms met trough cleanliness and track condition targets, and none met the criteria for shelter and shade. This was a sample of farms based on convenience, so more data are required to establish the representativeness of these results. Such testing should involve assessment of the repeatability and reliability of the measures in our protocol.

Venetoclax enhances the efficacy of therapeutic antibodies in B-cell malignancies by augmenting tumor cell phagocytosis.

Immunotherapy has evolved as a powerful tool for the treatment of B-cell malignancies, and patient outcomes have improved by combining therapeutic antibodies with conventional chemotherapy. Overexpression of antiapoptotic B-cell lymphoma 2 (Bcl-2) is associated with a poor prognosis, and increased levels have been described in patients with "double-hit" diffuse large B-cell lymphoma, a subgroup of Burkitt's lymphoma, and patients with pediatric acute lymphoblastic leukemia harboring a t(17;19) translocation. Here, we show that the addition of venetoclax (VEN), a specific Bcl-2 inhibitor, potently enhanced the efficacy of the therapeutic anti-CD20 antibody rituximab, anti-CD38 daratumumab, and anti-CD19-DE, a proprietary version of tafasitamab. This was because of an increase in antibody-dependent cellular phagocytosis by macrophages as shown in vitro and in vivo in cell lines and patient-derived xenograft models. Mechanistically, double-hit lymphoma cells subjected to VEN triggered phagocytosis in an apoptosis-independent manner. Our study identifies the combination of VEN and therapeutic antibodies as a promising novel strategy for the treatment of B-cell malignancies.

Combining daratumumab with CD47 blockade prolongs survival in preclinical models of pediatric T-ALL.

Acute lymphoblastic leukemia (ALL) is the most common malignant disease affecting children. Although therapeutic strategies have improved, T-cell acute lymphoblastic leukemia (T-ALL) relapse is associated with chemoresistance and a poor prognosis. One strategy to overcome this obstacle is the application of monoclonal antibodies. Here, we show that leukemic cells from patients with T-ALL express surface CD38 and CD47, both attractive targets for antibody therapy. We therefore investigated the commercially available CD38 antibody daratumumab (Dara) in combination with a proprietary modified CD47 antibody (Hu5F9-IgG2σ) in vitro and in vivo. Compared with single treatments, this combination significantly increased in vitro antibody-dependent cellular phagocytosis in T-ALL cell lines as well as in random de novo and relapsed/refractory T-ALL patient-derived xenograft (PDX) samples. Similarly, enhanced antibody-dependent cellular phagocytosis was observed when combining Dara with pharmacologic inhibition of CD47 interactions using a glutaminyl cyclase inhibitor. Phase 2-like preclinical in vivo trials using T-ALL PDX samples in experimental minimal residual disease-like (MRD-like) and overt leukemia models revealed a high antileukemic efficacy of CD47 blockade alone. However, T-ALL xenograft mice subjected to chemotherapy first (postchemotherapy MRD) and subsequently cotreated with Dara and Hu5F9-IgG2σ displayed significantly reduced bone marrow infiltration compared with single treatments. In relapsed and highly refractory T-ALL PDX combined treatment with Dara and Hu5F9-IgG2σ was required to substantially prolong survival compared with single treatments. These findings suggest that combining CD47 blockade with Dara is a promising therapy for T-ALL, especially for relapsed/refractory disease harboring a dismal prognosis in patients.

Enhancement of epidermal growth factor receptor antibody tumor immunotherapy by glutaminyl cyclase inhibition to interfere with CD47/signal regulatory protein alpha interactions.

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a "don't eat me" signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N-terminal pyro-glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell-mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab-mediated direct growth inhibition, complement-dependent cytotoxicity, or Ab-dependent cell-mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα-Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose-dependent manner, suggesting that pyro-glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab-dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte-mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell-mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.

Animal Welfare Assessment: Can We Develop a Practical, Time-Limited Assessment Protocol for Pasture-Based Dairy Cows in New Zealand?

Despite being a leading producer and exporter of dairy products, New Zealand has no industry-recognised welfare assessment protocol. A New Zealand-specific protocol is essential, as almost all dairy farms in New Zealand are pasture-based and housing is rarely used. Therefore, protocols developed for intensive cows are not suitable. The aim of this study was to develop a simple yet practical welfare assessment protocol that could be used to assess the welfare of a dairy herd during one visit timed to occur around milking. Six welfare assessment protocols and four studies of dairy cattle welfare assessments that had some focus on dairy cattle welfare at pasture were used, along with the New Zealand Dairy Cattle Code of Welfare, to identify potential assessments for inclusion in the protocol. Eighty-four potential assessments (20 record-based and 64 that needed assessing on-farm) were identified by this process of welfare assessments. After screening to exclude on-farm assessments that were not relevant, that had only limited practical application in pasture-based dairy cows or that required more time than available, 28 on-farm assessments remained, which were put together with the 20 record-based assessments and were tested for feasibility, practicality and time on two pasture-based dairy farms. Assessments were then identified as suitable, suitable after modification or not feasible. Suitable and modified assessments were then included in the final protocol alongside additional measures specific to New Zealand dairy farms. The final protocol included 24 on-farm assessments and eight record-based assessments. Further testing of these 32 assessments is needed on more dairy farms across New Zealand before the protocol can be used to routinely assess the welfare of dairy cows in New Zealand.

Modelling the transmission dynamics of bovine digital dermatitis in New Zealand pastoral dairy production systems.

Bovine digital dermatitis (DD) is an important infectious cause of cattle lameness worldwide that has become increasingly prevalent in New Zealand pastoral dairy herds. In this study, a simplified DD scoring system after considering both M and Iowa DD scoring systems was applied to explore the transmission dynamics of DD in a typical spring-calving pastoral New Zealand dairy herd. The modified model only included three compartments: normal skin, early stage lesions and advanced lesions. Lesions regressing after treatment were excluded as DD lesions are rarely treated in New Zealand. Furthermore, sub-classes within each lesion class were not defined due to the lack of variability in DD lesion presentations within New Zealand. The model was validated based on longitudinal field data from three dairy herds in the Waikato region during one lactation season (2017-18). The model suggested that in infected dairy herds, although DD prevalence will tend to increase year-on-year it is likely to remain relatively low (< 18%) even after 10 years of within-herd transmission. It is likely that the low transmission rate during the late lactation (model assumption) results in more cases resolving than developing during this period and therefore results in the low prevalence of infectious cattle at the start of each subsequent lactation. Cattle with advanced lesions had a stronger influence on the establishment and maintenance of DD than cattle with early stage lesions highlighting the importance of targeting these animals for intervention. On-going monitoring of DD is highly recommended to assess the long-term progression of the disease in affected dairy herds.

Bayesian analysis of herd-level risk factors for bovine digital dermatitis in New Zealand dairy herds.

BACKGROUND: Bovine digital dermatitis (BDD) is considered the most important infectious cause of lameness in dairy cattle worldwide, but has only recently been observed in New Zealand. Although many studies have investigated the risk factors for BDD in confined dairy systems, information on risk factors in pasture-based system is limited. Therefore a cross-sectional study including 59,849 animals from 127 dairy herds in four regions of New Zealand was conducted to identify the herd-level factors associated with the probability of a herd being BDD-lesion positive and with within-herd BDD prevalence. RESULTS: Purchasing heifers was associated with increased odds of a herd being BDD-lesion positive (odds ratio [OR]: 2.33, 95% probability interval [PI]: 1.26-4.42) and a cow being BDD affected (OR: 3.76, 95%PI: 1.73-8.38), respectively. Higher odds of a herd being BDD-lesion positive (OR: 2.06, 95%PI: 1.17-3.62) and a cow being BDD affected (OR: 2.87, 95%PI: 1.43-5.94) were also seen in herds where heifers co-grazed with cattle from other properties. In addition, using outside staff to treat lameness was associated with higher odds of a cow being BDD affected (OR: 2.18, 95%PI: 0.96-4.98). CONCLUSION: This study highlighted that movements of heifers are significantly associated with the spread of BDD within and between dairy herds in New Zealand. To minimise the risk of disease introductions in herds where moving heifers cannot be avoided, it is best to purchase heifers only from herds where BDD-freedom has been confirmed and, if heifers have to graze-off a farm, they should be reared as a single biosecure management group, especially since animals may be BDD-infected without having clinically obvious lesions.

Parasitism drives host genome evolution: Insights from the Pasteuria ramosa-Daphnia magna system.

Because parasitism is thought to play a major role in shaping host genomes, it has been predicted that genomic regions associated with resistance to parasites should stand out in genome scans, revealing signals of selection above the genomic background. To test whether parasitism is indeed such a major factor in host evolution and to better understand host-parasite interaction at the molecular level, we studied genome-wide polymorphisms in 97 genotypes of the planktonic crustacean Daphnia magna originating from three localities across Europe. Daphnia magna is known to coevolve with the bacterial pathogen Pasteuria ramosa for which host genotypes (clonal lines) are either resistant or susceptible. Using association mapping, we identified two genomic regions involved in resistance to P. ramosa, one of which was already known from a previous QTL analysis. We then performed a naïve genome scan to test for signatures of positive selection and found that the two regions identified with the association mapping further stood out as outliers. Several other regions with evidence for selection were also found, but no link between these regions and phenotypic variation could be established. Our results are consistent with the hypothesis that parasitism is driving host genome evolution.