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Monoclonal antibodies are extensively used as biotherapeutics for treatment of a variety of diseases. Glycosylation of therapeutic antibodies is considered a critical quality attribute as it influences the effector function, circulatory half-life, immunogenicity, and eventually efficacy and patient safety. During upstream process development, media components play a significant role in determining the glycosylation profile. In this study, we have evaluated 20 media additives (metal ions, vitamins, sugars, nucleosides). Six of the additives were shortlisted for their impact and then used to modulate the glycosylation profile of an in-house produced mAb (G0 2.38 ± 0.08%, G0F 75.58 ± 0.45%, G1F 10.07 ± 0.04%, G2F 0.54 ± 0.01%, G0F-N 5.84 ± 0.32%, sialylation 1.60 ± 0.33%, mannosylation 1.56 ± 0.39%) to achieve the glycan profile of a commercially available reference product (G0 2.49 ± 0.07%, G0F 37.83 ± 0.37%, G1F 34.77 ± 0.03%, G2F 4.87 ± 0.01%, G0F-N 2.34 ± 0.12%, sialylation 9.84 ± 0.30%, mannosylation 2.86 ± 0.29%). The proposed approach yielded us a glycan profile (G0 2.10 ± 0.07%, G0F 38.00 ± 0.49%, G1F 31.92 ± 0.09%, G2F 5.26 ± 0.54%, G0F-N 1.92 ± 0.02%, sialylation 10.28 ± 1.68%, mannosylation 3.12 ± 0.29%) that was near identical to that of the reference product. Equally importantly, other quality attributes including charge variants, aggregates, titer, and viability were not found to be significantly impacted by the addition of the additives under consideration. Key points • Screened 20 media additives to evaluate their effect on glycosylation of mAbs. • Developed glycosylation indices models to evaluate the effect of various additives. • Additive concentrations were optimized to target the reference product profile.

Both glial fibrillary acidic protein (GFAP) and S100β are found in glial cells and are released into serum following a traumatic brain injury (TBI), however, the clinical utility of S100β as a biomarker has been questioned because of its release from bone. This study examined the ability of GFAP and S100β to detect intracranial lesions on computed tomography (CT) in trauma patients and also assessed biomarker performance in patients with fractures and extracranial injuries on head CT. This prospective cohort study enrolled a convenience sample of adult trauma patients at a Level I trauma center with and without mild or moderate traumatic brain injury (MMTBI). Serum samples were obtained within 4 h of injury. The primary outcome was the presence of traumatic intracranial lesions on CT scan. There were 397 general trauma patients enrolled: 209 (53%) had a MMTBI and 188 (47%) had trauma without MMTBI. Of the 262 patients with a head CT, 20 (8%) had intracranial lesions. There were 137 (35%) trauma patients who sustained extracranial fractures below the head to the torso and extremities. Levels of S100β were significantly higher in patients with fractures, compared with those without fractures (p<0.001) whether MMTBI was present or not. However, GFAP levels were not significantly affected by the presence of fractures (p>0.05). The area under the receiver operating characteristics curve (AUC) for predicting intracranial lesions on CT for GFAP was 0.84 (0.73–0.95) and for S100β was 0.78 (0.67–0.89). However, in the presence of extracranial fractures, the AUC for GFAP increased to 0.93 (0.86–1.00) and for S100β decreased to 0.75 (0.61–0.88). In a general trauma population, GFAP out-performed S100β in detecting intracranial CT lesions, particularly in the setting of extracranial fractures.

Leukocyte trafficking to acute sites of injury or infection requires spatial and temporal cues that fine tune precise sites of firm adhesion and guide migration to endothelial junctions where they undergo diapedesis to sites of insult. Many detailed studies on the location and gradient of chemokines such as IL-8 and other CXCR ligands reveal that their recognition shortly after selectin-mediated capture and rolling exerts acute effects on integrin activation and subsequent binding to their ligands on the endothelium, which directs firm adhesion, adhesion strengthening, and downstream migration. In this process, G-protein coupled receptor (GPCR) signaling has been found to play an integral role in activating and mobilizing intracellular stores of calcium, GTPases such as Rap-1 and Rho and cytokeletal proteins such as Talin and F-actin to facilitate cell polarity and directional pseudopod formation. A critical question remaining is how intracellular Ca(2+) flux from CRAC channels such as Orai1 synergizes with cytosolic stores to mediate a rapid flux which is critical to the onset of PMN arrest and polarization. Our review will highlight a specific role for calcium as a signaling messenger in activating focal clusters of integrins bound to the cytoskeleton which allows the cell to attain a migratory phenotype. The precise interplay between chemokines, selectins, and integrins binding under the ubiquitous presence of shear stress from blood flow provides an essential cooperative signaling mechanism for effective leukocyte recruitment.

Innate-like B-1a lymphocytes rapidly redistribute to regional mediastinal lymph nodes (MedLNs) during influenza infection to generate protective IgM. Here we demonstrate that influenza infection-induced type I interferons directly stimulate body cavity B-1 cells and are a necessary signal required for B-1 cell accumulation in MedLNs. Vascular mimetic flow chamber studies show that type I interferons increase ligand-mediated B-1 cell adhesion under shear stress by inducing high-affinity conformation shifts of surface-expressed integrins. In vivo trafficking experiments identify CD11b as the non-redundant, interferon-activated integrin required for B-1 cell accumulation in MedLNs. Thus, CD11b on B-1 cells senses infection-induced innate signals and facilitates their rapid sequester into secondary lymphoid tissues, thereby regulating the accumulation of polyreactive IgM producers at sites of infection. Tissue-resident B-1 cells express CD11b, unlike their lymphoid organ-residing counterparts. Here the authors show that influenza-induced type I interferons activate CD11b on B-1 cells, facilitating entry to mediastinal lymph nodes, where they provide the first line of antibody-mediated host defense.

Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance.

Background We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p  = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP). Methods The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression. Results At baseline, a significantly higher level of expression ( p  < 0.05) of PD-L1 was identified in patient monocytes compared to HD. TCR modulation revealed dysfunction of circulating T-cells in patient baseline samples as compared to HD, and this was more pronounced in PD-1 + cells. Treatment with radiotherapy and fresolimumab did not resolve this dyfunctional signaling. However, in vitro PD-1 blockade enhanced TCR signaling in patient PD-1 + T cells and not in PD-1 - T cells or in PD-1 + T cells from HD. Conclusions Functional T cell analysis suggests that baseline T cell functionality is hampered in metastatic breast cancer patients, at least in part mediated by the PD-1 signaling pathway. These preliminary data support the rationale for investigating the possible beneficial effects of adding PD-1 blockade to improve responses to TGFβ blockade and radiotherapy. Trial registration NCT01401062 .

Objectives To attain a collective expert opinion on the use of air powder waterjet technology (APWT) with erythritol and glycine powders in the prophylaxis and therapy of periodontal and peri‐implant diseases. Material and Methods In the first step, a modified one‐round online Delphi survey including 44 five‐point Likert scale questions was conducted among a group of 10 expert clinicians and researchers with thorough knowledge and experience in this topic. In the second step, the single questions and the survey results were discussed during a meeting, and consensus statements were formulated, respectively. Results An agreement was reached on most items, especially opinions supporting glycine and erythritol powders as favorable with respect to efficiency, safety, and comfort. More scientific evidence is needed to support the improvement in clinical attachment on teeth and implants, especially when APWT with erythritol is used. In addition, APWT needs more long‐term evaluation and studies in terms of microbiome/microbiological effects as well as effects on the inflammatory response on natural teeth and implants, also in light of a guided biofilm therapy concept. Conclusions In line with the expert opinions and supported by the evidence, it was concluded that the use of APWT with erythritol and glycine powders in nonsurgical periodontal and peri‐implant therapy and prophylaxis is patient compliant and efficient.

As we approach the aftermath of a global pandemic caused by Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2), the importance of quickly developing rapid screening tests has become very clear from the point of view of containment and also saving lives. Here, we present an explorative study to develop a telepathology-based screening tool using peripheral blood smears (PBS) to identify Coronavirus Disease (COVID-19)-positive cases from a group of 138 patients with flu-like symptoms, consisting of 82 positive and 56 negative samples. Stained blood smear slides were imaged using an automated slide scanner (AI 100) and the images uploaded to the cloud were analyzed by a pathologist to generate semi-quantitative leukocyte morphology-related data. These telepathology data were compared with the data generated from manual microscopy of the same set of smear slides and also the same pathologist. Besides good correlation between the data from telepathology and manual microscopy, we were able to achieve a sensitivity and specificity of 0.83 and 0.71, respectively, for identifying positive and negative COVID-19 cases using a six-parameter combination associated with leukocyte morphology. The morphological features included plasmacytoid cells, neutrophil dysplastic promyelocyte, neutrophil blast-like cells, apoptotic cells, smudged neutrophil, and neutrophil-to-immature granulocyte ratio. Although Polymerase Chain Reaction (PCR) and antibody tests have a superior performance, the PBS-based telepathology tool presented here has the potential to be an interim screening tool in resource-limited settings in underdeveloped and developing countries.

Traumatic brain injury (TBI) is often associated with enduring impairments in high-level cognitive functioning, including working memory (WM). We examined WM function in predominantly chronic patients with mild, moderate and severe TBI and healthy comparison subjects behaviorally and, in a small subset of moderate-to-severe TBI patients, with event-related functional magnetic resonance imaging (fMRI), using a visual n-back task that parametrically varied WM load. TBI patients showed severity-dependent and load-related WM deficits in performance accuracy, but not reaction time. Performance of mild TBI patients did not differ from controls; patients with moderate and severe TBI were impaired, relative to controls and mild TBI patients, but only at higher WM-load levels. fMRI results show that TBI patients exhibit altered patterns of activation in a number of WM-related brain regions, including the dorsolateral prefrontal cortex and Broca's area. Examination of the pattern of behavioral responding and the temporal course of activations suggests that WM deficits in moderate-to-severe TBI are due to associative or strategic aspects of WM, and not impairments in active maintenance of stimulus representations. Overall, results demonstrate that individuals with moderate-to-severe TBI exhibit WM deficits that are associated with dysfunction within a distributed network of brain regions that support verbally mediated WM. (JINS, 2004, 10, 724–741.)

BackgroundNKTR-255 is an investigational IL-15Rα-dependent, polymer-conjugated, recombinant human IL-15 agonist designed to provide sustained pharmacodynamic (PD) responses without the need for daily dosing. NKTR-255 engages all IL-15 receptor binding complexes to expand, proliferate and activate natural killer (NK) and CD8+ T-cells. This Phase 1b/2 trial (NCT04616196) evaluates NKTR-255+cetuximab in highly refractory patients with head-and-neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC).MethodsSuccessive cohorts received escalating doses of NKTR-255 (q3w) +cetuximab (250mg/m< sup >2 weekly), 1 week after a loading dose of cetuximab alone. Safety (CTCAEv5.0; MTD/recommended Phase 2 dose [RP2D]) and efficacy (RECISTv1.1) were measured. PK/PD analyses were conducted, including assessment by flow cytometry/plasma cytokine analysis. Fold-change was calculated as treatment over baseline for NKTR-255 (baseline=1).ResultsAs of August 15, 2021, 12 patients had received ≥1 dose of NKTR-255+cetuximab; (37–70 years; 92% male; HNSCC n=4, CRC n=8; NKTR-255 1.5µg/kg n=7, NKTR-255 3.0µg/kg n=5). Patients had received a median 3.5 lines of prior therapy for metastatic disease. 11 patients had no response to the most recent prior therapy. Of the 12 patients, seven remain on treatment, with five not yet reaching first scan. RP2D has not yet been reached; dose escalation is ongoing.10 patients experienced an AE; one G5 AE occurred (due to progression). Seven patients reported NKTR-255-related AEs (all G1-2, except one G3 [which resolved in 24 hours]). Any-cause AEs in ≥20% were acneiform dermatitis, fatigue, and infusion-related reaction.Treatment-induced transient changes in inflammatory cytokines, including IFNy, MCP-1 and IL-6, at 1.5µg/kg (n=3) peaked 4 hours post-infusion and resolved by 24-48 hours. Mean T1/2 of NKTR-255 (1.5µg/kg dose, first cycle) was 27.8 hours.Dose-dependent expansion of NK and CD8+ T-cells was observed in peripheral blood. For NK cells, mean peak fold-change was ~4-fold and ~6-fold, and for CD8+ T-cells was ~2-fold and ~3-fold (1.5µg/kg and 3µg/kg dose levels, respectively). NK and CD8+ T-cells demonstrated increased Ki67+ proliferative ability.As of August 15, four patients in the 1.5µg/kg NKTR-255 dose cohort were response-evaluable: one CRC patient (4 prior metastatic treatment lines) reported a confirmed PR (–52%) after 3 cycles; two HNSCC patients reported SD.ConclusionsNKTR-255 was biologically active and led to expansion and proliferation of NK and CD8+ T-cells. Early dose-escalation data suggest that NKTR-255+cetuximab is safe and tolerable with preliminary anti-tumor activity. Updated data will be presented. NKTR-255, alone and in combination with daratumumab and rituximab, is also being evaluated in liquid tumors.AcknowledgementsThe authors thank the patients and their families involved in the trial.Trial RegistrationNCT04616196Ethics ApprovalThe study was approved by site IRBs.ConsentN/A