Phase 2 study of ibrutinib in classic and variant hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare B-cell malignancy, and there is a need for novel treatments for patients who do not benefit from purine analogs. Ibrutinib, an oral agent targeting Bruton tyrosine kinase in the B-cell receptor signaling pathway, is highly effective in several malignancies. Its activity in HCL was unknown, so we conducted a multisite phase 2 study of oral ibrutinib in patients with either relapsed classic or variant hairy cell leukemia. The primary outcome measure was the overall response rate (ORR) at 32 weeks, and we also assessed response at 48 weeks and best response during treatment. Key secondary objectives were characterization of toxicity and determination of progression-free survival (PFS) and overall survival (OS). Thirty-seven patients were enrolled at 2 different doses (24 at 420 mg, 13 at 840 mg). The median duration of follow-up was 3.5 years (range, 0-5.9 years). The ORR at 32 weeks was 24%, which increased to 36% at 48 weeks. The best ORR was 54%. The estimated 36-month PFS was 73% and OS was 85%. The most frequent adverse events were diarrhea (59%), fatigue (54%), myalgia (54%), and nausea (51%). Hematologic adverse events were common: anemia (43%), thrombocytopenia (41%), and neutropenia (35%). Ibrutinib can be safely administered to patients with HCL with objective responses and results in prolonged disease control. Although the initial primary outcome objective of the study was not met, the observation of objective responses in heavily pretreated patients coupled with a favorable PFS suggests that ibrutinib may be beneficial in these patients. This trial was registered at www.clinicaltrials.gov as #NCT01841723.

Labeling of endothelial cells with magnetic microbeads by angiophagy.

OBJECTIVES: Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism 'angiophagy'. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. RESULTS: In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001). CONCLUSION: We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.

Extraordinary Evasion of Neutralizing Antibody Response by Omicron XBB.1.5, CH.1.1 and CA.3.1 Variants.

Newly emerging Omicron subvariants continue to emerge around the world, presenting potential challenges to current vaccination strategies. This study investigates the extent of neutralizing antibody escape by new subvariants XBB.1.5, CH.1.1, and CA.3.1, as well as their impacts on spike protein biology. Our results demonstrated a nearly complete escape of these variants from neutralizing antibodies stimulated by three doses of mRNA vaccine, but neutralization was rescued by a bivalent booster. However, CH.1.1 and CA.3.1 variants were highly resistant to both monovalent and bivalent mRNA vaccinations. We also assessed neutralization by sera from individuals infected during the BA.4/5 wave of infection and observed similar trends of immune escape. In these cohorts, XBB.1.5 did not exhibit enhanced neutralization resistance over the recently dominant BQ.1.1 variant. Notably, the spike proteins of XBB.1.5, CH.1.1, and CA.3.1 all exhibited increased fusogenicity compared to BA.2, correlating with enhanced S processing. Overall, our results support the administration of new bivalent mRNA vaccines, especially in fighting against newly emerged Omicron subvariants, as well as the need for continued surveillance of Omicron subvariants.

Neutralization escape of Omicron XBB, BR.2, and BA.2.3.20 subvariants.

New Omicron subvariants continue to emerge throughout the world. In particular, the XBB subvariant, which is a recombinant virus between BA.2.10.1.1 and BA.2.75.3.1.1.1, as well as the BA.2.3.20 and BR.2 subvariants that contain mutations distinct from BA.2 and BA.2.75, are currently increasing in proportion of variants sequenced. Here we show that antibodies induced by 3-dose mRNA booster vaccination as well as BA.1- and BA.4/5-wave infection effectively neutralize BA.2, BR.2, and BA.2.3.20 but have significantly reduced efficiency against XBB. In addition, the BA.2.3.20 subvariant exhibits enhanced infectivity in the lung-derived CaLu-3 cells and in 293T-ACE2 cells. Overall, our results demonstrate that the XBB subvariant is highly neutralization resistant, which highlights the need for continued monitoring of the immune escape and tissue tropism of emerging Omicron subvariants.

Enhanced neutralization resistance of SARS-CoV-2 Omicron subvariants BQ.1, BQ.1.1, BA.4.6, BF.7, and BA.2.75.2.

The continued evolution of SARS-CoV-2 has led to the emergence of several new Omicron subvariants, including BQ.1, BQ.1.1, BA.4.6, BF.7, and BA.2.75.2. Here, we examine the neutralization resistance of these subvariants against sera from 3-dose vaccinated healthcare workers, hospitalized BA.1-wave patients, and BA.4/5-wave patients. We found enhanced neutralization resistance in all new subvariants, especially in the BQ.1 and BQ.1.1 subvariants driven by N460K and K444T mutations, as well as the BA.2.75.2 subvariant driven largely by its F486S mutation. All Omicron subvariants maintained their weakened infectivity in Calu-3 cells, with the F486S mutation driving further diminished titer for the BA.2.75.2 subvariant. Molecular modeling revealed the mechanisms of antibody-mediated immune evasion by R346T, K444T, F486S, and D1199N mutations. Altogether, these findings shed light on the evolution of newly emerging SARS-CoV-2 Omicron subvariants.

Enhanced evasion of neutralizing antibody response by Omicron XBB.1.5, CH.1.1, and CA.3.1 variants.

Omicron subvariants continuingly challenge current vaccination strategies. Here, we demonstrate nearly complete escape of the XBB.1.5, CH.1.1, and CA.3.1 variants from neutralizing antibodies stimulated by three doses of mRNA vaccine or by BA.4/5 wave infection, but neutralization is rescued by a BA.5-containing bivalent booster. CH.1.1 and CA.3.1 show strong immune escape from monoclonal antibody S309. Additionally, XBB.1.5, CH.1.1, and CA.3.1 spike proteins exhibit increased fusogenicity and enhanced processing compared with BA.2. Homology modeling reveals the key roles of G252V and F486P in the neutralization resistance of XBB.1.5, with F486P also enhancing receptor binding. Further, K444T/M and L452R in CH.1.1 and CA.3.1 likely drive escape from class II neutralizing antibodies, whereas R346T and G339H mutations could confer the strong neutralization resistance of these two subvariants to S309-like antibodies. Overall, our results support the need for administration of the bivalent mRNA vaccine and continued surveillance of Omicron subvariants.

COVID-19 Booster Uptake among First Responders and Their Household Members May Be Lower than Anticipated.

(1) Background: COVID-19 vaccination status varies widely among law enforcement and emergency medical services professionals. Though at high risk of exposure, these first responders have demonstrated significant vaccine hesitancy, with only 70% reportedly vaccinated. We sought to understand whether similar vaccine hesitancy exists for first responders and their household contacts around COVID-19 boosters. (2) Methods: In a prospective longitudinal cohort of first responders and their household contacts, survey data was collected, including demographics, medical history, COVID-19 exposure risks, and vaccination and/or booster status. The statistical analysis focused on primary vaccination and booster rates of both the first responders and their household contacts. (3) Results: Across 119 study participants, 73% reported having received some combination of vaccine and/or booster, and 26% were unvaccinated. Vaccinated individuals were older, reported less prior exposure to COVID-19 and had more comorbidities. Only 23% reported having received a COVID-19 booster. Pairing of the data for household contacts demonstrated a 60% agreement to receive primary vaccination but only a 20% agreement for boosters within households. (4) Conclusions: This study provides insight into the vaccination and booster rates of first responders and household contacts. Focused efforts to enhance vaccinations is essential for the protection and maintenance of this critical workforce.

Patient-driven research: Initial results from a prospective health-related quality of life study performed at the request of patients living with hairy cell leukemia.

A diagnosis of leukemia can have a profound effect on patients' health-related quality of life (HRQoL), however this has not been measured prospectively in patients with hairy cell leukemia (HCL). At the request of patients living with HCL who had identified this gap in knowledge about the disease, we conducted a longitudinal study of HRQoL among patients enrolled in the HCL Patient Data Registry (PDR). From September 1, 2018 to September 1, 2020, 165 patients were enrolled in the study and completed the baseline survey. The Functional Assessment of Cancer Therapy - Leukemia (FACT-Leu) was used to measure patients' HRQoL. Results show that newly diagnosed HCL patients reported the lowest HRQoL, followed by patients in relapse and those on "watch and wait." Factors associated with higher (better) FACT-Leu total scores in the multivariable analysis included older age, higher social support, and greater physical activity. These same factors were associated with lower levels of fatigue. In rare diseases where it is difficult to perform large prospective studies, patient/researcher collaborations are critical for the identification of studies that are of importance to patients and their families in order to maximize the benefits of the research and improve the lives of patients living with HCL.

Distinct Neutralizing Antibody Escape of SARS-CoV-2 Omicron Subvariants BQ.1, BQ.1.1, BA.4.6, BF.7 and BA.2.75.2.

Continued evolution of SARS-CoV-2 has led to the emergence of several new Omicron subvariants, including BQ.1, BQ. 1.1, BA.4.6, BF.7 and BA.2.75.2. Here we examine the neutralization resistance of these subvariants, as well as their ancestral BA.4/5, BA.2.75 and D614G variants, against sera from 3-dose vaccinated health care workers, hospitalized BA.1-wave patients, and BA.5-wave patients. We found enhanced neutralization resistance in all new subvariants, especially the BQ.1 and BQ.1.1 subvariants driven by a key N460K mutation, and to a lesser extent, R346T and K444T mutations, as well as the BA.2.75.2 subvariant driven largely by its F486S mutation. The BQ.1 and BQ.1.1 subvariants also exhibited enhanced fusogenicity and S processing dictated by the N460K mutation. Interestingly, the BA.2.75.2 subvariant saw an enhancement by the F486S mutation and a reduction by the D1199N mutation to its fusogenicity and S processing, resulting in minimal overall change. Molecular modelling revealed the mechanisms of receptor-binding and non-receptor binding monoclonal antibody-mediated immune evasion by R346T, K444T, F486S and D1199N mutations. Altogether, these findings shed light on the concerning evolution of newly emerging SARS-CoV-2 Omicron subvariants.

Development of a distributed international patient data registry for hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disorder, comprising only 2% of all leukemias. The Hairy Cell Leukemia Foundation (HCLF) has developed a patient data registry to enable investigators to better study the clinical features, treatment outcomes, and complications of patients with HCL. This system utilizes a centralized registry architecture. Patients are enrolled at HCL Centers of Excellence (COE) or via a web-based portal. All data are de-identified, which reduces regulatory burden and increases opportunities for data access and re-use. To date, 579 patients have been enrolled in the registry. Efforts are underway to engage additional COE's to expand access to patients across the globe. This international PDR will enable researchers to study outcomes in HCL in ways not previously possible due to the rarity of the disease and will serve as a platform for future prospective research.

Changes in surface topologies of chondrocytes subjected to mechanical forces: an AFM analysis.

The cartilage is composed of chondrocytes embedded in a matrix of collagen fibrils interspersed within a network of proteoglycans and is constantly exposed to biomechanical forces during normal joint movement. Characterization of the surface morphology, cytoskeletal structure, adherance and elastic properties of these mechanosensitive cells are crucial in understanding the effects of mechanical forces around a cell and how a cell responds to changes in its physical environment. In this work, we employed the atomic force microscope (AFM) to image cultured chondrocytes before and after subjecting them to mechanical forces in the presence or absence of interleukin-1beta to mimic inflammatory conditions. Nanoscale imaging and quantitative measurements from AFM data revealed that there are distinct changes in cell-surface topology and cytoskeleton arrangement in the cells following treatment with mechanical forces, IL-1beta or both. Our findings for the first time demonstrate that cultured chondrocytes are amenable to high-resolution AFM imaging and dynamic tensile forces may help overcome the effect of inflammatory factors on chondrocyte response.

Regulation of biomechanical signals by NF-kappaB transcription factors in chondrocytes.

Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic diseases and in the physiological restoration of joints. This report aims to summarize current peer-reviewed literature and available experimental data to explain how the signals generated by mechanical forces/joint mobilization generate beneficial effects on inflamed articular cartilage, and to propose the basis for using appropriate physical therapies for the optimal benefit to the patient suffering from joint associated injuries.

Detection of toxigenic Clostridium difficile colonization in patients admitted to the hospital for chemotherapy or haematopoietic cell transplantation.

Increasing evidence suggests that asymptomatic carriers are an important source of healthcare-associated Clostridium difficile infection. However, it is not known which test for the detection of C. difficile colonization is most sensitive in patients with haematological malignancies. We performed a prospective cohort study of 101 patients with haematological malignancies who had been admitted to the hospital for scheduled chemotherapy or haematopoietic cell transplantation. Each patient provided a formed stool sample. We compared the performance of five different commercially available assays, using toxigenic culture as the reference method. The prevalence of toxigenic C. difficile colonization as determined by toxigenic culture was 14/101 (14 %). The Cepheid Xpert PCR C. difficile/Epi was the most sensitive test for the detection of toxigenic C. difficile colonization, with 93 % sensitivity and 99 % negative predictive value. Our findings suggest that the Xpert PCR C. difficile/Epi could be used to rule out toxigenic C. difficile colonization in this population.

The antitumor effects of IFN-alpha are abrogated in a STAT1-deficient mouse.

IFN-alpha activates the signal transducer and activator of transcription (STAT) family of proteins; however, it is unknown whether IFN-alpha exerts its antitumor actions primarily through a direct effect on malignant cells or by stimulating the immune system. To investigate the contribution of STAT1 signaling within the tumor, we generated a STAT1-deficient melanoma cell line, AGS-1. We reconstituted STAT1 into AGS-1 cells by retroviral gene transfer. The resulting cell line (AGS-1STAT1) showed normal regulation of IFN-alpha-stimulated genes (e.g., H2k, ISG-54) as compared with AGS-1 cells infected with the empty vector (AGS-1MSCV). However, mice challenged with the AGS-1, AGS-1STAT1, and AGS-1MSCV cell lines exhibited nearly identical survival in response to IFN-alpha treatment, indicating that restored STAT1 signaling within the tumor did not augment the antitumor activity of IFN-alpha. In contrast, STAT1-/- mice could not utilize exogenous IFN-alpha to inhibit the growth of STAT1+/+ melanoma cells in either an intraperitoneal tumor model or in the adjuvant setting. The survival of tumor-bearing STAT1-/- mice was identical regardless of treatment (IFN-alpha or PBS). Additional cell depletion studies demonstrated that NK cells mediated the antitumor effects of IFN-alpha. Thus, STAT1-mediated gene regulation within immune effectors was necessary for mediating the antitumor effects of IFN-alpha in this experimental system.

Biomechanical strain regulates TNFR2 but not TNFR1 in TMJ cells.

We sought to examine whether cyclic tensile strain (CTS) regulates the gene expression of tumor necrosis factor (TNF)-alpha, its receptors TNFR1 and TNFR2, and inducible nitric oxide synthase (iNOS) under inflammatory conditions, and whether these effects of CTS are sustained. Rat temporomandibular joint disc cells (TDC) were exposed to CTS in the presence or absence of interleukin (IL)-1beta for 4 and 24h. Cells were also stimulated with IL-1beta for 24h while being subjected to CTS only for the initial 1, 2, 4, 8, and 12h or the entire 24h incubation time. Furthermore, cells were incubated with IL-1beta for 24, 36, or 48 h while being exposed to CTS only for the initial 8h. Gene expression of TNF-alpha, its receptors, and iNOS was analyzed by RT-PCR, whereas protein synthesis was determined by ELISA for TNF-alpha, immunofluorescence for TNFRs, and Griess reaction for nitric oxide. CTS inhibited the IL-1beta-stimulated synthesis of TNF-alpha, TNFR2, and iNOS. TNFR1 was constitutively expressed but not regulated by IL-1beta or CTS. Application of CTS for only 1 or 2h during a 24h incubation with IL-1beta was sufficient to inhibit IL-1beta-induced upregulation of TNF-alpha, TNFR2, and iNOS. However, for maximal inhibition of these genes a longer exposure of CTS was required. These findings are the first to show that biomechanical signals regulate the expression of TNFR2 but not TNFR1 under inflammatory conditions. Furthermore, the antiinflammatory effects of biomechanical signals on TDC are maintained for prolonged periods of time but are transient.

Mechanotransduction Effects on Endothelial Cell Proliferation via CD31 and VEGFR2: Implications for Immunomagnetic Separation.

Immunomagnetic separation is used to isolate circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) for diagnostics and tissue engineering. However, potentially detrimental changes in cell properties have been observed post-separation. Here, the effect of mechanical force, which is naturally applied during immunomagnetic separation, on proliferation of human umbilical vein endothelial cells (HUVEC), kinase insert domain-positive receptor (KDR) cells, and peripheral blood mononuclear cells (PBMCs). Cells are exposed to CD31 or Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) targeted MACSi beads at varying bead to cell ratios and compared to free antibody and unconjugated beads. A vertical magnetic gradient is applied to static 2D cultures, and a magnetic cell sorter is used to analyze cells in dynamic flow. No significant difference in EC proliferation is observed for controls or VEGFR2-targeting beads, whereas CD31-conjugated beads increase proliferation in a dose dependent manner in static 2-D cultures. This effect occurs in the absence of magnetic field, but is more pronounced with magnetic force. After flow sorting, similar increases in proliferation are seen for CD31 targeting beads. Thus, the effects of targeting antibody and magnetic force applied should be considered when designing immunomagnetic separation protocols for ECs.