Histopathological and Immunohistochemical Characterization of Sebaceous Adenoma and Epithelioma in Dogs.

Sebaceous gland tumors are neoplasms originating from the sebaceous gland and are the third most common type of skin tumor, accounting for 21-35% of all cutaneous neoplasms in dogs. According to their histopathological characteristics, sebaceous gland tumors can be classified into adenoma as a benign tumor and epithelioma as a malignant tumor. Sebaceous epithelioma is distinguished from sebaceous adenoma by containing 90% or more reserve cells. However, this simple numerical criterion is insufficient to histologically distinguish between epitheliomas and adenomas. In addition, sebaceoma in humans, a similar tumor to sebaceous epithelioma, is a term used for tumors with more than 50% of reserve cells, unlike epithelioma. Therefore, we aimed to compare and characterize the histological and immunohistochemical profiles of comprehensive sebaceous adenoma, epithelioma, and borderline tumors that have more than 50% but less than 90% of reserve cells. A total of 14 canine sebaceous tumors were diagnosed as seven adenomas, four borderline tumors, and three epitheliomas. Histologically, the sebaceous adenomas showed nodules consisting of mature sebocytes surrounded by monolayer basaloid cells. In contrast, the portion of the reserve cells was increased, the portion of lipidized cells was decreased, and the majority of lipidized cells were found to be immature in sebaceous epithelioma. In the sebaceous adenomas, necrosis was not observed and mitotic figures were rarely seen. However, necrosis and mitotic figures were highly frequent in both borderline tumor and sebaceous epithelioma. Immunohistochemistry revealed that borderline tumor and sebaceous epithelioma showed significantly higher expression against Ki-67 than sebaceous adenoma. We conclude that it is more accurate to employ the cut-off value of 50% reserve cells in humans rather than the current 90% reserve cells for classifying sebaceous gland tumors in dogs, thereby providing new insight into the characterization of the sebaceous gland tumors.

Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As.

Although the significance of chemical modifications on RNA is acknowledged, the evolutionary benefits and specific roles in human immunodeficiency virus (HIV-1) replication remain elusive. Most studies have provided only population-averaged values of modifications for fragmented RNAs at low resolution and have relied on indirect analyses of phenotypic effects by perturbing host effectors. Here we analysed chemical modifications on HIV-1 RNAs at the full-length, single RNA level and nucleotide resolution using direct RNA sequencing methods. Our data reveal an unexpectedly simple HIV-1 modification landscape, highlighting three predominant N6-methyladenosine (m6A) modifications near the 3' end. More densely installed in spliced viral messenger RNAs than in genomic RNAs, these m6As play a crucial role in maintaining normal levels of HIV-1 RNA splicing and translation. HIV-1 generates diverse RNA subspecies with distinct m6A ensembles, and maintaining multiple of these m6As on its RNAs provides additional stability and resilience to HIV-1 replication, suggesting an unexplored viral RNA-level evolutionary strategy.

The Perspectives of Platelet Proteomics in Health and Disease.

Cardiovascular thromboembolic diseases and cancer continue to be a leading cause of death and disability worldwide. Therefore, it is crucial to advance their diagnoses and treatment in the context of individualized medicine. However, the disease specificity of the currently available markers is limited. Based on analyses of a subset of peptides and matching proteins in disease vs. healthy platelets, scientists have recently shown that focused platelet proteomics enables the quantification of disease-specific biomarkers in humans. In this review, we explored the potential of accurate platelet proteomic research, which is required to identify novel diagnostic and pharmaceutical targets by comprehending the proteome variety of healthy individuals and patients for personalized and precision medicine.

Expression of sphingosine-1-phosphate receptor 1 in neuroinflammation of canine brains.

Sphingosine-1-phosphate (S1P) is a signaling lipid mediator that is involved in multiple biological processes. The S1P/S1P receptor (S1PR) signaling pathway has an important role in the central nervous system. It contributes to physiologic cellular homeostasis and is also associated with neuroinflammation. Therefore, this study was performed to evaluate the expression of S1PR in dogs with meningoencephalitis of unknown etiology (MUE) and experimental autoimmune encephalomyelitis (EAE). The analysis used 12 brain samples from three neurologically normal dogs, seven dogs with MUE, and two canine EAE models. Anti-S1PR1 antibody was used for immunohistochemistry. In normal brain tissues, S1PR1s were expressed on neurons, astrocytes, oligodendrocytes, and endothelial cells. In MUE and EAE lesions, there was positive staining of S1PR1 on leukocytes. Furthermore, the expression of S1PR1 on neurons, astrocytes, oligodendrocytes, and endothelial cells was upregulated compared to normal brains. This study shows that S1PR1s are expressed in normal brain tissues and leukocytes in inflammatory lesions, and demonstrates the upregulation of S1PR1 expression on nervous system cells in inflammatory lesions of MUE and EAE. These findings indicate that S1P/S1PR signaling pathway might involve physiologic homeostasis and neuroinflammation and represent potential targets for S1PR modulators to treat MUE.

Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS.

The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.

Case report: Fundic gland polyps caused by long-term omeprazole use in a Maltese dog.

Long-term use of proton-pump inhibitors can induce fundic gland polyps in the human stomach. However, this phenomenon has not been described in the veterinary literature. A 5-year-old intact female Maltese dog was referred to our hospital with chronic intermittent vomiting. The dog had been administered omeprazole (0.7-1.0 mg/kg PO q24 h) for the management of hydrocephalus for over 4 years; the omeprazole dose was increased to 10 mg/kg PO q24 h 8 months prior to presentation at referring hospital. Abdominal ultrasonography revealed marked thickening of the gastric wall with multi-lobulated, thickened folds. Subsequent endoscopy revealed marked polypoid lesions, and histological examination of the biopsy samples was consistent with the fundic gland polyps associated with proton-pump inhibitor use in humans. The lesions resolved after cessation of omeprazole, as assessed by ultrasonography. This report describes a case of fundic gland polyps following the long-term administration of omeprazole in a dog.

Expression of vitamin D receptor, CYP24A1, and CYP27B1 in normal and inflamed canine pancreases.

Vitamin D plays a role in anti-inflammatory processes, and the alteration of its metabolism is associated with the inflammatory processes of pancreatitis. This study was performed to evaluate the expression of the vitamin D receptor (VDR) and the two major enzymes that regulate vitamin D metabolism, 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1), in the canine pancreas and to compare their degrees of immunoreactivity between normal and inflamed pancreases. Five normal and inflamed pancreatic tissues each were obtained from six dogs. The expression of VDR, CYP24A1, and CYP27B1 were determined immunohistochemically, and the degree of immunostaining was assessed semiquantitatively. The VDR was expressed in the ducts, acini, and islets of Langerhans of normal pancreases and in the ducts and acini of inflamed ones. There was a significant difference in the immunoreactivity score for VDR in the islets of Langerhans between normal (median, 3 [interquartile range, 2-7.5] score) and inflamed pancreatic tissues (0 [0-0.5] score, p = 0.03). CYP24A1 was expressed in the ducts and islets of Langerhans in both normal and inflamed pancreases, whereas CYP27B1 was expressed in the ducts and acini in only some normal and inflamed pancreatic tissues. This study showed that VDR expression decreased in inflamed pancreases and demonstrated CYP24A1 and CYP27B1 expression in the canine pancreas for the first time. These findings indicate that the pancreas could regulate the metabolism and biological activity of vitamin D and suggest that a decrease in these might be related to the pathophysiology of pancreatitis.

CD3-immunotoxin mediated depletion of T cells in lymphoid tissues of rhesus macaques.

Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies. To date, a comprehensive evaluation of T-cell depletion and CD3 proliferation for all lymphoid tissues has not been conducted. Here, two rhesus macaques were administered A-dmDT390-SCFBdb (CD3-IT) intravenously at 25 µg/kg twice daily for four days. Samples were collected one day prior to and four days post administration. Flow cytometry and immunofluorescence staining were used to evaluate treatment efficiency accurately. Our preliminary results suggest that CD3-IT treatment may induce higher depletion of CD3 and CD4 T-cells in the lymph nodes and spleen, but is ineffective in the colon and thymus. The data showed a better elimination tendency of CD4 T-cells in the B-cell zone relative to the germinal center in the lymph nodes. Further, CD3-IT treatment may lead to a reduction in germinal center T follicular helper CD4 cells in the lymph nodes compared to healthy controls. The number of proliferating CD3 T-cell indicated that repopulation in different lymphoid tissues may occur four days post treatment. Our results provide insights into the differential efficacy of CD3-IT treatment and T-cell proliferation post treatment in different lymphoid tissues. Overall, CD3-IT treatment shows potential efficacy in depleting T-cells in the periphery, lymph nodes, and spleen, making it a viable preconditioning regimen for cell or organ transplantation. Our pilot study provides critical descriptive statistics and can contribute to the design of larger future studies.

Case report: Evaluation of hindlimb ischemia using 18F-fluorodeoxyglucose positron emission tomography in a cat with cardiogenic arterial thromboembolism.

A 12-year-old castrated male domestic shorthair cat weighing 6.7 kg presented with acute hindlimb paralysis and tachypnea. The femoral pulse was absent bilaterally. Thoracic radiography showed finding compatible with cardiogenic pulmonary edema. Echocardiography revealed hypertrophic cardiomyopathy phenotype and a spontaneous echocardiographic contrast in the left atrium, suggesting cardiogenic arterial thromboembolism. Oxygen supplementation, diuretics, and antithrombotic and thrombolytic agents were also administered. However, hindlimb motor function was not restored. Severely increased aspartate aminotransferase and creatinine phosphokinase, as well as neutropenia with a degenerative left shift were identified, and amputation was considered to prevent sepsis caused by necrosis of the ischemic tissues. 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography was performed to evaluate the metabolic activity of the muscle tissues and determine the level of amputation. There was no 18F-FDG uptake in the extremities of either the hind limbs or the caudal parts of the bilateral femoral muscle mass, suggesting a loss of metabolic activity in the area. Considering the wide affected area, a decreased quality of life was predicted postoperatively, and the cat was euthanized at the owner's request. Postmortem muscle biopsy confirmed weak atrophy of the left femoral muscle and prominent atrophy of the right calf. This case report describes the use of 18F-FDG PET in a cat with ischemia caused by cardiogenic arterial thromboembolism.

Shedding Light on the Cell Biology of Platelet-Derived Extracellular Vesicles and Their Biomedical Applications.

EVs are membranous subcellular structures originating from various cells, including platelets which consist of biomolecules that can modify the target cell's pathophysiological functions including inflammation, cell communication, coagulation, and metastasis. EVs, which are known to allow the transmission of a wide range of molecules between cells, are gaining popularity in the fields of subcellular treatment, regenerative medicine, and drug delivery. PEVs are the most abundant EVs in circulation, being produced by platelet activation, and are considered to have a significant role in coagulation. PEV cargo is extremely diverse, containing lipids, proteins, nucleic acids, and organelles depending on the condition that induced their release and can regulate a wide range of biological activities. PEVs, unlike platelets, can overcome tissue barriers, allowing platelet-derived contents to be transferred to target cells and organs that platelets cannot reach. Their isolation, characterization, and therapeutic efficacy, on the other hand, are poorly understood. This review summarizes the technical elements of PEV isolation and characterization methods as well as the pathophysiological role of PEVs, including therapeutic potential and translational possibility in diverse disciplines.

Role of Prednisolone in Platelet Activation by Inhibiting TxA2 Generation through the Regulation of cPLA2 Phosphorylation.

Glucocorticoids have been commonly used in the treatment of inflammation and immune-mediated diseases in human beings and small animals such as cats and dogs. However, excessive use can lead to Cushing's syndrome along with several thrombotic and cardiovascular diseases. Although it is well-known that glucocorticoids exert a significant effect on coagulation, the effect of cortisol on platelet function is much less clear. Thus, we aimed to study the effects of prednisolone, one of the commonly used glucocorticoids, on the regulation of platelet function using murine platelets. We first evaluated the concentration-dependent effect of prednisolone on 2-MeSADP-induced platelet function and found that the 2-MeSADP-induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by TxA2 generation, this result suggested a role of prednisolone in platelet TxA2 generation. Consistently, prednisolone did not affect the 2-MeSADP-induced aggregation in aspirinated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA2 generation by aspirin. In addition, thrombin-induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive-feedback effect of TxA2 generation on platelet function. Furthermore, prednisolone completely inhibited 2-MeSADP-induced TxA2 generation, confirming the role of prednisolone in TxA2 generation. Finally, Western blot analysis revealed that prednisolone significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets, while only cPLA2 phosphorylation, but not ERK phosphorylation, was significantly inhibited by prednisolone in aspirinated platelets. In conclusion, prednisolone affects platelet function by the inhibition of TxA2 generation through the regulation of cPLA2 phosphorylation, thereby shedding light on its clinical characterization and treatment efficacy in dogs with hypercortisolism in the future.

Abnormal Hyperphosphorylation of Tau in Canine Immune-mediated Meningoencephalitis.

BACKGROUND/AIM: Tau is a microtubule-associated protein involved in the assembly and stabilization of microtubules. In human medicine, hyperphosphorylation of tau is associated with microtubule instability and is considered to play a role in the progression of multiple sclerosis (MS). MS is an autoimmune neurological disease that shares many characteristics, including pathological mechanisms, with canine meningoencephalitis of unknown etiology (MUE). With this background, this study investigated the presence of hyperphosphorylated tau in dogs with MUE and experimental autoimmune encephalomyelitis (EAE). MATERIALS AND METHODS: In total, eight brain samples were examined from two neurologically normal dogs, three dogs with MUE, and three canine EAE models. Anti-(phospho-S396) tau antibody was used for immunohisto-chemistry, which stained hyperphosphorylated tau. RESULTS: In normal brain tissues, hyperphosphorylated tau was not found. In all the dogs with EAE and one of the dogs with MUE, immunoreactivity for S396 p-tau was observed in glial cell cytoplasm and the background in the periphery of the inflammatory lesion. CONCLUSION: These results suggest for the first time that tau pathology may be involved in the progression of neuroinflammation in dogs, similar to that in human MS.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells.

CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact of CD3e-IT treatment on these local cells, however, remains poorly understood. Here, using a new murine testing model, we demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment. Differential surface expression of CD3e among T-cell subsets appears to be a main driver of Treg enrichment in CD3e-IT treatment. The surviving Tregs in CD3e-IT-treated mice were mostly the CD3edimCD62Llo effector phenotype, but the levels of this phenotype markedly varied among different lymphoid and nonlymphoid organs. We also found notable variations in surface CD3e levels among tissue-resident T cells of different organs, and these variations drive CD3e-IT to uniquely reshape T-cell compositions in local organs. The functions of organs and anatomic locations (lymph nodes) also affected the efficacy of CD3e-IT. The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Therapeutic Efficacy of Weissella cibaria CMU and CMS1 on Allergic Inflammation Exacerbated by Diesel Exhaust Particulate Matter in a Murine Asthma Model.

Background and Objectives: Diesel exhaust particulate matter (DEPM) is an air pollutant that is associated with asthma. In this study, the therapeutic efficacy of Weissella cibaria strains CMU (Chonnam Medical University) and CMS (Chonnam Medical School) 1, together with the drug Synatura, an anti-tussive expectorant, was investigated in a murine asthma model exacerbated by DEPM. Materials and Methods: BALB/c mice were sensitized with ovalbumin (OVA) before intranasal challenge with OVA and DEPM. W. cibaria CMU, CMS1, and Synatura were administered orally for 21 days. Results: Neither Synatura nor W. cibaria strains affected spleen, liver, or lung weights. W. cibaria strains CMU and CMS1 significantly reduced the levels of interleukin (IL)-4, OVA-specific immunoglobulin E (IgE), and total lung collagen in bronchoalveolar lavage fluid (BALF), similar to those with Synatura, regardless of the oral dose concentration (p < 0.05). In addition, the W. cibaria CMU strain significantly alleviated IL-1ß, IL-6, IL-12, monocyte chemotactic protein-1, and tumor necrosis factor-α in BALF, whereas the CMS1 strain significantly alleviated IL-10 and IL-12 in BALF (p < 0.05); however, Synatura did not show any statistical efficacy against them (p > 0.05). All concentrations of W. cibaria CMU and low concentrations of W. cibaria CMS1 significantly reduced lung bronchiolar changes and inflammatory cell infiltration. Conclusions: In conclusion, W. cibaria CMU in asthmatic mice showed better efficacy than W. cibaria CMS1 in improving asthma exacerbated by DEPM exposure, as well as better results than pharmaceuticals.

Case Report: 18F-Fluoro-L-Phenylalanine Positron Emission Tomography Findings and Immunoreactivity for L-Type Amino Acid Transporter 1 in a Dog With Meningioma.

A 12-year-old intact female Miniature Pinscher dog weighing 5.4 kg presented with a history of seizures. On neurological examination, postural reactions were decreased in the left-sided limbs, and menace responses were bilaterally absent. Magnetic resonance imaging (MRI) of the brain was performed, and a solitary amorphous mass (2.7 × 1.9 × 2.2 cm) was observed on the right side of the frontal lobe. Based on the signalment, clinical signs, and MRI findings, a brain tumor was tentatively diagnosed, and meningioma was suspected. The dog was treated with hydroxyurea, prednisolone, and other antiepileptic drugs. One week after the treatment began, postural reactions returned to normal, and the menace response improved. At 119 days after treatment, 18F-fluoro-L-phenylalanine (18F-FDOPA) positron emission tomography (PET) was performed. Marked 18F-FDOPA uptake was observed in the lesion. The mean and maximal standardized uptake values of the lesion were 2.61 and 3.72, respectively, and the tumor-to-normal tissue ratio was 1.95. At 355 days after the initial treatment, a second MRI scan was performed and the tumor size had increased to 3.5 × 2.8 × 2.9 cm. The dog died 443 days after the initial treatment and was definitively diagnosed with grade 1 meningioma by histopathological examination. Immunohistochemical staining for Ki67 and L-type amino acid transporter 1 was positive and negative for p53, respectively. The labeling index of Ki67 was 2.4%. This is the first case to demonstrate 18F-FDOPA PET findings in a clinical case of a dog histologically diagnosed with a meningioma.

Protocol for the generation of HIV-1 genomic RNA with altered levels of N 6-methyladenosine.

N 6-methyladenosine (m6A) modification of human immunodeficiency virus type 1 (HIV-1) RNA plays a critical role in regulating viral replication and evasion of innate immunity. Here, we describe a protocol for the production of HIV-1 with altered m6A levels by manipulating the expression of m6A demethylases in HIV-1 producer cells. RNA from purified virions is analyzed by northern blot and dot blot for m6A levels prior to use in downstream assays to determine the function of m6A modification of viral RNA. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).

Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.

An Insight into Recent Advances on Platelet Function in Health and Disease.

Platelets play a variety of roles in vascular biology and are best recognized as primary hemostasis and thrombosis mediators. Platelets have a large number of receptors and secretory molecules that are required for platelet functionality. Upon activation, platelets release multiple substances that have the ability to influence both physiological and pathophysiological processes including inflammation, tissue regeneration and repair, cancer progression, and spreading. The involvement of platelets in the progression and seriousness of a variety of disorders other than thrombosis is still being discovered, especially in the areas of inflammation and the immunological response. This review represents an integrated summary of recent advances on the function of platelets in pathophysiology that connects hemostasis, inflammation, and immunological response in health and disease and suggests that antiplatelet treatment might be used for more than only thrombosis.

Comparison of CD3e Antibody and CD3e-sZAP Immunotoxin Treatment in Mice Identifies sZAP as the Main Driver of Vascular Leakage.

Anti-CD3-epsilon (CD3e) monoclonal antibodies (mAbs) and CD3e immunotoxins (ITs) are promising targeted therapy options for various T-cell disorders. Despite significant advances in mAb and IT engineering, vascular leakage syndrome (VLS) remains a major dose-limiting toxicity for ITs and has been poorly characterized for recent "engineered" mAbs. This study undertakes a direct comparison of non-mitogenic CD3e-mAb (145-2C11 with Fc-silentTM murine IgG1: S-CD3e-mAb) and a new murine-version CD3e-IT (saporin-streptavidin (sZAP) conjugated with S-CD3e-mAb: S-CD3e-IT) and identifies their distinct toxicity profiles in mice. As expected, the two agents showed different modes of action on T cells, with S-CD3e-mAb inducing nearly complete modulation of CD3e on the cell surface, while S-CD3e-IT depleted the cells. S-CD3e-IT significantly increased the infiltration of polymorphonuclear leukocytes (PMNs) into the tissue parenchyma of the spleen and lungs, a sign of increased vascular permeability. By contrast, S-CD3e-mAbs-treated mice showed no notable signs of vascular leakage. Treatment with control ITs (sZAP conjugated with Fc-silent isotype antibodies) induced significant vascular leakage without causing T-cell deaths. These results demonstrate that the toxin portion of S-CD3e-IT, not the CD3e-binding portion (S-CD3e-mAb), is the main driver of vascular leakage, thus clarifying the molecular target for improving safety profiles in CD3e-IT therapy.