Antiplatelet antibody-induced thrombocytopenia does not correlate with megakaryocyte abnormalities in murine immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to antibody-mediated autoimmunity, it is now clear that T cells also play a significant role in the disease. However, the exact interplay between platelet destruction, megakaryocyte dysfunction and the elements of both humoral and cell-mediated immunity in ITP remains incompletely defined. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the antiplatelet antibodies can also destroy bone marrow megakaryocytes. To address this, we negated the effects of T cells by utilizing an in vivo passive ITP model where BALB/c mice were administered various anti-αIIb, anti-ß3 or anti-GPIb antibodies or antisera and platelet counts and bone marrow megakaryocytes were enumerated. Our results show that after 24 hours, all the different antiplatelet antibodies/sera induced variable degrees of thrombocytopenia in recipient mice. Compared with naïve control mice, however, histological examination of the bone marrow revealed that only 2 antibody preparations (mouse-anti-mouse ß3 sera and an anti- αIIb monoclonal antibody (MWReg30) could affect bone marrow megakaryocyte counts. Our study shows that while most antiplatelet antibodies induce acute thrombocytopenia, the majority of them do not affect the number of megakaryocytes in the bone marrow. This suggests that other mechanisms may be responsible for megakaryocyte abnormalities seen during immune thrombocytopenia.

Synthesis and Characterization of Micelle-Forming PEG-Poly(Amino Acid) Copolymers with Iron-Hydroxamate Cross-Linkable Blocks for Encapsulation and Release of Hydrophobic Drugs.

Described is the development of a polymeric micelle drug delivery platform that addresses the physical property limitations of many nanovectors. The system employs triblock copolymers comprised of a hydrophilic poly(ethylene glycol) (PEG) block, and two poly(amino acid) (PAA) blocks: a stabilizing cross-linking central block, and a hydrophobic drug encapsulation block. Detailed description of synthetic strategies and considerations found to be critical are discussed. Of note, it was determined that the purity of the α-amino acid-N-carboxyanhydrides (NCA) monomers and PEG macroinitiator are ultimately responsible for impurities that arise during the polymerization. Also, contrary to current beliefs in the field, the presence of water does not adversely affect the polymerization of NCAs. Furthermore, we describe the impact of poly(amino acid) conformational changes, through the incorporation of d-amino acids to form mixed stereochemistry PAA blocks, with regard to the physical and pharmacokinetic properties of the resulting micelles.

Imaging the delivery of drug-loaded, iron-stabilized micelles.

Nanoparticle drug carriers hold potential to improve current cancer therapy by delivering payload to the tumor environment and decreasing toxic side effects. Challenges in nanotechnology drug delivery include plasma instability, site-specific delivery, and relevant biomarkers. We have developed a triblock polymer comprising a hydroxamic acid functionalized center block that chelates iron to form a stabilized micelle that physically entraps chemotherapeutic drugs in the hydrophobic core. The iron-imparted stability significantly improves the integrity of the micelle and extends circulation pharmacokinetics in plasma over that of free drug. Furthermore, the paramagnetic properties of the iron-crosslinking exhibits contrast in the tumors for imaging by magnetic resonance. Three separate nanoparticle formulations demonstrate improved anti-tumor efficacy in xenograft models and decreased toxicity. We report a stabilized polymer micelle that improves the tolerability and efficacy of chemotherapeutic drugs, and holds potential for non-invasive MRI to image drug delivery and deposition in the tumor.

HLA alloimmunization against platelet transfusions: pathophysiology, significance, prevention and management.

Approximately five decades ago, alloimmunization to human leukocyte antigens (HLA) and platelet refractoriness were recognized as potentially serious complications of platelet transfusions. The mechanisms that result in stimulating immunity against blood products are still incompletely understood but are related to both the composition of the donor product transfused and the immune status of the recipient. Based on murine studies of platelet immunity, platelets are inherently immunogenic and there are at least two independent levels of immunoregulation against platelet transfusions. The first level resides within the recipient and is related to antigen processing/presentation events and CD8+ T cell-mediated immunosuppression. The second level relates to the donor product and includes donor antigen presenting cells (APC) levels as well as age-induced changes in donor APC and/or platelets. Implementation of pre-storage leukoreduction of cellular blood components led to a marked reduction in platelet alloimmunization and its dreaded complication, platelet refractoriness. Platelet refractoriness is usually managed by transfusion of matched platelets, selected according to one of the many published methods. It is unclear which of these methods is superior, and given the difficulty of obtaining a perfectly matched product, perhaps the most logical approach is to use a combination of selection strategies. This review discusses the various aspects of platelet alloimmunization and the clinical consequences that may result. It highlights how animal studies have shed light on the immune mechanisms responsible for allogeneic platelet immunity and immunomodulation and reviews relevant literature on clinical and laboratory manifestations of immune platelet refractoriness.

Himalaya Air Quality Impacts From the COVID-19 Lockdown Across the Indo-Gangetic Plain.

Starting in January 2020, the novel coronavirus, now known as acute respiratory syndrome coronavirus (SARS-CoV-2) and the disease that it causes (COVID-19) has had significant impacts on human health, the environment, and the economy globally. The rapid lockdown that occurred as well as its well documented timing allows for an unprecedented opportunity to examine the impact of air pollution from densely populated regions has on adjacent and pristine environments. Here, we use in situ and satellite observations to show that there was a step function decrease in two key indicators of air quality, nitrogen dioxide and airborne particulates, in locations within the Indo-Gangetic Plan (IGP) as a result of the Spring 2020 lockdown. Based on anomaly patterns, we find a dipole response with a statistically significant reduction in air pollution along the western IGP and Himalaya and an increase in air pollution in the eastern IGP and Himalaya. We show that spatial variability in the reductions in economic activity across northern India and the adjoining countries of Nepal, Pakistan, and Bangladesh contributed to this dipole as did a persistent atmospheric circulation anomaly across the region during the lockdown.

Was an Avalanche Swarm Responsible for the Devastation at Mount Everest Base Camp During the April 2015 Nepal Earthquake?

Moore, G.W.K., Paolo Cristofanelli, Paolo Bonasoni, Gian Pietro Verza, and J.L. Semple. Was an avalanche swarm responsible for the devastation at Mount Everest Base Camp during the April 2015 Nepal earthquake? High Alt Med Biol. 21:352-359, 2020. Introduction: An avalanche triggered by an earthquake on April 25, 2015, struck the Mount Everest Base Camp (EBC) resulting in 15 deaths and over 70 injuries. Despite the common occurrence of avalanches in this region, little is known about their intensity and the stability of the glaciers that ring the Mount Everest massif. Here we present unique observations from a nearby automatic weather station (AWS) in the minutes just after the earthquake. Methods: Several (AWS) were deployed along the Khumbu Valley in Nepal. The site at Kala Patthar (elevation 5,613 m asl) 3.5 km from EBC and 4 km from the col along the ridge between Pumori and Lingtren was active from 2010 to 2015 and recorded temperature, relative humidity, pressure, solar radiation, and wind speed and direction. Results: The sequence of wind direction anomalies indicated that multiple air blasts passed the AWS, each associated with a distinct avalanche source, suggesting that earthquake likely caused a number of distinct avalanches from different source regions along this ridge. Discussion: Results suggest that a swarm of avalanches collectively lead to the death and destruction at EBC, suggesting the need for improvement in our understanding of avalanches in the region as well as in our ability to model and forecast such events.

Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome.

Apoptosis plays a crucial role in development and tissue homeostasis. Some key survival pathways, such as DNA damage checkpoints and DNA repair, have been described to be inactivated during apoptosis. Here, we describe the processing of the human checkpoint protein Claspin during apoptosis. We observed cleavage of Claspin into multiple fragments in vivo. In vitro cleavage with caspases 3 and 7 of various fragments of the protein, revealed cut sites near the N- and C-termini of the protein. Using mass spectrometry, we identified a novel caspase cleavage site in Claspin at Asp25. Importantly, in addition to cleavage by caspases, we observed a proteasome-dependent degradation of Claspin under apoptotic conditions, resulting in a reduction of the levels of both full-length Claspin and its cleavage products. This degradation was not dependent upon the DSGxxS phosphodegron motif required for SCF(beta-TrCP)-mediated ubiquitination of Claspin. Finally, downregulation of Claspin protein levels by short interfering RNA resulted in an increase in apoptotic induction both in the presence and absence of DNA damage. We conclude that Claspin has antiapoptotic activity and is degraded by two different pathways during apoptosis. The resulting disappearance of Claspin from the cells further promotes apoptosis.

Second-generation nitazoxanide derivatives: thiazolides are effective inhibitors of the influenza A virus.

AIM: The only small molecule drugs currently available for treatment of influenza A virus (IAV) are M2 ion channel blockers and sialidase inhibitors. The prototype thiazolide, nitazoxanide, has successfully completed Phase III clinical trials against acute uncomplicated influenza. RESULTS: We report the activity of seventeen thiazolide analogs against A/PuertoRico/8/1934(H1N1), a laboratory-adapted strain of the H1N1 subtype of IAV, in a cell culture-based assay. A total of eight analogs showed IC50s in the range of 0.14-5.0 µM. Additionally a quantitative structure-property relationship study showed high correlation between experimental and predicted activity based on a molecular descriptor set. CONCLUSION: A range of thiazolides show useful activity against an H1N1 strain of IAV. Further evaluation of these molecules as potential new small molecule therapies is justified.

Sulfated beef insulin treatment elicits CD8+ T cells that may abrogate immunologic insulin resistance in type I diabetes.

The in vitro responses of T cells from 13 insulin-nonresistant and 1 immunologically insulin-resistant (IIR) type I diabetes patients to sulfated beef insulin (SBI) were analyzed. Insulin A-loop specific CD4+ T cells from these patients did not respond to SBI. After 1 yr of treatment with SBI the IIR patient's T cell and antibody responses to beef, pork, and human insulin progressed from very high to nondetectable levels. This occurred in parallel to the appearance of her insulin-specific CD8+ T cells, which inhibited the response of her A-loop-specific CD4+ T cells to insulin. A transient increase in her CD8+ anti-insulin antibody activity coincided with a relative lack of her CD8+ T cell activity. CD8+ T cells that regulate T cell responsiveness to insulin are probably present but difficult to detect in most type I diabetes patients. These T cells were identified in only 2 of 13 insulin-nonresistant patients who presented with lipoatrophy and insulin allergy, respectively, and who possessed high-titered, anti-insulin antibodies. Our data demonstrate that CD8+ T cells play an important role in controlling peripheral tolerance to insulin and may abrogate IIR in a diabetic patient treated with SBI.

Synthesis and facile end-group quantification of functionalized PEG azides.

Azido-functionalized poly(ethylene glycol) (PEG) derivatives are finding ever-increasing applications in the areas of conjugation chemistry and targeted drug delivery by their judicious incorporation into nanoparticle-forming polymeric systems. Quantification of azide incorporation into such PEG polymers is essential to their effective use. 1H Nuclear Magnetic Resonance (NMR) analysis offers the simplest approach; however, the relevant adjacent azide-bearing methylene protons are often obscured by the PEG manifold signals. This study describes the synthesis of 1,2,3-triazole adducts from their corresponding PEG azides via a convenient, mild click reaction, which facilitates straightforward NMR-based quantitative end-group analysis.This method was found to be compatible with many examples of bifunctional azido PEGs with molecular weights ranging from 2 to 18 kDa bearing a variety of functional groups. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 2888-2895.

Stabilized Polymer Micelles for the Development of IT-147, an Epothilone D Drug-Loaded Formulation.

Epothilones have demonstrated promising potential for oncology applications but suffer from a narrow therapeutic window. Epothilone D stabilizes microtubules leading to apoptosis, is active against multidrug-resistant cells, and is efficacious in animal tumor models despite lack of stability in rodent plasma. Clinical development was terminated in phase II due to dose limiting toxicities near the efficacious dose. Taken together, this made epothilone D attractive for encapsulation in a stabilized polymer micelle for improved safety and efficacy. We have designed a library of triblock copolymers to develop IT-147, a lead formulation of epothilone D that extends plasma circulation for accumulation in the tumor environment, and potentially decrease systemic exposure to reduce dose limiting toxicities. The drug loading efficiency for IT-147 exceeds 90%, is 75 nm in diameter, and demonstrates pH-dependent release of epothilone D without chemical conjugation or enzymatic activation. Administration of IT-147 at 20 mg/kg increases exposure of epothilone D to the plasma compartment over 6-fold compared to free drug. At the same dose, 20 mg/kg epothilone D from IT-147 is considered the no observed adverse effect level (NOAEL) but is the maximum tolerated dose for free drug. Consequently, IT-147 is positioned to be a safer, more effective means to deliver epothilone D.

Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli. Kinetics of processing by antigen presenting cells.

An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence. The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion. Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques. The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart. Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min. These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.

Immune pathophysiology of autoimmune thrombocytopenic purpura.

Chronic autoimmune thrombocytopenic purpura (AITP) is an immune-mediated, bleeding disorder in which platelets are opsonized by autoantibodies and prematurely destroyed by phagocytic cells in the reticuloendothelial system. It is classed as an organ-specific autoimmune disease primarily mediated by immunoglobulin G (IgG) autoantibodies and its etiology appears to be similar to that observed for other organ-specific autoimmune diseases. Th1 cells are important in the process, and the costimulation of Th1 cells and B cells takes place in a cytokine milieu that is reminiscent of a proinflammatory process. Chronic AITP has classically been treated with nonspecific, immunosuppressive regimens (e.g., steroids). One of the most significant developments in the treatment of AITP in the last 20 years has been the use of intravenous immunoglobulin (IVIg) and anti-D preparations. These treatments confer benefit to patients with AITP by significantly raising platelet counts. Despite this, their exact mechanisms of action remain elusive. This review focuses on cell-mediated and cytokine abnormalities within AITP, and presents data related to the mechanism of action of anti-D.

Stimulation of muscle protein degradation by murine and human epidermal cytokines: relationship to thermal injury.

Accelerated muscle proteolysis is a characteristic of systemic reaction following trauma, sepsis, or extensive thermal injury. The factors involved in this accelerated muscle breakdown have not been fully described. However, recently leukocytic pyrogen or interleukin 1 (IL-1) have been implicated in the induction of muscle protein degradation in septicemia or trauma. The epidermal cytokine epidermal cell-derived thymocyte activating factor (ETAF) is biochemically and functionally similar to IL-1. Injury to skin can augment ETAF activity. Using a murine model, we found that thermal injury can significantly enhance ETAF/IL-1 activity in a dose-dependent fashion. In addition, ETAF can cause net muscle protein breakdown in vitro. Thus, increased amounts of ETAF produced by thermally injured skin may contribute to the accelerated muscle breakdown in extensive thermal injury.

Visuo-spatial short-term recognition memory and learning after temporal lobe excisions, frontal lobe excisions or amygdalo-hippocampectomy in man.

Three groups of neurosurgical patients with temporal lobe excisions, frontal lobe excisions or unilateral amygdalo-hippocampectomy were assessed on a computerized battery of tasks designed to investigate visuo-spatial short-term recognition memory and learning. A double dissociation is reported between deficits of pattern recognition memory and spatial recognition memory which were observed in the two posterior groups and frontal lobe patients, respectively. In addition, both the temporal lobe and amygdalo-hippocampectomy patients were also impaired on a delayed matching-to-sample paradigm whilst frontal lobe patients performed at an equivalent level to controls. Finally, whilst the impaired performance of the three groups was indistinguishable on a test of paired-associate learning, quite different patterns of deficit were observed on a test of spatial working memory. These results are discussed with reference to recent suggestions that visual recognition memory is mediated by a neural system which includes, as major components, the inferotemporal cortex, the medial temporal lobe structures and particular sectors of the frontal lobe, and are compared to previous findings from patients with idiopathic Parkinson's disease and dementia of the Alzheimer type.

A critical examination of current HIV therapies.

This review critically examines the current methods of eliminating and preventing human immunodeficiency virus (HIV) infection. It illustrates both the experimental and practical limitations that each approach faces, and how they may be overcome. An overview of the HIV, including its structure and life cycle is presented. Subsequently, the two main methods of post-infection treatment, drug and gene therapy are outlined. The development of HIV vaccination is discussed with an analysis of conventional vaccination techniques leading into the novel approaches. The final option examined describes the potential for a combined vaccination regimen. Finally, the question of why these approaches have met with little success is addressed. This includes practical research limitations, as well as an examination of the qualities of HIV that make it so elusive.

Cellular immune mechanisms in chronic autoimmune thrombocytopenic purpura (ATP).

Chronic autoimmune thrombocytopenic purpura (ATP) is a common autoimmune-mediated bleeding disease in which autoantibodies are directed against platelets, resulting in their enhanced Fc-mediated destruction by macrophages in the spleen. While there has been extensive studies relating to the autoantibodies in this autoimmune disorder, relatively few have dealt with cell-mediated immunoregulation of the anti-platelet autoantibody response. Nonetheless, there is accumulating evidence that suggests the production of these anti-platelet autoantibodies is under the influence of several abnormal lymphocyte-mediated mechanisms, i.e. enhanced anti-platelet T helper cell activity with concomitant reduced T suppressor cell activity. This review focuses on these cellular events and presents a working model which attempts to explain their close interrelationships.

Pleomorphic variant of invasive lobular carcinoma of the breast.

Infiltrating lobular carcinoma (ILC) of the classic type is well recognized; less well appreciated is a group of variant forms of ILC, which includes solid, alveolar, mixed, apocrine, signet-ring, histiocytoid, and tubulolobular variants. In addition, Page et al (Diagnostic Histopathology of the Breast, Churchill Livingstone, 1987, pp 219-226) recently have emphasized the pleomorphic variant of ILC, which demonstrates the infiltrating pattern of classic ILC; however, the nuclei are more pleomorphic and have features that may overlap with those of infiltrating duct carcinoma. To determine whether pleomorphic ILC shows significant prognostic differences from classic ILC, we reviewed the clinical courses of 25 patients with classic ILC and compared them with 16 patients with pleomorphic ILC. The major determinant in placing ILC into the pleomorphic category was the presence of nuclei of nuclear grade 2 or 3. All classic ILCs had nuclei of grade 1 by the Scarff-Bloom-Richardson criteria. Survival to recurrence was significantly worse (P less than or equal to .05) for the patients with pleomorphic ILC at lengths of survival greater than 30 months. In addition, node-negative patients with pleomorphic ILC were four times more likely to experience recurrence than node-negative patients with classic ILC; those with positive nodes and pleomorphic histology were 30 times more likely to experience recurrence. Although there appeared to be a trend toward decreased overall length of survival for those patients with pleomorphic ILC when compared with patients with classic ILC, this difference was not statistically significant.

Phenotypic characteristics of lymphoid populations of middle gestation human fetal liver, spleen and thymus.

Mononuclear cells isolated from liver, spleen and thymus of fetuses between 18 and 24 weeks gestational age were stained for a number of lymphoid cell markers by indirect immunofluorescence and analyzed by flow cytometry. Studies were carried out on freshly isolated mononuclear cell preparations and on cultured cells after selective expansion in interleukin 2 (IL2). Many mononuclear cells in fresh isolates of liver and spleen could not be identified with antibodies to mature T- and B-cell markers. An average of 3% of isolated liver cells and 34% of isolated spleen cells stained positively for CD3, and 19% of liver cells and 37% of spleen cells stained positively for CD20. Lymphoid cells of the fetal thymus were an average 67% CD3+, 76% CD4+, 84% CD8+, and showed greater CD45RO staining (93%) than mononuclear cells of other tissues. Propagation of liver and spleen cell populations in culture favored CD3 phenotypes and CD8 phenotypes. Propagated T cell populations of liver and spleen were primarily TCR alpha/beta+ (81% in liver, 85% in spleen), suggesting a selective advantage in IL2 expansion of alpha/beta T cells over gamma/delta T cells. Propagated gamma/delta T cells of liver and spleen were predominantly TCR gamma/delta 2+. Whereas propagated cells of liver and spleen consisted of approximately 10% gamma/delta+ cells, thymus-derived cells expanded in culture were only an average of 2% TCR gamma/delta+, demonstrating a rarity of IL2-responsive gamma/delta T cells in middle gestation fetal thymus.