Refine
Has Fulltext
- yes (12)
Is part of the Bibliography
- yes (12)
Document Type
- Journal article (12) (remove)
Language
- English (12) (remove)
Keywords
- flow cytometry (2)
- phosphorylation (2)
- platelet physiology (2)
- platelets (2)
- Bullous pemphigoid (1)
- Immunoadsorption (1)
- Immunoapheresis (1)
- PRRT (1)
- T-cell reactivity (1)
- adenosine (1)
- adenosine diphosphate (1)
- apheresis technologies (1)
- apheresis-therapeutic (1)
- blood (1)
- blood plasma (1)
- blood processing (1)
- ceftriaxone (1)
- cell-cell interactions (1)
- clinical use (1)
- cold storage (1)
- cold temperature (1)
- convalescent plasma (1)
- drug-induced immune hemolytic anemia (1)
- fibrinogen (1)
- haemostasis (1)
- hemolysis (1)
- immunity (1)
- inhibitory signaling (1)
- melanoma (1)
- pandemic (1)
- peptide receptor (1)
- peptide vaccination (1)
- plasma (1)
- platelet activation (1)
- platelet aggregation (1)
- platelet responsiveness (1)
- purinergic receptors (1)
- radionuclide therapy (1)
- receptors (1)
- regulation (1)
- sarcoidosis (1)
- short‑term refrigeration (1)
- somatostatin receptors (1)
- specimen storage (1)
- statistical data (1)
- survivin (1)
- therapy (1)
Institute
- Institut für Klinische Transfusionsmedizin und Hämotherapie (12) (remove)
The pandemic spread of an infectious disease poses a plethora of challenges to society, clinicians, health care providers and regulating authorities. In order to mount a rapid response and to provide hope in a potentially catastrophic situation as the current COVID-19 pandemic, emergency plans, regulations and funding strategies have to be developed on regional, national and international levels. The speed needed to establish rapid response programs is challenged by the dynamics of the spread of the disease, the concurrent and competing development of different and potentially more effective treatment options, and not the least by regulatory uncertainty. Convalescent plasma, that is plasma collected from patients who have recovered from COVID-19 infections, has emerged as one of the first potential treatment options in the absence of drugs or vaccines with proven efficacy against SARS-CoV-2. The societal aspects of convalescent plasma and the public awareness gave an additional boost to the rapid employment of convalescent plasma donation platforms immediately after the SARS-CoV-2 outbreak. At the same time, uncertainty remains as to the efficacy of convalescent plasma. With evidence mostly limited to empirical reports, convalescent plasma has been used for decades for the prophylaxis and treatment of various infectious diseases. Clinical trials have addressed different infectious agents, stages of disease, target groups of patients and yielded sometimes inconclusive results. The aim of this short review is to delineate the regulatory background for the emergency use of convalescent plasma in the USA, in the European Union and in Germany, and the transition to the setting of clinical trials. In addition, we describe observations made in the process of collecting COVID-19 convalescent plasma (herein referred to as CCP), and formulate proposals to further improve the framework for rapid responses in future emergency situations.
Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors
(2021)
Background
Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells.
Methods
Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM.
Results
Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8+ T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors.
Conclusions
These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.
Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands
(2022)
Background
Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets.
Materials and Methods
Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H2DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet–neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging.
Results
Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet–neutrophil contacts.
Conclusion
Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils.
Storage of platelet concentrates (PC) at cold temperature (CT) is discussed as an alternative to the current standard of storage at room temperature (RT). Recently, we could show that cold-induced attenuation of inhibitory signaling is an important mechanism promoting platelet reactivity. For developing strategies in blood banking, it is required to elucidate the time-dependent onset of facilitated platelet activation. Thus, freshly prepared platelet-rich-plasma (PRP) was stored for 1 and 2 h at CT (2–6 °C) or at RT (20–24 °C), followed by subsequent comparative analysis. Compared to RT, basal and induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation levels were decreased under CT within 1 h by approximately 20%, determined by Western blot analysis and flow cytometry. Concomitantly, ADP- and collagen-induced threshold aggregation values were enhanced by up to 30–40%. Furthermore, platelet-covered areas on collagen-coated slides and aggregate formation under flow conditions were increased after storage at CT, in addition to induced activation markers. In conclusion, a time period of 1–2 h for refrigeration is sufficient to induce an attenuation of inhibitory signaling, accompanied with an enhancement of platelet responsiveness. Short-term refrigeration may be considered as a rational approach to obtain PC with higher functional reactivity for the treatment of hemorrhage.
Background and Objectives
Immunoadsorptions (IA) are used to remove autoantibodies from the plasma in autoimmune disorders. In this study, we evaluated the effects of a single-use, recombinant staphylococcal protein A-based immunoadsorber on blood composition of the patient.
Materials and Methods
In a cohort of patients with myasthenia gravis or stiff-person syndrome, essential parameters of blood cell count, coagulation, clinical chemistry or plasma proteins and immunoglobulins (Ig) were measured before and after IA (n = 11).
Results
In average, IA reduced the levels of total IgG, IgG1, IgG2 and IgG4 by approximately 60%, the acetylcholine receptor autoantibody levels by more than 70%. IgG3, IgA or IgM were diminished to a lower extent. In contrast to fibrinogen or other coagulation factors, the column markedly removed vitamin K-dependent coagulation factors II, VII, IX and X by approximately 40%–70%. Accordingly, international normalized ratio and activated partial thromboplastin time were increased after IA by 59.1% and 32.7%, respectively. Coagulation tests almost returned to baseline values within 24 h. Blood cell count, electrolytes, total protein or albumin were not essentially affected. No clinical events occurred.
Conclusion
The single-use, multiple-pass protein A adsorber column is highly efficient to remove IgG1, IgG2 and IgG4 or specific acetylcholine receptor autoantibodies from the plasma. Coagulation parameters should be monitored, since the column has the capacity to largely reduce vitamin K-dependent factors.
Elimination of pathogenic autoantibodies by immunoadsorption (IA) has been described as an effective adjuvant treatment in severe bullous autoimmune diseases, especially in pemphigus. There is much less experience in the treatment of bullous pemphigoid (BP). BP was diagnosed in a 62-year-old Caucasian woman presenting a pruritic rash with multiple tense blisters. Standard treatments with topical and oral corticosteroids, steroid-sparing agents including dapsone, azathioprine, mycophenolate mofetil (MMF) and intravenous immunoglobulins were ineffective or had to be discontinued due to adverse events. An immediate clinical response could be achieved by two treatment cycles of adjuvant protein A immunoadsorption (PA-IA) in addition to continued treatment with MMF (2 g/day) and prednisolone (1 mg/kg/day). Tolerance was excellent. Clinical improvement remained stable after discontinuation of IA and went along with sustained reduction of circulating autoantibodies. Our data demonstrate that PA-IA might be a safe and effective adjuvant treatment in severe and recalcitrant BP.
Introduction
Cold storage of platelets is considered to contribute to lower risk of bacterial growth and to more efficient hemostatic capacity. For the optimization of storage strategies, it is required to further elucidate the influence of refrigeration on platelet integrity. This study focused on adenosine diphosphate (ADP)-related platelet responsiveness.
Materials and Methods
Platelets were prepared from apheresis-derived platelet concentrates or from peripheral whole blood, stored either at room temperature or at 4°C. ADP-induced aggregation was tested with light transmission. Activation markers, purinergic receptor expression, and P2Y12 receptor function were determined by flow cytometry. P2Y1 and P2X1 function was assessed by fluorescence assays, cyclic nucleotide concentrations by immunoassays, and vasodilator-stimulated phosphoprotein (VASP)-phosphorylation levels by Western blot analysis.
Results
In contrast to room temperature, ADP-induced aggregation was maintained under cold storage for 6 days, associated with elevated activation markers like fibrinogen binding or CD62P expression. Purinergic receptor expression was not essentially different, whereas P2Y1 function deteriorated rapidly at cold storage, but not P2Y12 activity. Inhibitory pathways of cold-stored platelets were characterized by reduced responses to nitric oxide and prostaglandin E1. Refrigeration of citrated whole blood also led to the attenuation of induced inhibition of platelet aggregation, detectable within 24 hours.
Conclusion
ADP responsiveness is preserved under cold storage for 6 days due to stable P2Y12 activity and concomitant disintegration of inhibitory pathways enabling a higher reactivity of stored platelets. The ideal storage time at cold temperature for the highest hemostatic effect of platelets should be evaluated in further studies.
Background:
Drug induced immune hemolytic anemia (DIIHA) is a rare complication and often underdiagnosed. DIIHA is frequently associated with a bad outcome, including organ failure and even death. For the last decades, ceftriaxone has been one of the most common drugs causing DIIHA, and ceftriaxone-induced immune hemolytic anemia (IHA) has especially been reported to cause severe complications and fatal outcomes.
Case Presentation:
A 76-year-old male patient was treated with ceftriaxone for cholangitis. Short time after antibiotic exposure the patient was referred to intensive care unit due to cardiopulmonary instability. Hemolysis was observed on laboratory testing and the patient developed severe renal failure with a need for hemodialysis for 2 weeks. Medical history revealed that the patient had been previously exposed to ceftriaxone less than 3 weeks before with subsequent hemolytic reaction. Further causes for hemolytic anemia were excluded and drug-induced immune hemolytic (DIIHA) anemia to ceftriaxone could be confirmed.
Conclusions:
The case demonstrates the severity of ceftriaxone-induced immune hemolytic anemia, a rare, but immediately life-threatening condition of a frequently used antibiotic in clinical practice. Early and correct diagnosis of DIIHA is crucial, as immediate withdrawal of the causative drug is essential for the patient prognosis. Thus, awareness for this complication must be raised among treating physicians.
Sarcoidosis is a multisystem granulomatous disorder of unknown etiology that can involve virtually all organ systems. Whereas most patients present without symptoms, progressive and disabling organ failure can occur in up to 10% of subjects. Somatostatin receptor (SSTR)-directed peptide receptor radionuclide therapy (PRRT) has recently received market authorization for treatment of SSTR-positive neuroendocrine tumors.
Methods:
We describe the first case series comprising two patients with refractory multi-organ involvement of sarcoidosis who received 4 cycles of PRRT.
Results:
PRRT was well-tolerated without any acute adverse effects. No relevant toxicities could be recorded during follow-up. Therapy resulted in partial response accompanied by a pronounced reduction in pain (patient #1) and stable disease regarding morphology as well as disease activity (patient #2), respectively.
Conclusion:
Peptide receptor radionuclide therapy in sarcoidosis is feasible and might be a new valuable tool in patients with otherwise treatment-refractory disease. Given the long experience with and good tolerability of PRRT, further evaluation of this new treatment option for otherwise treatment-refractory sarcoidosis in larger patient cohorts is warranted.
Background:
Platelets are important for effective hemostasis and considered to be involved in pathophysiological processes, e.g. in cardiovascular diseases. Platelets provided for research or for therapeutic use are frequently separated from citrated whole blood (WB) stored for different periods of time. Although functionally intact platelets are required, the stability of platelet integrity, e.g. adenosine diphosphate (ADP) mediated responsiveness, has never been thoroughly investigated in citrated WB under ex vivo conditions.
Objectives:
Platelet integrity was evaluated at different time points in citrated WB units, collected from healthy donors and stored for 5 days at ambient temperature. The analysis included the measurement of activation markers, of induced light transmission aggregometry and of purinergic receptor expression or function. Inhibitory pathways were explored by determination of basal vasodilator-stimulated phosphoprotein (VASP)-phosphorylation, intracellular cyclic nucleotide levels and the content of phosphodiesterase 5A. Fresh peripheral blood (PB) samples served as controls.
Results:
On day 5 of storage, thrombin receptor activating peptide-6 (TRAP-6) stimulated CD62P expression and fibrinogen binding were comparable to PB samples. ADP induced aggregation continuously decreased during storage. Purinergic receptor expression remained unchanged, whereas the P2Y1 activity progressively declined in contrast to preserved P2Y12 and P2X1 function. Inhibitory pathways were unaffected except for a slight elevation of VASP phosphorylation at Ser\(^{239}\) on day 5.
Conclusion:
After 5 days of storage in citrated WB, platelet responsiveness to TRAP-6 is sufficiently maintained. However, ADP-mediated platelet integrity is more sensitive to deterioration, especially after storage for more than 2 days. Decreasing ADP-induced aggregation is particularly caused by the impairment of the purinergic receptor P2Y1 activity. These characteristics should be considered in the use of platelets from stored citrated WB for experimental or therapeutic issues.