The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion.

BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase life-span of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested. STUDY DESIGN AND METHODS: A pool-and-split design of buffy coat derived platelet concentrates (PCs) was used. One arm was treated with cumulative doses of NR-triflate, the control arm with sodium triflate. Storage lesion was monitored for 23 days. Platelet metabolic and functional parameters were tested. Clearance of human platelets was measured in a mouse model of transfusion. RESULTS: Total intracellular NAD levels in platelets decreased two-fold from 4.8 ± 0.5 fmol (mean ± SD, n = 6) to 2.1 ± 1.8 fmol per 103 control cells, but increased almost 10-fold to 41.5 ± 4.1 fmol per 103 NR treated platelets. This high intracellular NAD level had no significant impact on platelet count, mean platelet volume, swirling, nor on lactate and glucose levels. Platelet aggregation and integrin αIIb ß3 activation declined steadily and comparably in both conditions. GPIbα levels were slightly lower in NR-treated platelets compared to control, but this was not caused by reduced receptor shedding because glycocalicin increased similarly. Apoptotic markers cytochrome c, Bcl-xL, cleaved caspase-3, and Bak were not different throughout storage for both conditions. Platelet survival in a mouse model of transfusion was not different between NR-treated and control platelets. CONCLUSION: Platelets carry the cellular machinery to metabolize NR into NAD at rates comparable to other eukaryotic cells. Unlike those cells, platelet life-span cannot be prolonged using this strategy.

Pretargeted PET Imaging Using a Bioorthogonal 18F-Labeled trans-Cyclooctene in an Ovarian Carcinoma Model.

In cancer research, pretargeted positron emission tomography (PET) imaging has emerged as an effective two-step approach that combines the excellent target affinity and selectivity of antibodies with the advantages of using short-lived radionuclides such as fluorine-18. One possible approach is based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between tetrazines and trans-cyclooctene (TCO) derivatives. Here, we report the first successful use of an 18F-labeled small TCO compound, [18F]1 recently developed in our laboratory, to perform pretargeted immuno-PET imaging. The study was performed in an ovarian carcinoma mouse model, using a trastuzumab-tetrazine conjugate.

Glioma-derived galectin-1 regulates innate and adaptive antitumor immunity.

Galectin-1 is a glycan-binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin-1 has also been demonstrated to play a pivotal role in tumor-mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin-1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high-grade glioma model. Stable galectin-1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin-1-targeting miRNA. We demonstrated that the absence of tumor-derived but not of host-derived galectin-1 significantly prolonged the survival of glioma-bearing mice as such and in combination with dendritic cell (DC)-based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma-derived galectin-1 significantly decreased the amount of brain-infiltrating macrophages and myeloid-derived suppressor cells (MDSC) in tumor-bearing mice. Additionally, we revealed a pro-angiogenic role for galectin-1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma-derived galectin-1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high-grade gliomas. Furthermore, the prolonged survival observed in untreated and DC-vaccinated glioma-bearing mice upon the silencing of tumor-derived galectin-1 strongly suggest that the in vivo targeting of tumor-derived galectin-1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM.

Foxp3+ regulatory T cells are activated in spite of B7-CD28 and CD40-CD40L blockade.

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.

Depletion of regulatory T cells in a mouse experimental glioma model through anti-CD25 treatment results in the infiltration of non-immunosuppressive myeloid cells in the brain.

The recruitment and activation of regulatory T cells (Tregs) in the micro-environment of malignant brain tumors has detrimental effects on antitumoral immune responses. Hence, local elimination of Tregs within the tumor micro-environment represents a highly valuable tool from both a fundamental and clinical perspective. In the syngeneic experimental GL261 murine glioma model, Tregs were prophylactically eliminated through treatment with PC61, an anti-CD25 mAb. This resulted in specific elimination of CD4+CD25hiFoxp3+ Treg within brain-infiltrating lymphocytes and complete protection against subsequent orthotopic GL261 tumor challenge. Interestingly, PC61-treated mice also showed a pronounced infiltration of CD11b+ myeloid cells in the brain. Phenotypically, these cells could not be considered as Gr-1+ myeloid-derived suppressor cells (MDSC) but were identified as F4/80+ macrophages and granulocytes.

Lens epithelium-derived growth factor/p75 qualifies as a target for HIV gene therapy in the NSG mouse model.

Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF(325-530)) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD(4)(+) T-cells were transduced with lentiviral vectors encoding LEDGF(325-530), the interaction-deficient mutant LEDGF(325-530)D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF(325-530) overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD(4)(+) T-cells expressing LEDGF(325-530) in comparison with engraftment with LEDGF(325-530)D366N cells. Four weeks after transplantation with LEDGF(325-530)D366N cells, 70% of the CD(4)(+) cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF(325-530) cells only a 20% decrease in CD(4)(+) cells was measured. Liver and spleen sections of LEDGF(325-530) mice contained less HIV than LEDGF(325-530)D366N mice as measured by p24 antigen detection. LEDGF(325-530) overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD(4)(+) T-cells.

Complementarity determining regions in SARS-CoV-2 hybrid immunity.

Pre-vaccination SARS-CoV-2 infection can boost protection elicited by COVID-19 vaccination and post-vaccination breakthrough SARS-CoV-2 infection can boost existing immunity conferred by COVID-19 vaccination. Such 'hybrid immunity' is effective against SARS-CoV-2 variants. In order to understand 'hybrid immunity' at the molecular level we studied the complementarity determining regions (CDR) of anti-RBD (receptor binding domain) antibodies isolated from individuals with 'hybrid immunity' as well as from 'naive' (not SARS-CoV-2 infected) vaccinated individuals. CDR analysis was done by liquid chromatography/mass spectrometry-mass spectrometry. Principal component analysis and partial least square differential analysis showed that COVID-19 vaccinated people share CDR profiles and that pre-vaccination SARS-CoV-2 infection or breakthrough infection further shape the CDR profile, with a CDR profile in hybrid immunity that clustered away from the CDR profile in vaccinated people without infection. Thus, our results show a CDR profile in hybrid immunity that is distinct from the vaccination-induced CDR profile.

Integration of autologous dendritic cell-based immunotherapy in the standard of care treatment for patients with newly diagnosed glioblastoma: results of the HGG-2006 phase I/II trial.

PURPOSE: Dendritic cell (DC)-based tumor vaccination has rendered promising results in relapsed high-grade glioma patients. In the HGG-2006 trial (EudraCT 2006-002881-20), feasibility, toxicity, and clinical efficacy of the full integration of DC-based tumor vaccination into standard postoperative radiochemotherapy are studied in 77 patients with newly diagnosed glioblastoma. PATIENTS AND METHODS: Autologous DC are generated after leukapheresis, which is performed before the start of radiochemotherapy. Four weekly induction vaccines are administered after the 6-week course of concomitant radiochemotherapy. During maintenance chemotherapy, 4 boost vaccines are given. Feasibility and progression-free survival (PFS) at 6 months (6mo-PFS) are the primary end points. Overall survival (OS) and immune profiling, rather than monitoring, as assessed in patients' blood samples, are the secondary end points. Analysis has been done on intent-to-treat basis. RESULTS: The treatment was feasible without major toxicity. The 6mo-PFS was 70.1 % from inclusion. Median OS was 18.3 months. Outcome improved significantly with lower EORTC RPA classification. Median OS was 39.7, 18.3, and 10.7 months for RPA classes III, IV, and V, respectively. Patients with a methylated MGMT promoter had significantly better PFS (p = 0.0027) and OS (p = 0.0082) as compared to patients with an unmethylated status. Exploratory "immunological profiles" were built to compare to clinical outcome, but no statistical significant evidence was found for these profiles to predict clinical outcome. CONCLUSION: Full integration of autologous DC-based tumor vaccination into standard postoperative radiochemotherapy for newly diagnosed glioblastoma seems safe and possibly beneficial. These results were used to power the currently running phase IIb randomized clinical trial.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood.

The ongoing COVID-19 pandemic has emphasized the urgent need for rapid, accurate, and large-scale diagnostic tools. Next to this, the significance of serological tests (i.e., detection of SARS-CoV-2 antibodies) also became apparent for studying patients' immune status and past viral infection. In this work, we present a novel approach for not only measuring antibody levels but also profiling of binding kinetics of the complete polyclonal antibody response against the receptor binding domain (RBD) of SARS-CoV-2 spike protein, an aspect not possible to achieve with traditional serological tests. This fiber optic surface plasmon resonance (FO-SPR)-based label-free method was successfully accomplished in COVID-19 patient serum and, for the first time, directly in undiluted whole blood, omitting the need for any sample preparation. Notably, this bioassay (1) was on par with FO-SPR sandwich bioassays (traditionally regarded as more sensitive) in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) had a significantly shorter time-to-result of only 30 min compared to >1 or 4 h for the FO-SPR sandwich bioassay and the conventional ELISA, respectively. Finally, the label-free approach revealed that no direct correlation was present between antibody levels and their kinetic profiling in different COVID-19 patients, as another evidence to support previous hypothesis that antibody-binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Taking all this into account, the presented work positions the FO-SPR technology at the forefront of other COVID-19 serological tests, with a huge potential toward other applications in need for quantification and kinetic profiling of antibodies.

High Incidence of SARS-CoV-2 Variant of Concern Breakthrough Infections Despite Residual Humoral and Cellular Immunity Induced by BNT162b2 Vaccination in Healthcare Workers: A Long-Term Follow-Up Study in Belgium.

To mitigate the massive COVID-19 burden caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccination campaigns were initiated. We performed a single-center observational trial to monitor the mid- (3 months) and long-term (10 months) adaptive immune response and to document breakthrough infections (BTI) in healthcare workers (n = 84) upon BNT162b2 vaccination in a real-world setting. Firstly, serology was determined through immunoassays. Secondly, antibody functionality was analyzed via in vitro binding inhibition and pseudovirus neutralization and circulating receptor-binding domain (RBD)-specific B cells were assessed. Moreover, the induction of SARS-CoV-2-specific T cells was investigated by an interferon-γ release assay combined with flowcytometric profiling of activated CD4+ and CD8+ T cells. Within individuals that did not experience BTI (n = 62), vaccine-induced humoral and cellular immune responses were not correlated. Interestingly, waning over time was more pronounced within humoral compared to cellular immunity. In particular, 45 of these 62 subjects no longer displayed functional neutralization against the delta variant of concern (VoC) at long-term follow-up. Noteworthily, we reported a high incidence of symptomatic BTI cases (17.11%) caused by alpha and delta VoCs, although vaccine-induced immunity was only slightly reduced compared to subjects without BTI at mid-term follow-up.

Potent neutralizing anti-SARS-CoV-2 human antibodies cure infection with SARS-CoV-2 variants in hamster model.

Treatment with neutralizing monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to COVID-19 management. Unfortunately, SARS-CoV-2 variants escape several of these recently approved mAbs, highlighting the need for additional discovery and development. In a convalescent patient with COVID-19, we identified six mAbs, classified in four epitope groups, that potently neutralized SARS-CoV-2 D614G, beta, gamma, and delta infection in vitro, with three mAbs neutralizing omicron as well. In hamsters, mAbs 3E6 and 3B8 potently cured infection with SARS-CoV-2 Wuhan, beta, and delta when administered post-viral infection at 5 mg/kg. Even at 0.2 mg/kg, 3B8 still reduced viral titers. Intramuscular delivery of DNA-encoded 3B8 resulted in in vivo mAb production of median serum levels up to 90 µg/mL, and protected hamsters against delta infection. Overall, our data mark 3B8 as a promising candidate against COVID-19, and highlight advances in both the identification and gene-based delivery of potent human mAbs.

Experimental immunotherapy for malignant glioma: lessons from two decades of research in the GL261 model.

Nearly twenty years of experimental immunotherapy for malignant glioma yielded important insights in the mechanisms governing glioma immunology. Still considered promising, it is clear that immunotherapy does not on its own represent the magic bullet in glioma therapy. In this review, we summarize the major immunotherapeutic achievements in the mouse GL261 glioma model, which has emerged as the gold standard syngeneic model for experimental glioma therapy. Gene therapy, monoclonal antibody treatment, cytokine therapy, cell transfer strategies and dendritic cell therapy were hereby considered. Apart from the considerable progress made in understanding glioma immunology in this model, we also addressed its most pertinent issues and shortcomings. Despite these, the GL261 model will remain indispensable in glioma research since it is a fast, highly reproducible and easy-to-establish model system.

Haptoglobin deficiency facilitates the development of autoimmune inflammation.

Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL-6. HP has been demonstrated to modulate the immune response and to have anti-inflammatory activities. To analyze HP's effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp(-/-)) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). Hp(-/-)mice suffered from a more severe disease that was associated with increased expression of IL-17A, IL-6, and IFN-gamma mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL-17+ CD4+ and IFN-gamma+ CD4+ cells persisted in the CNS of Hp(-/-) mice. Absence of HP affected the priming and differentiation of T cells after MOG(35-55) immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp(-/-) T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.

Integration of autologous dendritic cell-based immunotherapy in the primary treatment for patients with newly diagnosed glioblastoma multiforme: a pilot study.

Despite resection, radiochemotherapy, and maintenance temozolomide chemotherapy (TMZm), the prognosis of patients with glioblastoma multiforme (GBM) remains poor. We integrated immunotherapy in the primary standard treatment for eight pilot adult patients (median age 50 years) with GBM, to assess clinical and immunological feasibility and toxicity in preparation of a phase I/II protocol HGG-2006. After maximum, safe resection, leukapheresis was performed before radiochemotherapy, and four weekly vaccinations with autologous GBM lysate-loaded monocyte-derived dendritic cells were given after radiochemotherapy. Boost vaccines with lysates were given during TMZm. During the course of vaccination, immunophenotyping showed a relative increase in CD8+CD25+ cells in six of the seven patients, complying with the prerequisites for implementation of immunotherapy in addition to postoperative radiochemotherapy. In five patients, a more than twofold increase in tumor antigen-reacting IFN-gamma-producing T cells on Elispot was seen at the fourth vaccination compared with before vaccination. In three of these five patients this more than twofold increase persisted after three cycles of TMZm. Quality of life during vaccination remained excellent. Progression-free survival at six months was 75%. Median overall survival for all patients was 24 months (range: 13-44 months). The only serious adverse event was an ischemic stroke eight months postoperatively. We conclude that tumor vaccination, fully integrated within the standard primary postoperative treatment for patients with newly diagnosed GBM, is feasible and well tolerated. The survival data were used to power a currently running phase I/II trial.

Selective inhibition of TGF-ß1 produced by GARP-expressing Tregs overcomes resistance to PD-1/PD-L1 blockade in cancer.

TGF-ß1, ß2 and ß3 bind a common receptor to exert vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Global TGF-ß inhibition thus bears the risk of undesired tumor-promoting effects. We show that selective blockade of TGF-ß1 production by Tregs with antibodies against GARP:TGF-ß1 complexes induces regressions of mouse tumors otherwise resistant to anti-PD-1 immunotherapy. Effects of combined GARP:TGF-ß1/PD-1 blockade are immune-mediated, do not require FcγR-dependent functions and increase effector functions of anti-tumor CD8+ T cells without augmenting immune cell infiltration or depleting Tregs within tumors. We find GARP-expressing Tregs and evidence that they produce TGF-ß1 in one third of human melanoma metastases. Our results suggest that anti-GARP:TGF-ß1 mAbs, by selectively blocking a single TGF-ß isoform emanating from a restricted cellular source exerting tumor-promoting activity, may overcome resistance to PD-1/PD-L1 blockade in patients with cancer.

DC vaccination with anti-CD25 treatment leads to long-term immunity against experimental glioma.

We studied the feasibility, efficacy, and mechanisms of dendritic cell (DC) immunotherapy against murine malignant glioma in the experimental GL261 intracranial (IC) tumor model. When administered prophylactically, mature DCs (DCm) ex vivo loaded with GL261 RNA (DCm-GL261-RNA) protected half of the vaccinated mice against IC glioma, whereas treatment with mock-loaded DCm or DCm loaded with irrelevant antigens did not result in tumor protection. In DCm-GL261-RNA-vaccinated mice, a tumor-specific cellular immune response was observed ex vivo in the spleen and tumordraining lymph node cells. Specificity was also shown in vivo on the level of tumor challenge. Depletion of CD8(+) T-cells by anti-CD8 treatment at the time of tumor challenge demonstrated their essential role in vaccine-mediated antitumor immunity. Depletion of CD25(+) regulatory T-cells (Tregs) by anti-CD25 (aCD25) treatment strongly enhanced the efficacy of DC vaccination and was itself also protective, independently of DC vaccination. However, DC vaccination was essential to protect the animals from IC tumor rechallenge. No long-term protection was observed in animals that initially received aCD25 treatment only. In mice that received DC and/or aCD25 treatment, we retrieved tumor-specific brain-infiltrating cytotoxic T-lymphocytes. These data clearly demonstrate the effectiveness of DC vaccination for the induction of long-lasting immunological protection against IC glioma. They also show the beneficial effect of Treg depletion in this kind of glioma immunotherapy, even combined with DC vaccination.

Wilms' tumor gene 1 (WT1) in endometrial carcinoma.

OBJECTIVE: Wilms' tumor gene (WT1), located on chromosome 11, encodes a transcription factor that contributes to the carcinogenesis of uterine sarcomas. To expand the knowledge on the biological role of WT1 in other uterine cancers, we focused on its detection in endometrial carcinoma. METHODS: In total, 36 paraffin-embedded tumors were available for WT1 immunohistochemical (IHC) analysis including endometrial endometrioid carcinoma (n=24), serous carcinoma (n=9) and clear cell carcinoma (n=3). Three slides from different sites of the tumor were analysed. Of these tumors, 32 snap frozen tissue samples were available for RT-PCR (endometrioid carcinoma (23), serous carcinoma (7) and clear cell carcinoma (2)). To compare, WT1 expression was also evaluated by IHC in benign endometrium (12) and benign endometrial polyps (5). RESULTS: WT1 positivity was noticed in tumor cells and endothelial cells, lining the intratumoral blood vessels. Overall, 72% (26/36) of tumors stained positive for WT1. RT-PCR results showed WT1 positivity in 75% (24/32) of samples. Comparing the staining patterns of the 3 different bioptic sites, tumor heterogeneity was demonstrated in the majority (72%) of samples. CONCLUSION: Although WT1 is expressed in a majority of endometrial carcinomas, a heterogeneous staining pattern is observed. This information is important for WT1-directed immunotherapy.

The Introduction of a Full Medication Review Process in a Local Hospital: Successes and Barriers of a Pilot Project in the Geriatric Ward.

For the majority of Belgian hospitals, a pharmacist-led full medication review process is not standard care and, therefore, challenging to introduce. With this study, we aimed to evaluate the successes and barriers of the implementation of a pharmacist-led full medication review process in the geriatric ward at a local Belgian hospital. To this end, we carried out an interventional study, performing a full medication review on older patients (≥70 years) with polypharmacy (≥5 drugs) who had an unplanned admission to the geriatric ward. The process consisted of 3 steps: (1) medication reconciliation upon admission; (2) medication review using an explicit reviewing tool (STOPP/START criteria or GheOP³S tool), followed by a discussion between the pharmacist and the geriatrician; and (3) medication reconciliation upon discharge. Ethical approval was obtained from the Ethical Commission of the Ghent University Hospital. Outcomes included objective data on the interventions (e.g., number of drug discrepancies; number of potentially inappropriate prescriptions (PIP)); as well as subjective experiences (e.g., satisfaction with service; opinion on inter-professional communication). There was a special focus on communication aspects within the introduction of this process. In total, 52 patients were included in the study, taking a median of 10 drugs (IQR 8-12). Upon admission, 122 drug discrepancies were detected. During medication review, 254 PIPs were detected and discussed, leading to an improvement in the appropriateness of medication use. The satisfaction of community pharmacists concerning additional communication and the satisfaction of the patients after counselling at discharge were positive. However, several barriers were encountered, such as the time-consuming process to gather necessary information from different sources, the non-continuity of the service due to the lack of trained personnel or the lack of safe, electronic platforms to share information. The communicative and non-communicative successes and hurdles encountered during this project need to be addressed in order to improve the full medication review process and to strengthen the role of the clinical pharmacist.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

BACKGROUND: Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. OBJECTIVES: To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. METHODS: Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. RESULTS: Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. CONCLUSIONS: Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells.

Milk protein-derived peptides with angiotensin-converting enzyme (ACE) inhibitory activity can reduce blood pressure in hypertensive subjects. The lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) is an ACE-inhibitory peptide released by tryptic digestion from the milk protein beta-lactoglobulin. Its ACE-inhibitory activity is 100 times lower than that of captopril. The latter is known to inhibit the release of the vasoconstrictor endothelin-1 (ET-1) by endothelial cells. The effects of ALPMHIR on the endothelium are currently unknown. In this study, the influence of ALPMHIR on release of ET-1 by endothelial cells was investigated. The basal ET-1 release of the cells was reduced by 29% (p<0.01) in the presence of 1 mM ALPMHIR, compared to 42% (p<0.01) for 0.1 mM captopril. Addition of 10 U/ml thrombin to the incubation medium increased the release of ET-1 by 66% (p<0.01). Co-incubation of 10 U/ml thrombin with 1 microM captopril or with 0.1 mM ALPMHIR inhibited the stimulated ET-1 release by 45% (p<0.01) and by 32% (p<0.01), respectively. These data indicate that dietary peptides, such as ALPMHIR, can modulate ET-1 release by endothelial cells. These effects, among other mechanisms, may play a role in the anti-hypertensive effect of milk protein-derived peptides.