Significant Decrease In Platelets Research Articles

Metabolomic Insight into Implications of Induction Chemotherapy Followed by Concomitant Chemoradiotherapy in Locally Advanced Head and Neck Cancer.

The present study compares two groups of locally advanced patients with head and neck squamous cell carcinoma (LA-HNSCC) undergoing concurrent chemoradiotherapy (cCHRT), specifically those for whom it is a first-line treatment and those who have previously received induction chemotherapy (iCHT). The crucial question is whether iCHT is a serious burden during subsequent treatment for LA-HNSCC and how iCHT affects the tolerance to cCHRT. Of the 107 LA-HNSCC patients, 54 received cisplatin-based iCHT prior to cCHRT. The patients were clinically monitored at weekly intervals from the day before until the completion of the cCHRT. The 843 blood samples were collected and divided into two aliquots: for laboratory blood tests and for nuclear magnetic resonance (NMR) spectroscopy (a Bruker 400 MHz spectrometer). The NMR metabolites and the clinical parameters from the laboratory blood tests were analyzed using orthogonal partial least squares analysis (OPLS) and the Mann-Whitney U test (MWU). After iCHT, the patients begin cCHRT with significantly (MWU p-value < 0.05) elevated blood serum lipids, betaine, glycine, phosphocholine, and reticulocyte count, as well as significantly lowered NMR inflammatory markers, serine, hematocrit, neutrophile, monocyte, red blood cells, hemoglobin, and CRP. During cCHRT, a significant increase in albumin and psychological distress was observed, as well as a significant decrease in platelet, N-acetyl-cysteine, tyrosine, and phenylalanine, in patients who received iCHT. Importantly, all clinical symptoms (except the decreased platelets) and most metabolic alterations (except for betaine, serine, tyrosine, glucose, and phosphocholine) resolve until the completion of cCHRT. In conclusion, iCHT results in hematological toxicity, altered lipids, and one-carbon metabolism, as well as downregulated inflammation, as observed at the beginning and during cCHRT. However, these complications are temporary, and most of them resolve at the end of the treatment. This suggests that iCHT prior to cCHRT does not pose a significant burden and should be considered as a safe treatment option for LA-HNSCC.

Preclinical and Clinical Use of AB1, a DNMT1 Protein Depleter, to Upregulate Fetal Hemoglobin in Townes Sickle Cell Disease (SCD) Mice and Patients with SCD

Background: Sickle Cell Disease (SCD) is an inherited hematologic disorder characterized by lifelong hemolysis, vaso-occlusion and severe complications including pain, acute chest syndrome and stroke among others. Its complex pathophysiology is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells. Fetal hemoglobin (HbF) has been well documented to interfere with HbS polymerization, however, γ-globin gene transcription is silenced after birth through the action of a repressor complex including DNA methyltransferase 1 (DNMT1) during hemoglobin switching. We previously demonstrated the ability of AB1, a potent non-cytotoxic agent which depletes DNMT1 protein, to induce HbF in sickle erythroid progenitors. To expand these findings, we aimed to 1) perform preclinical studies in the Townes SCD mouse model to determine the ability of AB1 to induce HbF and deplete DNMT1 protein; and 2) evaluate the safety of oral AB1 in patients with SCD through an open-label, dose escalating phase 1/2 study (NCT05261711). Methods: Preclinical. Townes SCD mice, 4 months old (6 mice per group) were treated by intraperitoneal injections of escalating AB1 doses of 0.5, 1.5 and 3.0 mg/dose, hydroxyurea (HU; 100 mg/kg/dose) alone or HU combined with AB1 (0.5mg/dose) and water control, 5 days/week for 6 weeks. Every 2 weeks, complete blood counts with differentials were performed along with flow cytometry for HbF positive cells (F-cells) and reticulocytes; RT-qPCR for γ- and β-globin mRNA levels was completed and the spleen was harvested for weights and DNMT1 protein levels by Western blot. Clinical Trial. After informed consent, patients with confirmed HbSS or HbSb 0 thalassemia, on stable HU doses (if on treatment), and no recent/chronic transfusions or pain crisis were approached for the study. Patients were enrolled at Duke and Augusta University, starting on 2 mg of AB1 with 4 additional cohorts (4mg, 8mg, 16mg, and 32mg) to be recruited. Doses were taken daily by mouth for 8 weeks. Safety and tolerability (primary outcomes), as well as pharmacokinetics, pharmacodynamics and HbF response (secondary outcomes) were assessed. The F-cells and mean fluorescence intensity were measured by flow cytometry and HbF% by HPLC. Results: Preclinical. In general, SCD mice treated with AB1 showed good weight gain without toxicity. Three deaths occurred in the HU and combination treatment groups along with a significant decrease in platelets, granulocytes, and monocytes; by contrast, one death occurred in the 3mg/kg AB1 group, but the blood or reticulocyte counts were not affected. By week 6, HU increased F-cells by 2.1-fold (p=0.02) compared to a 2.2-fold (p=0.006) and 1.5-fold (p=0.03) increase by AB1 1.5mg/kg and 3.0 mg/kg respectively. Likewise, γ-globin mRNA increased, and β-globin mRNA decreased at the highest dose. Spleen analysis showed decreased weights and an 80% decrease in reactive oxygen species by AB1. Finally, DNMT1 protein levels were depleted by AB1 in spleen tissue. Clinical Trial. The clinical trial opened for enrollment in the winter of 2022. Two patients completed cohort 1 and two patients have enrolled and started cohort 2. Both cohort 1 patients had HbSS with a median age of 29.5 years (range 28-31). No change in total hemoglobin or reticulocyte counts was observed in either subject treated with 2mg AB1 daily. During the treatment period two adverse events occurred (rash-drug related and elevated liver enzymes-unrelated), however there were no serious adverse events. AB1 increased F-cell levels in subject 1 by 5.7-fold (2.9% to 16.4%). Expanded analysis of subject 2 showed an increase in F-cells 3-fold (4.8% to 14.4%) and 3-fold increase in F-reticulocytes. At the lowest 2mg dose, by HPLC analysis both patients had an increase in HbF% from a mean of 6.3% to 7% supporting protein induction by AB1. Discussion: The goal of this project is to develop an oral-active HbF inducer for the treatment of individuals with SCD. Data generated in the SCD mouse showed a significant increase in F-cells and γ-globin gene transcription, along with DNMT1 protein depletion. Subsequent oral AB1 treatment of patients with HbSS confirmed increased F-cells and HbF protein levels without clinical toxicities. These findings support continuing the ongoing phase 1/2 study, which will provide further insights into the ability of AB1 to increase HbF without hematologic side effects in patients with SCD.

Abstract P1172: Shear-mediated Remodeling Of Platelet Surface Glycosylation: How Can Platelets Trim Their Sugar Coats?

Mechanical circulatory support (MCS) is associated with bleeding complications often requiring multiple transfusions or reoperation. Hypershear stress within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, impaired aggregation, and microvesiculation - all contributors to bleeding. Recent studies showed that glycosylation of surface receptors, i.e. “ sugar coat ,” plays a major role in the regulation of platelet function and lifespan. We tested the hypothesis that shear stress promotes platelet surface deglycosylation by increasing platelet glycosidase activity. Methods: Human platelets were obtained from ACD-anticoagulated blood via gel filtration on Sepharose 2B. Platelets were exposed to neuraminidase and continuous shear stress in a hemodynamic shearing device. Platelets were stained with fluorophore-conjugated lectins binding terminal sialic acids (SNA I, MAL II) and galactose (ECA), anti-CD41 and anti-CD42a a/b. Multi-colored flow cytometry was used to quantify platelet surface glycosylation. Neuraminidase and galactosidase activities of intact and lysed platelets were measured using enzyme-specific fluorogenic substrates. Results: Exposure to shear stress and neuraminidase induced a significant decrease in platelet 2,6-sialylation and galactosylation, while 2,3-sialylation remained unaltered. Neuraminidase reinforced shear-mediated platelet desialylation, causing a prominent decrease in SNA binding even at low shear, and increased platelet galactose levels by unmasking the galactose residues capped by sialic acids. Shear stress resulted in a 9.5-fold increase in platelet neuraminidase and a 1.4-fold increase in galactosidase activities vs their basal levels. Neutral neuraminidase and galactosidase were upregulated by shear, while acidic isoforms did not increase, suggesting that other than lysosomal glycosidase pools are involved. Shear stress promotes platelet deglycosylation via upregulation of platelet neuraminidase and galactosidase activities. Developing therapeutic strategies for the preservation of platelet glycosylation offers significant translational potential for the pharmacologic management of MCS platelet dysfunction and bleeding.

Novel blood product transfusion regimen to prevent clotting and citrate accumulation during continuous renal replacement therapy with regional citrate anticoagulation in children.

Introduce a novel protocol to prevent clotting and citrate accumulation (CA) from blood product transfusion (BPT) during continuous renal replacement therapy (CRRT) with regional citrate anticoagulation (RCA) in children. We prospectively compared fresh frozen plasma (FFP) and platelet transfusions between the two BPT protocols, direct transfusion protocol (DTP) and partial replacement of citrate transfusion protocol (PRCTP), in terms of the risks of clotting, citric accumulation (CA), and hypocalcemia. For DTP, blood products were directly transfused without any adjustment to the original RCA-CRRT regimen. For PRCTP, the blood products were infused into the CRRT circulation near the sodium citrate infusion point, and the dosage of 4% sodium citrate was reduced depending on the dosage of sodium citrate in the blood products. Basic information and clinical data were recorded for all children. Heart rate, blood pressure, ionized calcium (iCa) and various pressure parameters were recorded before, during and after BPT, as well as coagulation indicators, electrolytes, and blood cell counts before and after BPT. Twenty-six children received 44 PRCTPs and 15 children received 20 DTPs. The two groups had similar in vitro ionized calcium (iCa) concentrations (PRCTP: 0.33 ± 0.06 mmol/L, DTP: 0.31 ± 0.04 mmol/L), total filter lifespan (PRCTP: 49.33 ± 18.58, DTP: 50.65 ± 13.57 h), and filter lifespan after BPT (PRCTP: 25.31 ± 13.87, DTP: 23.39 ± 11.34 h). There was no visible filter clotting during BPT in any of the two groups. The two groups had no significant differences in arterial pressure, venous pressure, and transmembrane pressure before, during, or after BPT. Neither treatment led to significant decreases in WBC, RBC, or hemoglobin. The platelet transfusion group and the FFP group each had no significant decrease in platelets, and no significant increases in PT, APTT, and D-dimer. The most clinically significant changes were in the DTP group, in which the ratio of total calcium to ionized calcium (T/iCa) increased from 2.06 ± 0.19 to 2.52 ± 0.35, the percentage of patients with T/iCa above 2.5 increased from 5.0% to 45%, and the level of in vivo iCa increased from 1.02 ± 0.11 to 1.06 ± 0.09 mmol/L (all p < 0.05). Changes in these three indicators were not significant in the PRCTP group. Neither protocol was associated with filter clotting during RCA-CRRT. However, PRCTP was superior to DTP because it did not increase the risk of CA and hypocalcemia.

ОЦЕНКА ЭФФЕКТИВНОСТИ ЛЕЧЕНИЯ ЭКСПЕРИМЕНТАЛЬНЫХ ОЖОГОВЫХ РАН ПО РЕЗУЛЬТАТАМ ЛАБОРАТОРНЫХ И МОРФОЛОГИЧЕСКИХ ИССЛЕДОВАНИЙ

Purpose of the study: to study the dynamics of laboratory blood parameters, as well as the morphological characteristics of the healing of experimental burn wounds when using bacterial cellulose (BC) wound dressings compared to traditional treatment. Materials and methods. 60 rats of both sexes of the Wistar breed were studied, in which skin burn of the third degree was simulated in the experiment. The animals were divided into 3 groups, of which group 1 included individuals who used BC-based wound dressing (n = 20) in the treatment of burn wounds, in group 2 (n=20) BC plates were used after exposure to a 1% chlorhexidine solution, and in group 3 (comparisons, n=20) treatment was carried out using the traditional method (daily dressings with Levomekol ointment). On the fifth, 10th, 15th, 20th, and 28th days, blood was taken from 5 animals to determine the detailed analysis, the main biochemical constants, and the wound tissue was excised for morphological and morphometric evaluation according to the developed criteria. Results. When analyzing laboratory blood parameters, an increase in hemoglobin level was noted, more pronounced in group 3, which is associated with blood clotting due to experimental burn injury. There is a more significant decrease in platelets in group 3 by the end of the study, which characterizes the process of consumption in the open management of an infected burn wound. In groups 1 and 2, compared with traditional treatment in group 3, differences were noted, consisting in a more effective restoration of hemoglobin levels, a decrease in the level of leukocytes, alanine transaminase, and a decrease in azotemia (p&lt;0,05). According to the morphological and morphometric criteria for the healing of burn wounds on days 20 and 28, in 18 (90%) out of 20 rats of groups 1 and 2, the wounds were at the stage of almost complete epithelialization (criterion 4), while in the comparison group this was noted only in 4 (40 %) out of 10 individuals (p&lt;0,01), and in 60% the wounds were in the stage of initial or moderate epithelialization (criteria 2 and 3).

Efficacy and safety of hepatic artery infusion chemotherapy with mFOLFOX in primary liver cancer patients with hyperbilirubinemia and ineffective drainage: a retrospective cohort study.

BackgroundHyperbilirubinemia is a major barrier to anti-tumor treatment in patients with end-stage primary liver cancer. However, no research has demonstrated the efficacy and safety of hepatic artery infusion chemotherapy (HAIC) in primary liver cancer patients with hyperbilirubinemia. This study investigated HAIC with a modified oxaliplatin, fluorouracil, and leucovorin (mFOLFOX) regimen. The efficacy and safety of the treatment regimen was evaluated and the optimal conditions for further anti-tumor treatments were identified.MethodsA total of 34 patients with hyperbilirubinemia (with an elevation of more than three times the upper limit of normal range in total bilirubin) who were not candidates for surgery, transplantation, tyrosine kinase inhibitor therapy, nor immune checkpoint inhibitor (ICI) therapy, and who received HAIC with the mFOLFOX regimen were enrolled in this study. The laboratory indexes (total bilirubin, tumor markers, and blood count), quality of life [World Health Organization Quality of Life (WHOQOL)-100 score], adverse reactions [Common Terminology Criteria for Adverse Events (CTCAE)-5.0], and overall survival were analyzed.ResultsBetween June 2017 and January 2021, 34 patients received a total of 81 cycles of HAIC after percutaneous transhepatic biliary drainage (PTBD). The total bilirubin (TBIL) decreased significantly at 1 month after the last cycle of HAIC (127.8 vs. 68.3 µmol/L; P<0.01). There was no significant decrease in platelet, white blood cell, nor red blood cell counts, suggesting that HAIC had limited toxicity on the hematopoietic system. The WHOQOL-100 score significantly increased at 3 months after HAIC (78.16 vs. 69.26; P<0.05). The median overall survival was 9.5 months (range, 2–24 months), the objective response rate was 14.7%, and the disease control rate was 61.8% in HAIC-treated patients. A total of 14 patients received targeted or immunological therapy after HAIC, and 2 of these patients achieved complete remission.ConclusionsHAIC improved the liver function and the quality of life in patients with liver cancer and hyperbilirubinemia, which provided options for further anti-tumor treatments in such patients.

MELATONIN CAN NOT SIGNIFICANTLY INCREASE PLATELET COUNT

Background: Burn injury defines as skin or tissue damage caused by heat, radioactivity, electricity, or chemicals. Burn injury have an impact on physiological homeostasis. Burn injury will cause an increase in free radicals, it can also cause changes in the platelet count. Melatonin has been proposed as burn supportive therapy because it acts as an antioxidant that can eliminate free radicals. Melatonin can increase the number of the platelets through the mechanism of increasing megakaryocyte fragmentation and modulating the cytokines involved in platelet production. Aim: Proving the effects of melatonin supplementation on the number of platelets in male Wistar rats with third degree of burn injury. Methods: This research was an experimental with randomized control group pre-post test design. Samples are 12 healthy male Wistar rats then randomly divide into two groups, control and experimental group. Each rat was induced 30% burn injury under anesthesia. Rats in control group were given placebo at 0, 8, and 16 hours after burn injury, while rats in experimental group were treated with melatonin intraperitoneal at 0, 8 and 16 hours after burn injury. Blood samples were collected from the retroorbital sinuses at 0, 3, and 24 hours. Data were analyzed statistically by Paired t-Test and Independent t-Test. Results: In control group, the number of platelets at 0-3 hours (p=0,024) and 0-24 hours (p=0,039) showed a significant decrease in platelet count. In experimental group, the number of platelets at 0-3 hours (p=0,047), 0-24 hours (0,015), 3-24 hours (p=0,04) showed a significant decrease in platelet count.Conclusions: Melatonin administration did not cause a significant increase of platelets number. Keywords: Burns, Platelet count, Melatonin

Investigating the acute and sub-acute toxicity of medicinal Cuscuta chinensis Lam plant

Ethnopharmacology relevanceCuscuta chinensis Lam. (Convolvulaceae) had received growing attention as a traditional medicinal herb widely used for treating female impotence, abortion, male reproductive system disease and cardiovascular diseases, respectively. Aim of the studyThe present study investigated the acute and sub-acute toxicities of C. chinensis water extract (CLW) in the ICR mice model. Materials and methodsVarious doses of CLW (1250, 2500, and 5000 mg/kg) were administered consecutively for 14 days to evaluate the acute toxicity level with examine mortality, general behavior, body weight, food and water intake of the mice. At the end of treatmet, macroscopic observation of the skin and major internal organs in the abdominal part and organ coefficients were taken. The same doses were administered daily for 28 days to determine the sub-acute toxicity level with examine mortality, general behavior, body weight, food and water intake of the mice. At the end of treatmet, macroscopical examination of organs, tissues, cavities, organ coefficients, pathology, hematological and biochemical parameters were carried out. ResultsThe acute toxicity test results revealed an LD 50 of over 5000 mg/kg for CLW. Similarly, no CLW-related mortality and severe toxicities were experienced in the sub-acute study. However, the treatment of CLW had a reducing effect on body weight of both male and female mice, and feed intake in female mice at the all tested doses (1250, 2500 and 5000 mg/kg). Moreover, significant effects in organ coefficients of brain, liver, lung, testis and thymus became apparent due to CLW mainly at the 2500 and 5000 mg/kg. The hematological analysis result showed a significant decrease in platelets, lymphocytes, and hematocrit. In contrast, a significant increase in the neutrophils was observed in the CLW treated groups (2500 and 5000 mg/kg). Biochemical test results showed a significant increase in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels while decreasing albumin, total cholesterol and triglyceride levels after treatment of CLW mostly at the doses of 2500 and 5000 mg/kg. Mild liver toxicity in both sexes treated with 5000 mg/kg of CLW was recorded in the histopathological analysis. ConclusionsOverall, our results suggested that CLW is safe at its dose lower than 1250 mg/kg, although liver toxicity from daily use may be a matter of concern.

Safety and Clinical Impact of a Single Red Light Irradiation on Breast Tumor-Bearing Mice.

Low-level light therapy has been used in health care as a therapeutic strategy for different diseases. However, its effects on cancer are controversial. This work evaluated the effects of three energies on breast cancer-bearing mice after a single red light-emitting diode (LED) irradiation. 4T1 cells were inoculated into the mammary fat pad of female BALB/c mice. When tumor volume reached 100 mm3 , animals were irradiated by a LED irradiator (660±11nm) with energies of 1.2, 3.6, and 6.0J. Control without irradiation and healthy animals were also evaluated. Mice were monitored regarding tumor volume and total blood count. After euthanasia, their organs were examined. We observed that a single irradiation does not increase tumor volume. All irradiated groups exhibited better clinical conditions than control, which presented a significant decrease in platelet and red blood cell levels compared with healthy mice. The energy of 3.6J arrested neutrophil-lymphocyte rate besides promoting longer survival and a lower number of metastatic nodules in the lungs. These findings suggest that a single red LED irradiation causes no impact on the course of the disease. Besides, the intermediary dose-effect should be further investigated since it seems to promote better outcomes on breast cancer-bearing mice.

Acute and Subacute Toxicity Studies of the Aqueous Extract from Haloxylon scoparium Pomel (Hammada scoparia (Pomel)) by Oral Administration in Rodents.

Materials and Methods Acute toxicity test was performed on Swiss albino mice at a single oral dose of 1-10 g/kg for 14 consecutive days. General behavioral adverse effects, mortality, and latency of mortality were determined. In the subacute study, the Haloxylon scoparium Pomel extract was administered orally at doses of 500, 1000, and 2000 mg/kg daily for 30 days to Wistar rats. Body weight and selected biochemical and hematological parameters were determined at the end of the experiment. Sections of livers and kidneys were removed for histological studies. Results Acute toxicity study showed that the oral LD50 value of Haloxylon scoparium Pomel extract was 5000 mg/kg. The subacute toxicity study of Haloxylon scoparium Pomel extract at doses 500, 1000, and 2000 mg/kg did not produce any observable symptoms of toxicity and no significant variation in body weight, organ weights, food, and water consumption or mortality in all treated rats. However, the administration of the Haloxylon scoparium Pomel extract to rats at 500 mg/kg and 1000 mg/kg showed a significant decrease in platelets. Moreover, only at the highest dose (2000 mg/kg), the extract caused a significant increase in red blood cells and hemoglobin. Our results showed that subacute treatments with Haloxylon scoparium Pomel extract at doses of 1000 mg/kg and 2000 mg/kg significantly elevated alkaline phosphatase and triglycerides. Histological studies showed that the subacute treatments of rats with Haloxylon scoparium Pomel extracts, at the doses 1000 and 2000 mg/kg, induced some histopathological changes in the livers but a slight changing in kidneys. Conclusion Our results indicated low acute toxicity of the aqueous extract of Haloxylon scoparium Pomel. Furthermore, daily oral administration of Haloxylon scoparium Pomel extract caused some damages to the livers of rats treated with high doses, expressed by an increase in some enzyme activities such as ALP. Regarding the renal function, we did not find remarkable toxicity in the subacute treatment with Haloxylon scoparium Pomel extracts at doses 1000 and 2000 mg/kg. However, further toxicity assessments should be done to ascertain the safety or the toxicity of this valuable plant species “Haloxylon scoparium pomel” in subchronic treatments.

Examination of hepato-renal functions and lipid panel among pregnant women with preeclampsia in Sétif, Algeria

Preeclampsia (PE) is a multisystem pregnancy disease inflecting maternal, fetal mortality and morbidity. This case-control study aims to determine the biochemical changes in lipid profile, hepatic and renal functions among sixty preeclamptic women compared to sixty age-matching normotensive pregnant women from the Maternity Hospital in Sétif, Algeria. Several biomarkers were collected and have been determined by appropriate kits. Association between various parameters was evaluated using Pearson’s correlation coefficient, with significance at P&lt;0.05. The PE women showed a significant increase in serum aminotransferase, gamma-glutamyl-transferase, alkaline phosphatase, as well as total cholesterol, triglycerides, urea, uric acid, and creatinine levels. However, a significant decrease in platelets, total protein, albumin, and plasma sodium levels were found in PE group. A positive correlation between aminotransferases and renal biomarkers was observed.PE has deleterious effects on liver and renal functions, and could be predicted by evaluating hepatic and renal biomarkers as well as lipid profile in the early stage of disease, for targeted prevention strategies in our population. Further studies on genetic assessment in cases of PE are needed for a better understanding of this disease’s pathophysiology in Algeria.

Examination of hepato-renal functions and lipid panel among pregnant women with preeclampsia in Sétif, Algeria

Preeclampsia (PE) is a multisystem pregnancy disease inflecting maternal and fetal mortality and morbidity. This case-control study aims to determine the biochemical changes in lipid profile, hepatic and renal functions among sixty late-onset preeclamptic women compared to sixty age-matching normotensive pregnant women from the Maternity Hospital in Sétif, Algeria. Several biomarkers were collected and have been determined by appropriate kits. Association between various parameters was evaluated using Pearson’s correlation coefficient, with significance at p&lt;0.05. The PE women showed a significant increase in serum aminotransferase, gamma-glutamyl-transferase, alkaline phosphatase, as well as total cholesterol, triglycerides, urea, uric acid, and creatinine levels. However, a significant decrease in platelets, total protein, albumin, and plasma sodium levels was found in PE group. A positive correlation between aminotransferases and renal biomarkers was observed. PE has deleterious effects on liver and renal functions, and complication’s progression could be predicted by evaluating hepatic and renal biomarkers as well as lipid profile. Further studies on maternal and neonatal outcomes in cases of PE are needed for a better understanding of this disease’s pathophysiology in Algeria.

Infusion-related thrombogenesis by liver-derived mesenchymal stem cells controlled by anticoagulant drugs in 11 patients with liver-based metabolic disorders

BackgroundMesenchymal stem cell (MSC) transplantation is a fast-developing therapy in regenerative medicine. However, some concerns have been raised regarding their safety and the infusion-related pro-coagulant activity. The aim of this study is to analyze the induced thrombogenic risk and the safety of adding anticoagulants during intraportal infusions of liver-derived MSCs (HepaStem), in patients with Crigler-Najjar (CN) and urea cycle disorders (UCD).MethodsEleven patients (6 CN and 5 UCD patients) were included in this partially randomized phase 1/2 study. Three cell doses of HepaStem were investigated: low (12.5 × 106 cells/kg), intermediate (50 × 106 cells/kg), and high doses (200 × 106 cells/kg). A combination of anticoagulants, heparin (10 I.U./5 × 106cells), and bivalirudin (1.75 mg/kg/h) were added during cell infusions. The infusion-related thrombogenic risk and anticoagulation were evaluated by clinical monitoring, blood sampling (platelet and D-dimer levels, activated clotting time, etc.) and liver Doppler ultrasound. Mixed effects linear regression models were used to assess statistically significant differences.ResultsOne patient presented a thrombogenic event such as a partial portal vein thrombus after 6 infusions. Minor adverse effects such as petechiae, epistaxis, and cutaneous hemorrhage at the site of catheter placement were observed in four patients. A significant decrease in platelet and increase in D-dimer levels were observed at the end of the infusion cycle, normalizing spontaneously after 7 days. No significant and clinically relevant increase in portal vein pressure could be observed once the infusion cycle was completed.ConclusionsThe safety- and the infusion-related pro-coagulant activity remains a concern in MSC transplantation. In our study, a combination of heparin and bivalirudin was added to prevent the thrombogenic risk induced by HepaStem infusions in 11 patients. A significant decrease in platelet and increase in D-dimer levels were observed, suggesting the activation of coagulation in these patients; however, this was spontaneously reversible in time. We can conclude that adding this combination of anticoagulants is safe and limits infusion-related thrombogenesis to subclinical signs in most of the patients.Trial registrationClinicalTrials.gov identifier: NCT01765283—January 10, 2013

Bone-Marrow Suppression in Elderly Patients Following Empiric Radioiodine Therapy: Real-Life Data.

Background: Elderly patients with differentiated thyroid cancer (DTC) tend to have more advanced disease at presentation, for which high activities of radioiodine (RAI) are often recommended. However, the 2015 American Thyroid Association guidelines recommend that empirically administered activities of RAI >150 mCi should be avoided in patients >70 years of age, based on calculated bone-marrow exposure according to two dosimetry-based studies. This study aimed to evaluate the effect of RAI treatment on bone-marrow function in elderly DTC patients. Methods: DTC patients ≥70 years of age who received RAI treatment and on whom a complete blood count was performed before and after treatment were included. Blood counts within one year before RAI and one year following treatment were compared in order to assess for marrow suppression. The impact of demographic, clinical, and laboratory variables on complete blood count were assessed. Results: One hundred fifty-three treatments in 122 patients met inclusion criteria, with a mean patient age of 76 ± 4.3 years, and 75% were women. High-risk features at presentation included T4 disease in 17%, lymph node metastases in 34%, and distant metastases in 14%. Mean RAI activity was 136.8 ± 48 mCi (82% ≥ 100 mCi, 66% ≥ 150 mCi). Of 153 RAI treatments analyzed, 114 (74%) were first treatments, 28 (18%) second treatments, seven (5%) third treatments, and four (3%) fourth treatments. At 0-3 months after RAI treatment, there was a statistically significant decrease in platelets (238 ± 66 vs. 216 ± 69 × 109/L, 10% decrease; p < 0.001), white blood cells (WBC; 6.9 ± 2 vs. 6.1 ± 1.9 × 109/L, 13% decrease; p < 0.001), and hemoglobin (Hb) in women (12.8 ± 1.1 vs. 12.4 ± 1.1 g/dL, 3% decrease; p = 0.01). Mean platelets, WBC, Hb in women, and lymphocytes remained decreased (but within the reference range) one year after treatment. Subgroup analysis demonstrated platelet suppression only with activities ≥100 mCi, and WBC and Hb suppression only with activities ≥150 mCi, with mean values within the reference ranges. There were no clinically significant cytopenia events during follow-up. Conclusions: Empiric RAI treatment in elderly patients causes mild bone-marrow suppression, with little clinical significance. Activities of 150-200 mCi can be safely used when indicated.

Effect of leukapheresis on blood coagulation in patients with hyperleukocytic acute myeloid leukemia

IntroductionLeukapheresis has been proposed to reduce white blood cell (WBC) count in hyperleukocytic acute myeloid leukemia (AML). However, no survival benefit has been proven and leukapheresis can potentially affect coagulation and worsen bleeding and disseminated intravascular coagulation (DIC). We analyzed the effect of leukapheresis on coagulation tests in a cohort of hyperleukocytic AML patients. MethodsRetrospective chart review of hyperleukocytic AML patients who underwent leukapheresis between 2003 and 2014. Blood coagulation tests (platelets, PT, INR, aPTT, fibrinogen, D-Dimers and fibrin degradation products (FDP)) were collected before and after each procedure and DIC score was computed. Transfusions of platelets and coagulation factors were collected. ResultsNinety patients and 129 leukapheresis sessions were screened. After exclusion of the sessions associated with transfusions, we observed in 44 patients a significant decrease in platelets (from 75.69±89.48 to 44.59±47.71.109/L, p=0.001) and fibrinogen (from 4.05±1.29 to 3.35±1.37g/L, p<0.0005) along with an increase in PT (from 14.62±2.73 to 15.62±3.63s, p=0.001), aPTT (from 33.70±6.32 to 39.24±13.53s, p=0.009) and INR (from 1.33±0.2 to 1.45±0.34, p=0.002) after the first procedure. Bleeding complications, all intracerebral hemorrhages, were documented in 3 patients within 24h of leukapheresis. After combining 73 repeat procedures, we observed similar significant results except for the aPTT prolongation. The platelets and PT components of the DIC score, but not the fibrinogen component, were significantly increased after leukapheresis. ConclusionsIn hyperleukocytic AML patients, leukapheresis is associated with clinically significant decreases in platelets and fibrinogen and prolonged clotting times.

The immunomodulatory effects induced by dietary Zearalenone in pregnant rats

Context: Zearalenone (ZEN) is a common contaminant that is present in feedstuff of high humidity and high temperatures.Objective: The aim of this study was to investigate the effects of diets contaminated with different concentrations of ZEN on immunomodulation in early pregnant rats.Materials and methods: Forty-eight pregnant Sprague Dawley (SD) rats were randomly divided into four treatment groups fed on a diet supplemented with one of four concentrations of ZEN: 0 mg/kg (ZEN 0), 50 mg/kg (ZEN 50), 100 mg/kg (ZEN 100) and 150 mg/kg (ZEN 150). The pregnant rats were fed ZEN-treated diets from gestation days 0 to 7 and a basal diet from gestation days 8 to 20.Results: ZEN exposure (ZEN 100 and 150) caused significant decreases in splenic coefficients, viability of splenocyte and T-cell proliferation and induced histopathological damage in the spleen of early pregnant rats compared with other groups. Levels of IgG and IgA were decreased, while IgM was increased, in high doses of ZEN (ZEN 100 and ZEN 150) compared with other groups. ZEN 150 caused increases in white blood cells and hemoglobin and induced a significant decrease in platelets in blood of the pregnant rats compared with other groups. ZEN 150 increased the mRNA expression levels of interleukin (IL)-6, IL-18 and IL-1β and decreased the mRNA expression levels of interferon-γ, tumor necrosis factor-α and IL-10 in the spleen of pregnant rats compared with ZEN 0.Conclusion: High doses of ZEN-induced immunomodulatory effects on early pregnant rats by altering immunological parameters.

Spatial and Temporal Fluctuations In Marrow SDF-1 Following Radiation Injury Regulate Megakaryocyte-Vascular Niche Interactions and Circulating Platelet Levels

The development of megakaryocytes (MKs) in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine SDF-1 (CXCL12), signaling through receptor CXCR4, is produced by stromal cell populations throughout the marrow and is implicated in the maturational chemotaxis of MKs to the sinusoids. Understanding the regulation of MK localization has significance not only for optimal platelet production and the development of therapies for thrombocytopenia, but also in light of the recently proposed role for MKs in supporting hematopoietic stem cells (Heazlewood et al. 2013). In the injury setting of lethal total body irradiation (TBI), it was observed that radioresistant mature MKs relocate to the endosteal niche (Dominici et al. 2009, Olson et al. 2013). Complicating the study of marrow niches post-TBI is the vascular dilation that accompanies the drastic loss of marrow cells. Having confirmed that MKs relocate to the endosteum in our model of sublethal radiation-induced thrombocytopenia (4Gy TBI), we asked whether this localization is due to changes in the spatial distribution of the vasculature or to altered microenvironmental SDF-1.In agreement with other TBI models, we find a significant elevation in SDF-1 transcript levels in the marrow at days 1-3 following 4Gy TBI. Radioresistant MKs, which do not decrease in number until after 3 days, have significantly increased CXCR4 surface expression, a finding we also observe following SDF-1 stimulation of MKs both in vitro and in vivo. In situ hybridization was used to localize the spatial distribution of SDF-1 RNA in femoral marrow. At 2 days post-4Gy, a significant SDF-1 gradient develops with 30% higher SDF-1 message adjacent to the endosteum than in the central marrow. However, this gradient is dynamically eliminated 24 hours later at 3 days post-TBI. These shifts in SDF-1 expression are accompanied by parallel changes in the spatial distribution of MKs by immunohistochemistry. At 2 days post-TBI, there is over a 40% increase in MK in the endosteal niche. In contrast, MKs in the endosteal niche decrease by more than 15% at 3 days, coincident with a significant increase in the MKs associated with vascular endothelium. Thus, these data suggest that the spatial distribution of MKs is dependent upon the localization of SDF-1 in the rapidly fluctuating post-injury bone marrow.To determine if SDF-1 functionally contributes to MK niche changes, we stabilized endogenously-produced SDF-1 using Diprotin A, an inhibitor of SDF-1-inactivating protease DPP4. In uninjured marrow, Diprotin A treatment causes over a 30% rise in MK association with vasculature and a 20% increase in circulating platelets 24 hours later, with no change in MK number. Elevation of vascular SDF-1 by intravenous (IV) administration yields similar results. These data indicate that an endogenous SDF-1 gradient toward the vasculature contributes to homeostatic megakaryopoiesis and thrombopoiesis.At 2 days post-TBI, when endosteal SDF-1 message is increased, stabilization with Diprotin A results in a 40% decrease in MKs associated with vasculature and a small but significant decrease in platelets 24 hours later. Further supporting a role for altered SDF-1 gradients, elevating vascular levels with IV SDF-1 at 2 days causes the opposite effect of Diprotin A, with more MKs found in the vascular niche and a rise in peripheral platelet count. In contrast, at 3 days post-TBI, stabilization of endogenous SDF-1 with Diprotin A causes a further 25% increase in MKs in the vascular niche and a 10% rise in circulating platelets, consistent with the rapid loss of the endosteal SDF-1 gradient.Taken together, our data demonstrate that changes in microenvironmental SDF-1 regulate the spatial distribution of MKs in the post-TBI bone marrow. Importantly, the observed SDF-1 changes have functional consequences for platelet production, as the movement of MKs toward the endosteum decreases circulating platelets, while MK association with the vasculature increases circulating platelets. This knowledge will ultimately lead to improved therapeutic strategies to enhance platelet output in the setting of thrombocytopenia and highlights the need to carefully optimize the timing of therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Fhl2 Regulates The Function Of Hematopoietic Stem/Progenitor Cells

Fhl2 is a member of four and one half LIM domain protein family. It acts as a transcriptional modulator, regulating proliferation and apoptosis in a cell-context dependent manner. The role of endogenous Fhl2 in hematopoietic stem/progenitor cells (HSCs/HPCs) is still unknown. To determine the in vivo function of Fhl2 in hematopoiesis, we characterized the HSCs/HPCs in Fhl2-null mice. At age of 3 months, Fhl2-null mice revealed normal hematopoietic parameters. As shown by flow cytometric analysis, Fhl2-null mice had a normal distribution of Mac-1+Gr+ myeloid cells, B220+ B cells and CD3+ T cells in bone marrow and spleen, suggesting that Fhl2 loss does not affect mature cell differentiation in young mice. The frequency and total number of stem cell enriched population LSK (Lin-Sca+c-Kit+) and Long-term HSCs (LT-HSCs) (Lin-Sca+c-Kit+CD48-CD150+) were marginally increased in Fhl2-/- mice as compared to wildtype mice, whereas the frequency and the number of hematopoietic progenitor cells was reduced in Fhl2-/- mice as compared to Fhl2+/+ mice.Transplantation exposes HSCs to various stresses including replicative and oxidative stresses. To examine whether Fhl2 regulates the function of HSCs/HPCs under stress, we transplanted bone marrow cells from Fhl2-/- or control mice into lethally-irradiated wildtype mice to generate chimeric mice. 7 months after transplantation, the Fhl2-/- chimeric mice developed MDS-like disease with a significant decrease in platelet, red blood cell (RBC) counts and Hemoglobin (Hb) level as compared to control chimeric mice. The number of LT-HSC, LSK and HPCs in Fhl2-/- chimeric mice were significantly less than they were in control chimeric mice, as determined by flow cytometric analysis. However, the frequency of Mac-1+Gr-1+ myeloid cells, B220+ B cells and CD3+ T cells were comparable in Fhl2-/- and Fhl2+/+ chimeric mice, suggesting that Fhl2 loss reduces repopulation capacity of LT-HSCs but does not affect HPCs differentiation in recipient mice. As evidenced by the reduced G0 population of HSCs and LSKs in Fhl2-null chimeric mice as compared to control chimeric mice, Fhl2 may regulate HSCs self-renewal under stress by controlling HSC quiescence. To further determine the function of HSCs, we performed the competitive repopulation assay. In agreement with the observation that Fhl2-/- HSCs have a reduced repopulation capacity, the ratio of Fhl2-/- derived total Peripheral Blood (PB) cells vs. competitor-derived PB cells was significantly decreased as compared to Fhl2+/+-derived recipients at secondary transplantation, and the ratio gradually decreased up to 4-5-fold as compared to Fhl2+/+-derived recipients at tertiary transplantation.Previously, we showed that FHL2 is down-regulated in subsets of Therapy-related Myelodyplastic Syndrome or Acute Myeloid Leukemia (t-MDS/t-AML patients). Together, these data suggest that Fhl2 is required for maintenance of HSCs/HPCs, and its downregulation may contribute to the development of t-MDS/t-AML in patients. Disclosures:No relevant conflicts of interest to declare.

A Dose-Dependent Hematological Evaluation of Whole-Body Gamma-Irradiation in the Göttingen Minipig.

There is a great deal of interest in the establishment of a standardized animal model for the acute radiation syndrome to allow development of diagnostic approaches and countermeasure treatments following radiological terrorist events. Due to physiological, anatomical, and biochemical similarities to humans, the minipig is an attractive large animal model for evaluating countermeasure efficacy. This study was conducted in order to aid in the establishment of the minipig, and the Göttingen minipig in particular, as an animal model for the hematopoietic acute radiation syndrome. Animals were exposed whole-body to Co at doses of 0 (sham control), 0.25, 0.5, 0.75, 1.0, and 2.0 Gy, and hematological parameters followed in time from pre-irradiation to post-irradiation Day 7. Following irradiation, a dose-dependent decrease in total white blood cells was observed, which was determined to be statistically different as compared to control animals at all dose levels above 0.25 Gy at 24 h post-irradiation. Similarly, a dose-dependent reduction in both absolute lymphocyte count and absolute neutrophil count occurred by the earliest time point measured for all exposed animals. A significant decrease in platelets was observed at post-irradiation Day 7 in animals exposed only at the highest (2.0 Gy) level. The platelet-to-lymphocyte ratio generated for exposures ranging from 0.25-2.0 Gy was able to differentiate response between high and low exposure levels even at 7 d post exposure. In conclusion, the present study supports the development of the Göttingen minipig as a suitable large animal model to study radiation-induced hematopoietic syndrome.

Mineral constituents and toxicological profile of Jateorhiza macrantha (Menispermaceae) aqueous extract

Ethnopharmacological relevanceJateorhiza macrantha is a medicinal plant used in popular medicine to treat several diseases, including cardiovascular disorders. To date, there is no documented report available on the toxicological profile of this medicinal plant. The aim of the studyThe present study was carried out to investigate the mineral content and the safety of the oral administration of the leaf aqueous extract of Jateorhiza macrantha in mice. Materials and methodsThe presence of Ca, Mg, K, Na, Cu, Mn, Fe and Zn in the extract was analyzed using Atomic Absorption Spectrophotometer. In the acute toxicity study, Male and female Balb-C mice were administered single doses of 2.5 and 5g/kg of body weight by gavage, and were monitored for 7 days. In the subacute toxicity study, the extract was administered by gavage at doses of 150, 300 and 600mg/kg body weight daily for 6 weeks to mice (8 mice/dose/sex). After this period of treatment, 5 mice per sex and per group were sacrificed. The rest of animals per group were observed without any treatment for 2 weeks. After the treatments, some biochemical and hematological parameters were analyzed. ResultsOur findings indicate the presence of all mineral elements tested. Acute study indicates no signs of toxicity at the doses used. The LD50 value is >5.00g/kg body weight, since there was no death registered at that dose. In subacute study, hematological and biochemical parameters showed a significant decrease in platelet and significant increases in ALT and AST in both sexes and creatinin levels only in male suggesting that some compounds of the plant extract were responsible of disturbances of hematopoiesis, liver and kidney functions. ConclusionsOur results show that Jateorhiza macrantha acute toxicity towards animals is very low. However, in subacute administration, this extract induces slight injuries on hematopoiesis, liver and kidney functions, suggesting its use with caution.