The IL-10 polarized cytokine pattern in innate and adaptive immunity cells contribute to the development of FVIII inhibitors

Background

Hemophilia A (HA) is an X-linked inherited bleeding disorder, resulting from a qualitative or quantitative deficiency of clotting factor VIII (FVIII). Antibodies against FVIII, also called inhibitors, block the procoagulant activity of FVIII; thus, impairing hemostatic activity in patients with HA. The exact mechanism underlying the immunological events behind the development of inhibitors remains unknown. This study aimed to understand immune response to FVIII in patients with HA who were either positive [HAα-FVIII(+)] or negative [HAα-FVIII(−)] for inhibitors.

Methods

Cytokine profiles [interferon-γ (IFN − γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-5, and IL-10] of innate and adaptive immune cells present in the peripheral blood of participants were characterized.

Results

Presence of inhibitors was significantly associated with decreased frequencies of TNF-α-positive monocytes and neutrophils, IL-5-positive monocytes, IL-4-positive neutrophils, and increased frequencies of IL-10-positive neutrophils and T cells. T cells from HAα-FVIII(−) patients expressed increased levels of almost all cytokines. In contrast, HAα-FVIII(+) patients showed lower levels of all cytokines in CD4⁺ and CD8⁺ T cells, except IL-10. B cells from HAα-FVIII(−) patients expressed increased levels of IL-4 while those from HAα-FVIII(+) patients expressed increased levels of IL-10.

Conclusions

The global cytokine profiles of innate and adaptive immune cells showed an anti-inflammatory/regulatory pattern in HAα-FVIII(+) patients and a mixed pattern, with a bias toward inflammatory cytokine profile, in HAα-FVIII(−) patients. The occurrence of these profiles seems to be associated with presence FVIII inhibitors.     

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.     

CD98: a type II transmembrane glycoprotein expressed from the beginning of primitive and definitive hematopoiesis may play a critical role in the development of hematopoietic cells

In our search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells we found that the Joro 177 monoclonal antibody (MoAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 12 fetal liver, and day 13 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 MoAb stimulated tyrosine phosphorylation of an integral of 124-kD protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 177 MoAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid, and erythroid lineage cells in vitro from early Lin- hematopoietic precursors. Joro 177 MoAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 177 MoAb recognizes a type II transmembrane protein, which is the mouse homologue of the human CD98 heavy chain gene. We suggest that CD98 is a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.     

Approach to idiopathic recurrent pancreatitis

Acute pancreatitis is caused by acute or chronic alcohol intake or choledocholithiasis in approximately 80% of cases. In the absence of alcohol abuse or gallstones, a variety of established and putative factors must be considered, any of which can cause a single or recurrent attacks of acute pancreatitis. When the underlying cause eludes detection following an initial thorough search and leads to a second attack, the term idiopathic acute recurrent pancreatitis (IARP) is applied. This article discusses IARP and its work-up.     

Intra-Arterial Thrombolysis Within Three Hours of Stroke Onset in Middle Cerebral Artery Strokes

Background and Purpose

The Prolyse in Acute Cerebral Thromboembolism II (PROACT II) trial showed improved outcomes in patients with proximal middle cerebral artery (MCA) occlusions treated with intra-arterial (IA) thrombolysis within 6 h of stroke onset. We analyzed outcomes of patients with proximal MCA occlusions treated within 3 h of stroke onset in order to determine the influence of time-to-treatment on clinical and angiographic outcomes in patients receiving IA thrombolysis.

Methods

Thirty-five patients from three academic institutions with angiographically demonstrated proximal MCA occlusions were treated with IA thrombolytics within 3 h of stroke onset. Outcome measures included outcomes at 30–90 day follow-up, recanalization rates, incidence of symptomatic intracranial hemorrhage, and mortality in the first 90 days. The endpoints were compared to the IA treated and control groups of the PROACT II trial.

Results

The median admission National Institutes of Health Stroke Scale (NIHSS) score was 16 (range 4–24). The mean time to initiation of treatment was 106 min (range 10–180 min). Sixty-six percent of patients treated, had a modified Rankin Scale (mRS) score of 2 or less at 1–3 month follow-up compared to 40% in the PROACT II trial. The recanalization rate was 77% (versus 66% in PROACT II). The symptomatic intracranial hemorrhage rate was 11% (versus 10% in PROACT II) and the mortality rate was 23% (versus 25% in PROACT II).

Conclusion

Time-to-treatment is just as important in IA thrombolysis as it is in IV thrombolysis, both for improving clinical outcomes and recanalization rates as well.     

Minimally invasive neuroradiologic model of preclinical transient middle cerebral artery occlusion in canines

Stroke is currently the third leading cause of death in the United States, with approximately 780,000 Americans affected by a new or recurring stroke each year. Although a variety of therapeutic approaches have shown promise in small-animal models of stroke, the vast majority of clinical trials to test the efficacy of such modalities have failed. To bridge the translational gap between laboratory and clinical research, we developed a preclinical model of acute ischemic stroke in dogs. Using a minimally invasive endovascular approach, a platinum coil was intravascularly guided through the vertebrobasilar system under C-arm fluoroscopy to occlude the M1 segment of the middle cerebral artery (MCA) for 1 h. The approach included femoral artery catheterization to access the MCA and therefore eliminated the occurrence of head trauma associated with other preclinical stroke models relying on transorbital or craniectomy approaches. After 1 h of focal MCA ischemia, the coil was retrieved to cause reperfusion, which was verified by arteriograms. At 24 h, T2-weighted coronal magnetic resonance (MR) images were acquired and processed for three-dimensional reconstruction of the brain and its vasculature. Infarction, limited to the area at risk, was noted. Two independent observers calculated the mean percentage hemispherical lesion volumes as follows: observer 1, 30.9 ± 2.1%; observer 2, 31.2 ± 4.3%. Infarct-affected changes in histology were determined by hematoxylin and eosin as well as by Fluoro-Jade staining. This work reports the successful development of a powerful preclinical model of stroke that lends itself to the study of biologic mechanisms as well as to testing experimental therapeutics.